Sequence-based taxonomic framework for the classification of uncultured single-stranded DNA viruses of the family Genomoviridae Arvind Varsani, 1,2,† and Mart Krupovic 3, * ‡ 1 The Biodesign Center for Fundamental and Applied Microbiomics, School of Life sciences, Center for Evolution and Medicine, Arizona State University, Tempe, AZ 85287, USA, 2 Structural Biology Research Unit, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory 7700, South Africa and 3 Unite ´ Biologie mole ´ culaire du Ge ` ne chez les Extr^ emophiles, Department of Microbiology, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, France *Corresponding authors: E-mails: [email protected]; [email protected]† http://orcid.org/0000-0003-4111-2415 ‡ http://orcid.org/0000-0001-5486-0098 Abstract With the advent of metagenomics approaches, a large diversity of known and unknown viruses has been identified in various types of environmental, plant, and animal samples. One such widespread virus group is the recently established family Genomoviridae which includes viruses with small (2–2.4 kb), circular ssDNA genomes encoding rolling-circle replication initia- tion proteins (Rep) and unique capsid proteins. Here, we propose a sequence-based taxonomic framework for classification of 121 new virus genomes within this family. Genomoviruses display 47% sequence diversity, which is very similar to that within the well-established and extensively studied family Geminiviridae (46% diversity). Based on our analysis, we establish a 78% genome-wide pairwise identity as a species demarcation threshold. Furthermore, using a Rep sequence phylogeny-based analysis coupled with the current knowledge on the classification of geminiviruses, we establish nine genera within the Genomoviridae family. These are Gemycircularvirus (n ¼ 73), Gemyduguivirus (n ¼ 1), Gemygorvirus (n ¼ 9), Gemykibivirus (n ¼ 29), Gemykolovirus (n ¼ 3), Gemykrogvirus (n ¼ 3), Gemykroznavirus (n ¼ 1), Gemytondvirus (n ¼ 1), Gemyvongvirus (n ¼ 1). The presented taxonomic framework offers rational classification of genomoviruses based on the sequence information alone and sets an ex- ample for future classification of other groups of uncultured viruses discovered using metagenomics approaches. Key words: Genomoviridae; CRESS DNA viruses; replication-associated protein; ssDNA viruses. 1. Introduction Viral metagenomics, fostered by powerful high-throughput se- quencing methods, has recently revolutionized our perception of virus diversity in the environment. Many novel groups of unculti- vated viruses have been discovered during the past decade, in- cluding viruses with small, moderately-sized, and even large genomes (Yau et al. 2011; Roux et al. 2012; Labonte and Suttle, 2013; Dutilh et al. 2014; Yutin et al. 2015; Zhou et al. 2015et al.; Dayaram et al. 2016; Steel et al. 2016). Many of these virus groups remain unclassified. To embrace the constantly growing output from viral metagenomics studies, virus taxonomy is increasingly switching from the traditional classification guided by biological features, such as serology, virion morphology or host range, to predominantly sequence-guided practices (Simmonds et al. 2017). Sequence-guided virus classification is relatively straightforward V C The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]1 Virus Evolution, 2017, 3(1): vew037 doi: 10.1093/ve/vew037 Reflections
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Sequence-based taxonomic framework for the
classification of uncultured single-stranded DNA
viruses of the family GenomoviridaeArvind Varsani12dagger and Mart Krupovic3Dagger
1The Biodesign Center for Fundamental and Applied Microbiomics School of Life sciences Center forEvolution and Medicine Arizona State University Tempe AZ 85287 USA 2Structural Biology Research UnitDepartment of Clinical Laboratory Sciences University of Cape Town Observatory 7700 South Africa and3Unite Biologie moleculaire du Gene chez les Extremophiles Department of Microbiology Institut Pasteur 25rue du Docteur Roux Paris 75015 France
With the advent of metagenomics approaches a large diversity of known and unknown viruses has been identified in varioustypes of environmental plant and animal samples One such widespread virus group is the recently established familyGenomoviridae which includes viruses with small (2ndash24 kb) circular ssDNA genomes encoding rolling-circle replication initia-tion proteins (Rep) and unique capsid proteins Here we propose a sequence-based taxonomic framework for classification of121 new virus genomes within this family Genomoviruses display47 sequence diversity which is very similar to thatwithin the well-established and extensively studied family Geminiviridae (46 diversity) Based on our analysis we establish a78 genome-wide pairwise identity as a species demarcation threshold Furthermore using a Rep sequence phylogeny-basedanalysis coupled with the current knowledge on the classification of geminiviruses we establish nine genera within theGenomoviridae family These are Gemycircularvirus (nfrac1473) Gemyduguivirus (nfrac141) Gemygorvirus (nfrac149) Gemykibivirus (nfrac1429)Gemykolovirus (nfrac143) Gemykrogvirus (nfrac143) Gemykroznavirus (nfrac141) Gemytondvirus (nfrac141) Gemyvongvirus (nfrac141) The presentedtaxonomic framework offers rational classification of genomoviruses based on the sequence information alone and sets an ex-ample for future classification of other groups of uncultured viruses discovered using metagenomics approaches
Key words Genomoviridae CRESS DNA viruses replication-associated protein ssDNA viruses
1 Introduction
Viral metagenomics fostered by powerful high-throughput se-quencing methods has recently revolutionized our perception ofvirus diversity in the environment Many novel groups of unculti-vated viruses have been discovered during the past decade in-cluding viruses with small moderately-sized and even largegenomes (Yau et al 2011 Roux et al 2012 Labonte and Suttle
2013 Dutilh et al 2014 Yutin et al 2015 Zhou et al 2015et alDayaram et al 2016 Steel et al 2016) Many of these virus groupsremain unclassified To embrace the constantly growing outputfrom viral metagenomics studies virus taxonomy is increasinglyswitching from the traditional classification guided by biologicalfeatures such as serology virion morphology or host range topredominantly sequence-guided practices (Simmonds et al 2017)Sequence-guided virus classification is relatively straightforward
VC The Author 2017 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc40) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly citedFor commercial re-use please contact journalspermissionsoupcom
1
Virus Evolution 2017 3(1) vew037
doi 101093vevew037Reflections
when the new viruses fall into existing taxa with well-defined de-marcation criteria However in the absence of isolated representa-tives and established taxonomic framework rational definition ofappropriate taxonomic ranks such as families genera and spe-cies for novel groups of uncultured viruses might be considerablymore complex Solutions to this problem are perhaps most ur-gently needed in the case of single-stranded (ss) DNA viruseswhich are extremely widespread in nature Due to their small ge-nomes sizes high mutation and recombination rates (Duffy andHolmes 2008 Duffy and Holmes 2009 Firth et al 2009 Harkinset al 2009 2014 Grigoras et al 2010 Martin et al 2011 Streck et al2011 Nguyen et al 2012 Cadar et al 2013 Roux et al 2013) andrelative ease of genome amplification an incredible diversity ofthese viruses has been discovered through metagenomics studiesin all conceivable habitats ssDNA viruses infect cells from allthree domains of life and are currently classified by theInternational Committee on Taxonomy of Viruses (ICTV) intoeleven families and one unassigned genus Members of the fami-lies Microviridae and Inoviridae infect bacteria viruses of the fami-lies Spiraviridae and Pleolipoviridae prey on archaea whereaseukaryotes host viruses classified into the families AnelloviridaeBidnaviridae Circoviridae Geminiviridae Genomoviridae Nanoviridaeand Parvoviridae and the unassigned genus Bacilladnavirus In addi-tion several widespread groups of uncultured viruses discoveredby viral metagenomics remain unclassified predominantly thosethat are circular replication-associated protein encoding single-stranded (CRESS) DNA viruses (Simmonds et al 2017)
The Genomoviridae family is one of the most recently establishedfamilies of ssDNA viruses (Adams et al 2016 Krupovic et al 2016)The family currently includes a single genus Gemycircularviruswhich contains a single species Sclerotinia gemycircularvirus 1 en-compassing a single isolate Sclerotinia sclerotiorumhypovirulence-associated DNA virus 1 (SsHADV-1) SsHADV-1 wasisolated from a plantndashpathogenic fungus Sclerotinia sclerotiorum andis the only ssDNA virus known to infect fungi (Yu et al 2010 2013)Recently Liu et al (2016) have shown that SsHADV-1 is able to in-fect a mycophagous insect (Lycoriella ingenua) which acts as a trans-mission vector SsHADV-1 virions are non-enveloped isometric20ndash22 nm in diameter and assembled from a single capsid protein(CP) (Yu et al 2010) The genome is a circular ssDNA molecule of2166 nucleotides and contains two genesmdashfor CP and rolling-circlereplication initiation protein (Rep) Like in many other ssDNA vi-ruses with circular genomes the large intergenic region ofSsHADV-1 contains a potential stem-loop structure with a nonanu-cleotide (TAATATTAT) motif at its apex which is likely to be im-portant for rolling-circle replication initiation The CP of SsHADV-1is not recognizably similar to the corresponding proteins from vi-ruses in other taxa Although SsHADV-1 remains the only isolatedand classified member of the Genomoviridae 121 viral genomeswith varying degree of similarity to that of SsHADV-1 have been re-covered and sequenced from various environmental plant- andanimal-associated samples indicating that these viruses are wide-spread and abundant in the environment (Table 1) However aproper taxonomic framework and demarcation criteria necessaryto accommodate these viruses within the family Genomoviridae arelacking Here we explore the diversity and evolution of unculturedSsHADV-1-like viruses and attempt to establish a framework fortheir classification based on sequence data alone
2 Genomoviridae diversity and speciesclassification
At the time of the analysis (August 2016) there were 121SsHADV-1-like genome sequences in the GenBank database
Each of these genomes encodes two putative proteins homolo-gous to the CP and Rep of SsHADV-1 highlighting strongcoherence of this virus assemblage Nevertheless there is a con-siderable sequence divergence within the group (SupplementaryFig S1) To investigate the extent of genomoviral sequence diver-sity we analyzed the distribution of genome-wide pairwiseidentities (one minus Hamming distances of pairwise alignedsequences with pairwise deletion of gaps) across all 121 availablegenomes (Fig 1A) using SDT v12 (Muhire Varsani and Martin2014) Most of the virus genomes in our dataset share 56ndash66genome-wide pairwise identities and only a handful containednearly identical relatives (98 identity) indicating that se-quence diversity among SsHADV-1-like viruses remains largelyunexplored
Pairwise comparison of the Rep and CP protein sequencesrevealed a broader distribution of identity values (Fig 1B and C)Notably the CPs were considerably more divergent that theReps with the highest proportion of pairwise identities being33 (versus 48 for the Rep) This observation is in line withfunctional differences of the two proteins and the fact that viralCPs often encompass host recognition determinants which areunder constant pressure to co-evolve with the cellular receptors(Kolawole et al 2014 Shangjin Cortey and Segales 2009) Basedon the analysis of distribution of the pairwise identities acrossgenomes CPs and Reps we consider a threshold of 78 to be aconservative value for species demarcation Thus all viral ge-nomes showing identities higher than this value should be con-sidered as variants of the existing species Nonetheless theremay be situations where it is difficult to assign species becausea particular new sequence is
1 gt78 similar to sequences from a particular species butislt 78 similar to other variants of that same species
2 gt78 similar to sequences from two or more differentspecies
To resolve the above conflicts we suggest adopting a similarapproach proposed for geminiviruses (Muhire et al 2013Varsani et al 2014a b Brown et al 2015) To resolve conflict 1we suggest that the new sequence be classified within any spe-cies in which it sharesgt78 identity to any one variant for-merly classified as belonging to that species even if it islt78identical to other viruses within that species To resolve conflict2 we suggest that the new sequence be considered as belongingto the species with sequences with which it shares the highestdegree of similarity
3 Rep-based approach for creation of genera
Maximum likelihood phylogenetic analyses based on the Rep of121 genomoviruses revealed several well-supported clades thatcould be considered as genera within the family (Fig 2) Wenote that the clades obtained in the Rep-based phylogeny arenot fully consistent with those obtained in the phylogeneticanalysis of the full genome or the more diverse CP sequences(Figs 3 and 4) This is most explicit in the case of the newly pro-posed genus Gemykolovirus (see below) In the Rep-based treecorresponding sequences form a sister clade to the single repre-sentative of the genus Gemyduguivirus (Fig 2) In contrast in thewhole-genome-based phylogeny gemykoloviruses form a sistergroup to members of the genus Gemycircularvirus (Fig 3) Thereason for this incongruence is likely to be intra-familial recom-bination between different genomovirus genomes resulting inchimeric entities encoding Rep and CP with different evolution-ary histories (Kraberger et al 2015a) Indeed in the CP-based
2 | Virus Evolution 2017 Vol 3 No 1
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myc
ircu
larv
irus
2K
T73
2793
Tba
tA
1037
91Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
3K
T73
2797
Tba
tA
1038
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
4K
T73
2814
Tba
tH
1038
06Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
5K
T73
2801
Tba
t12
377
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
5K
T73
2802
Tba
tH
1237
7Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
6K
T73
2796
Tba
tH
1036
39Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
6K
T73
2803
Tba
t10
3951
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
7K
T73
2807
Tba
tA
1037
46Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
7K
T73
2808
Tba
tA
1039
09Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
) (co
nti
nu
ed)
A Varsani and M Krupovic | 3
Tab
le1
Co
nti
nu
ed
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
09T
bat
H10
3746
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
10T
bat
H10
3909
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
11T
bat
L10
3746
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
12T
bat
L10
3909
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us8
KT
7328
06T
bat
3157
9Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us9
KT
7327
95T
bat
2138
3Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us10
KT
7327
94T
bat
H10
3958
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Rat
asso
ciat
edge
myc
ircu
larv
irus
1K
R91
2221
Ch
-zjr
at-0
1R
attu
sno
rveg
icus
Rat
Blo
od
Ch
ina
Liet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
GQ
3657
09Ss
HA
DV
-1C
NSc
lero
tini
asc
lero
tior
umSc
lero
tin
iaM
ycel
ials
amp
les
Ch
ina
Yu
etal
(20
10)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
5Ss
HA
DV
-1N
ZH
620
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
6Ss
HA
DV
-1N
ZSR
120
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
7Ss
HA
DV
-1N
ZSR
320
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
8Ss
HA
DV
-1N
ZSR
520
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
82Ss
HA
DV
-1-U
S-54
9LB
-12
Isch
nura
ram
buri
iD
amse
lfly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
83Ss
HA
DV
-1-U
S-54
9DFS
-12
Eryt
hem
issi
mpl
icic
ollis
Dra
gon
fly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
84Ss
HA
DV
-1-U
S-54
9SR
-12
Pant
ala
hym
enae
aD
rago
nfl
yA
bdo
men
USA
Day
aram
etal
(20
15)
Sew
age
deri
ved
gem
ycir
cula
rvir
us1
KJ5
4763
8B
S391
7Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us1
KM
8217
47Sa
Gm
V-1
NZ
-BS3
970-
2012
Sew
age
oxid
atio
npo
ndndash
Sew
age
New
Zea
lan
dK
rabe
rger
etal
(20
15a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us2
KJ5
4764
1B
S411
7Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us3
KJ5
4763
6B
S401
4Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us4
KJ5
4763
7B
S393
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us4
KJ5
4764
0B
S397
2Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us5
KJ5
4763
9B
S397
0Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Shee
pas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
4947
Fec8
0064
shee
pO
vis
arie
sSh
eep
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Shee
pas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
5151
Fec8
0064
shee
pO
vis
arie
sSh
eep
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Soyb
ean
asso
ciat
edge
myc
ircu
larv
irus
1K
T59
8248
SlaG
emV
1-1
Gly
cine
max
Soyb
ean
Leaf
USA
Mar
zan
oan
dD
om
ier
(201
6)
Gem
ydug
uiv
irus
Dra
gonfl
yas
soci
ated
gem
ydug
uivi
rus
1JX
1854
28T
O-D
FS3B
2-20
10Pa
ntal
afla
vesc
ens
Dra
gon
fly
Abd
om
enT
on
gaR
osa
rio
etal
(20
12)
Gem
ygor
viru
sC
anin
eas
soci
ated
gem
ygor
viru
s1
KT
8622
5453
Fec7
do
gC
anis
lupu
sfa
mili
aris
Do
gFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1JN
7046
10V
S470
0006
Mel
esm
eles
Euro
pea
nba
dge
rR
ecta
lsw
abN
eth
erla
nd
sva
nd
enB
ran
det
al(
2012
)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1K
T86
2238
4Fe
c7d
uck
Ana
spl
atyr
hync
hos
Du
ckFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1K
T86
2239
24Fe
c7d
uck
Ana
spl
atyr
hync
hos
Du
ckFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Pter
opus
asso
ciat
edge
myg
orvi
rus
1K
T73
2790
Tba
tA
1039
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myg
orvi
rus
1K
T73
2791
Tba
tH
1039
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Sew
age
deri
ved
gem
ygor
viru
s1
KJ4
1314
434
9H
omo
sapi
ens
Hu
man
Cer
vica
lsam
ple
Sou
thA
fric
a
Sew
age
deri
ved
gem
ygor
viru
s1
KJ5
4763
5B
S396
3Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Star
ling
asso
ciat
edge
myg
orvi
rus
1K
F371
632
P14
Stur
nus
vulg
aris
Euro
pea
nst
arli
ng
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Gem
ykib
ivir
usBa
dger
asso
ciat
edge
myk
ibiv
irus
1K
P263
543
588t
Mel
esm
eles
Euro
pea
nba
dge
rFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Blac
kro
bin
asso
ciat
edge
myk
ibiv
irus
1K
F371
634
P21
Petr
oica
trav
ersi
Ch
ath
amIs
lan
d
blac
kro
bin
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Blac
kbir
das
soci
ated
gem
ykib
ivir
us
1K
F371
633
P22
Tur
dus
mer
ula
Bla
ckbi
rdFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Bovi
neas
soci
ated
gem
ykib
ivir
us1
LK93
1483
HC
BI8
215
Bos
taur
usC
ow
Seru
mG
erm
any
Lam
bert
oet
al(
2014
)
Dra
gonfl
yas
soci
ated
gem
ykib
ivir
us1
JX18
5430
FL1-
2X-2
010
Mia
thyr
iam
arce
llaD
rago
nfl
yA
bdo
men
USA
Ro
sari
oet
al(
2012
)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KJ5
4764
4B
S398
0Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KJ5
4764
5B
S384
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KP9
7469
4D
B2
Hom
osa
pien
sH
um
anPl
asm
aG
erm
any
Zh
ang
etal
(20
16)
Hum
anas
soci
ated
gem
ykib
ivir
us1
LK93
1485
MSS
I22
25H
omo
sapi
ens
Hu
man
Blo
od
Ger
man
yLa
mbe
rto
etal
(20
14)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
5SL
1H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
6SL
2H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
7SL
3H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
(co
nti
nu
ed)
4 | Virus Evolution 2017 Vol 3 No 1
Tab
le1
Co
nti
nu
ed
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
8B
Z1
Hom
osa
pien
sH
um
anFa
eces
Bra
zil
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
9B
Z2
Hom
osa
pien
sH
um
anFa
eces
Bra
zil
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3308
0N
PU
ntre
ated
sew
age
ndashSe
wag
eN
epal
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us3
KP2
6354
654
1cH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us3
KP9
8788
7G
emyC
1cH
omo
sapi
ens
Hu
man
Plas
ma
Fran
ceU
chet
al(
2015
)
Hum
anas
soci
ated
gem
ykib
ivir
us4
KT
3638
39G
eTz1
Hom
osa
pien
sH
um
anC
ereb
rosp
inal
flu
idC
hin
aZ
ho
uet
al(
2015
)
Hum
anas
soci
ated
gem
ykib
ivir
us5
KU
3431
37H
V-G
cV2
Hom
osa
pien
sH
um
anPe
rica
rdia
lflu
idFr
ance
Hal
ary
etal
(20
16)
Mon
goos
eas
soci
ated
gem
ykib
ivir
us1
KP2
6354
516
0bH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Pter
opu
sas
soci
ated
gem
ykib
ivir
us1
KT
7328
13T
bat
A64
418
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
1K
J641
737
BtR
h-C
V-6
Tib
et20
13R
hino
loph
ushi
ppos
ider
osB
atPh
aryn
geal
amp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
1K
P263
544
181a
Her
pest
esic
hneu
mon
Egyp
tian
mo
ngo
ose
Faec
esPo
rtu
gal
Co
nce
icao
-Net
oet
al(
2015
)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
2K
J641
726
BtR
f-C
V-8
NM
2013
Rhi
nolo
phus
ferr
umeq
uinu
mB
atPh
aryn
geal
amp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Sew
age
deri
ved
gem
ykib
ivir
us1
KJ5
4764
3B
S414
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
4027
BS1
4149
chic
ken
Gal
lus
gallu
sdo
mes
ticu
sC
hic
ken
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
5252
BS1
4149
cow
Bos
taur
usC
ow
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
5556
BS1
4149
har
eLe
pus
euro
paeu
sH
are
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us2
KJ5
4764
2B
S391
1Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Gem
ykol
ovir
us
Pter
opu
sas
soci
ated
gem
ykol
ovir
us1
KT
7327
98T
bat
A10
3779
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ykol
ovir
us1
KT
7327
99T
bat
H10
3779
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myk
olov
irus
2K
T73
2800
Tba
tH
1039
21Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Gem
ykro
gvir
usB
ovin
eas
soci
ated
gem
ykro
gvir
us1
LK93
1484
HC
BI9
212
Bos
taur
usC
ow
Seru
mG
erm
any
Lam
bert
oet
al(
2014
)
Car
ibou
asso
ciat
edge
myk
rogv
irus
1K
J938
717
FaG
mC
V-1
3R
angi
fer
tara
ndus
Car
ibou
Faec
esC
anad
aN
get
al(
2014
)
Sew
age
deri
ved
gem
ykro
gvir
us1
KJ5
4763
4B
S391
3Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Gem
ykro
znav
iru
sR
abbi
tas
soci
ated
gem
ykro
znav
irus
1K
F371
631
as35
Ory
ctol
agus
cuni
culu
sR
abbi
tFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Gem
yton
dvir
usO
stri
chas
soci
ated
gem
yton
dvir
us1
KF3
7163
0as
3St
ruth
ioca
mel
usO
stri
chFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Gem
yvon
gvir
usH
um
anas
soci
ated
gem
yvon
gvir
us1
KP9
7469
3D
B1
Hom
osa
pien
sH
um
anPl
asm
aG
erm
any
Zh
ang
etal
(20
16)
A Varsani and M Krupovic | 5
tree gemykoloviruses are firmly nested within the large cladeincluding the majority of gemycircularviruses (Fig 4) Giventhat CP sequences of genomoviruses are considerably more di-vergent than the Rep sequences (Fig 1) it appears reasonable toestablish a higher (ie above the species level) taxonomicframework using the Rep (Fig 2) The latter protein is also
conserved in other eukaryotic ssDNA viruses (which is not thecase for the CP) and can thus be used to assess the place ofgenomoviruses within the larger community of ssDNA viruses
To evaluate the taxonomic structure of the Genomoviridaewe took advantage of the fact that in Rep-based phylogeneticanalyses genomoviruses consistently form a sister group to
Figure 1 Distribution of (A) genome-wide (B) Rep and (C) CP pairwise identities (121 taxa) of genomoviruses calculated using SDT v12 (Muhire Varsani and Martin
2014)
6 | Virus Evolution 2017 Vol 3 No 1
members of the Geminiviridae (Krupovic et al 2016) a compre-hensively characterized family of plant viruses with circularssDNA genomes (Varsani et al 2014b) Thus using the estab-lished taxonomic framework of the Geminiviridae overlaid on theRep-based phylogeny as a guide we could define five cladesand four additional singletons within the Genomoviridae branch(Fig 2) The defined groups displayed equivalent intra-family di-vergence as the established genera within the familyGeminiviridae (Varsani et al 2014b) The nine groups were sup-ported in both nucleotide and protein sequence inferred phylog-enies (Supplementary Fig S2) Consequently in addition to theexisting genus Gemycircularvirus we propose establishing eightnew genera within the family Genomoviridae The details of thenine genera are summarized in Fig 5 and briefly outlinedbelow
31 Gemycircularvirus
This genus has the largest number of new species (nfrac14 43 sev-enty-three genomes Table 1) and includes SsHADV-1 thefounding member of the family Members of the genus display44 diversity Viruses within the forty-three species clusterwith 99 and 96 branch support values in phylogenetic treesconstructed from either Rep or full genome sequences respec-tively (Figs 2 and 3)
32 Gemykibivirus
This is the second most populated genus (nfrac14 16 twenty-nine ge-nomes Table 1) in the family with 43 diversity among its mem-bers The name of the genus is an acronym of words geminivirus-like and myco-like kibi virus (kibi means circular in Amharic)Sequences within the fifteen species cluster with 93 branch sup-port within phylogenetic trees constructed from Rep (Fig 2) andtwo well-supported clades (100 and 96) within trees constructedfrom full genome sequences (Fig 3) suggesting that recombinationhas played an important role in the evolution of this group
33 Gemygorvirus
Members of this genus (nfrac14 5 nine genomes Table 1) display49 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like gor virus (gor means round inHindi) Sequences within the five species cluster with 100 and99 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
34 Gemykolovirus
Members of this genus (nfrac14 2 three genomes Table 1) display37 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like kolo virus (kolo means round inCzech) Sequences within the two species cluster with 100 and89 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
35 Gemykrogvirus
Members of this genus (nfrac14 3 three genomes Table 1) display33 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like krog virus (krog means round inSlovenian) Sequences within the three species cluster with 99and 100 branch support within phylogenetic trees constructedfrom either Rep or full genome sequences respectively (Figs 2and 3)
KP133079 Human associated gemykibivirus 1 KP133080 Human associated gemykibivirus 1 KP133078 Human associated gemykibivirus 1 KP133077 Human associated gemykibivirus 1 KP133075 Human associated gemykibivirus 1 KP133076 Human associated gemykibivirus 1
JX185428 Dragonfly associated gemyduguivirus 1 KP974693 Human associated gemyvongvirus 1
Figure 3 Maximum likelihood phylogenetic tree of the genomes of viruses in the Genomoviridae family The tree was inferred using FastTree (Price Dehal and Arkin
2010) (GTRthornCAT) The numbers at the branches indicate SH-like support values The topology of tree supports the proposed genera demarcation at the genome level
despite there being evidence of recombination within the genomes Branches withlt75 SH-like branch support have been collapsed
8 | Virus Evolution 2017 Vol 3 No 1
35 Gemyvongvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like vong virus (vong means circular in Lao) The sin-gle species Human associated gemyvongvirus 1 (Table 1) within thegenus shares between 56 and 62 genome-wide sequence simi-larity with viruses in other genera and is a divergent taxon inthe phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
36 Gemytondvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like tond virus (tond means round in Maltese) Thesingle species Ostrich associated gemytondvirus 1 (Table 1) withinthe genus shares between 53 and 61 genome-wide sequencesimilarity with viruses in other genera and is a divergent taxonin the phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
37 Gemykroznavirus
The name of the genus is an acronym of words geminivirus-likeand myco-like krozna virus (krozna means circular in Slovenian)The single species Rabbit associated gemykroznavirus 1 (Table 1)
KP974693 Human associated gemyvongvirus 1 KF371631 Rabbit associated gemykroznavirus 1
KP133075 Human associated gemykibivirus 2 KP133080 Human associated gemykibivirus 2 KP133079 Human associated gemykibivirus 2 KP133078 Human associated gemykibivirus 2 KP133076 Human associated gemykibivirus 2 KP133077 Human associated gemykibivirus 2
KJ413144 Human associated gemygorvirus 1 KJ547635 Sewage derived gemygorvirus 1
100
100
100
80
94
86
100
100100
10099
97
9986
8688
95
99
10091
89
100
100
80
96
84
100100
100
87
10083
79
89 95
100
91
9391
100
93
92
94
88
93100
10094
77
10096
90
94
9284
100
10089
9696
99
80
96
95
100
100
94
100
10099
89
94100
100
93100
93
100
9895
100
100
87
02 amino acid subs per site
GemyduguivirusGemycircularvirus
GemykibivirusGemygorvirus
Gemyvongvirus
Gemykolovirus
GemytondvirusGemykroznavirusGemykrogvirus
Figure 4 Maximum likelihood phylogenetic tree of the CP amino acid sequences
inferred using PHYML (Guindon et al 2010) with LGthornGthornI substitution models
and rooted with geminivirus sequences Branches withlt75 SH-like branch sup-
port have been collapsed
0 10 20 30 40 50 60 70 80
Percentage diversity
Number of isolates
Number of species
Gemyvongvirus
Gemytondvirus
Gemykroznavirus
Gemykrogvirus
Gemykolovirus
Gemykibivirus
Gemygorvirus
Gemyduguivirus
Gemycircularvirus
Figure 5 Summary of genera and the associated species and their diversity
(within genera) within the Genomoviridae family
A Varsani and M Krupovic | 9
within the genus shares between 56 and 61 genome-widesequence similarity with other sequences in other genera and isa divergent taxon in the phylogenetic trees constructed fromeither Rep or full genome sequences (Figs 2 and 3)
38 Gemyduguivirus
The name of the genus is an acronym of words geminivirus-likeand myco-like dugui virus (dugui means circular in Mongolian)The single species Dragonfly associated gemyduguivirus 1 (Table 1)within the genus shares between 57 and 62 genome-wide se-quence similarity with viruses in other genera and is a divergenttaxon in the phylogenetic trees constructed from either Rep orfull genome sequences (Figs 2 and 3)
4 Conserved sequence motifs in theGenomoviridae
CRESS DNA viruses replicate through the rolling circle replica-tion (RCR) mechanism which is similar to that used by bacterialplasmids (Khan 1997 Chandler et al 2013 Ruiz-Maso et al2015) RCR is initiated by the Rep encoded by CRESS DNAviruses cleaving the dsDNA between positions 7 and 8 of anonanucleotide sequence located at a putative stem-loopstructure at the origin of replication (Heyraud-Nitschke et al1995 Laufs et al 1995b Timchenko et al 1999 RosarioDuffy and Breitbart 2012) In the case of genomoviruses this
nonanucleotide is variable (lsquoTAWWDWRNrsquo) with lsquoTAATWYATrsquobeing the consensus nonanucleotide for gemycircularviruseswhereas gemykibiruses display the greatest variation inthis motifmdashlsquoWATAWWHANrsquo (Fig 6 Supplementary Data S1)In contrast we note that within the Geminiviridae familyincluding all recently described geminiviruses (Varsani et al2009 Briddon et al 2010 Krenz et al 2012 Loconsole et al 2012Bernardo et al 2013 Heydarnejad et al 2013 Ma et al 2015Bernardo et al 2016) the consensus nonanucleotide motif islsquoTRAKATTRCrsquo
The N terminus of the Rep contains motifs that are impor-tant for initiating RCR and it is not surprising that some of thesemotifs are well conserved across many ssDNA viruses phagesand plasmids that replicate using the RCR mechanism (Ilyinaand Koonin 1992 Vega-Rocha et al 2007a Rosario Duffy andBreitbart 2012 Krupovic 2013) The presence of a single cata-lytic tyrosine residue in the RCR motif III classifies genomovi-rus geminivirus bacilladnavirus circovirus and nanovirus Repsas members of superfamily II (Ilyina and Koonin 1992Krupovic 2013)
In genomoviruses the conserved sequence of the RCRmotif I which is thought to be involved in the recognition ofiterative sequences associated with the origin of replicationis predominantly lsquouuTYxQrsquo (u denotes hydrophobic residuesand x any residue) (Fig 6 Supplementary Data S1) with theexception of the Reps of currently known gemykolovirusesand gemykrogviruses The genomovirus RCR motif II lsquoxHxHxrsquo
Figure 6 Summary of conserved motifs that is nonanucleotide and Rep motifs illustrated using WebLogo3 (Crooks et al 2004) identified in the family Genomoviridae as
a whole and its nine genera separately Note the highly derived Walker A motif (GPHRRRRT) in the sole member of the genus Gemytondvirus
10 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
when the new viruses fall into existing taxa with well-defined de-marcation criteria However in the absence of isolated representa-tives and established taxonomic framework rational definition ofappropriate taxonomic ranks such as families genera and spe-cies for novel groups of uncultured viruses might be considerablymore complex Solutions to this problem are perhaps most ur-gently needed in the case of single-stranded (ss) DNA viruseswhich are extremely widespread in nature Due to their small ge-nomes sizes high mutation and recombination rates (Duffy andHolmes 2008 Duffy and Holmes 2009 Firth et al 2009 Harkinset al 2009 2014 Grigoras et al 2010 Martin et al 2011 Streck et al2011 Nguyen et al 2012 Cadar et al 2013 Roux et al 2013) andrelative ease of genome amplification an incredible diversity ofthese viruses has been discovered through metagenomics studiesin all conceivable habitats ssDNA viruses infect cells from allthree domains of life and are currently classified by theInternational Committee on Taxonomy of Viruses (ICTV) intoeleven families and one unassigned genus Members of the fami-lies Microviridae and Inoviridae infect bacteria viruses of the fami-lies Spiraviridae and Pleolipoviridae prey on archaea whereaseukaryotes host viruses classified into the families AnelloviridaeBidnaviridae Circoviridae Geminiviridae Genomoviridae Nanoviridaeand Parvoviridae and the unassigned genus Bacilladnavirus In addi-tion several widespread groups of uncultured viruses discoveredby viral metagenomics remain unclassified predominantly thosethat are circular replication-associated protein encoding single-stranded (CRESS) DNA viruses (Simmonds et al 2017)
The Genomoviridae family is one of the most recently establishedfamilies of ssDNA viruses (Adams et al 2016 Krupovic et al 2016)The family currently includes a single genus Gemycircularviruswhich contains a single species Sclerotinia gemycircularvirus 1 en-compassing a single isolate Sclerotinia sclerotiorumhypovirulence-associated DNA virus 1 (SsHADV-1) SsHADV-1 wasisolated from a plantndashpathogenic fungus Sclerotinia sclerotiorum andis the only ssDNA virus known to infect fungi (Yu et al 2010 2013)Recently Liu et al (2016) have shown that SsHADV-1 is able to in-fect a mycophagous insect (Lycoriella ingenua) which acts as a trans-mission vector SsHADV-1 virions are non-enveloped isometric20ndash22 nm in diameter and assembled from a single capsid protein(CP) (Yu et al 2010) The genome is a circular ssDNA molecule of2166 nucleotides and contains two genesmdashfor CP and rolling-circlereplication initiation protein (Rep) Like in many other ssDNA vi-ruses with circular genomes the large intergenic region ofSsHADV-1 contains a potential stem-loop structure with a nonanu-cleotide (TAATATTAT) motif at its apex which is likely to be im-portant for rolling-circle replication initiation The CP of SsHADV-1is not recognizably similar to the corresponding proteins from vi-ruses in other taxa Although SsHADV-1 remains the only isolatedand classified member of the Genomoviridae 121 viral genomeswith varying degree of similarity to that of SsHADV-1 have been re-covered and sequenced from various environmental plant- andanimal-associated samples indicating that these viruses are wide-spread and abundant in the environment (Table 1) However aproper taxonomic framework and demarcation criteria necessaryto accommodate these viruses within the family Genomoviridae arelacking Here we explore the diversity and evolution of unculturedSsHADV-1-like viruses and attempt to establish a framework fortheir classification based on sequence data alone
2 Genomoviridae diversity and speciesclassification
At the time of the analysis (August 2016) there were 121SsHADV-1-like genome sequences in the GenBank database
Each of these genomes encodes two putative proteins homolo-gous to the CP and Rep of SsHADV-1 highlighting strongcoherence of this virus assemblage Nevertheless there is a con-siderable sequence divergence within the group (SupplementaryFig S1) To investigate the extent of genomoviral sequence diver-sity we analyzed the distribution of genome-wide pairwiseidentities (one minus Hamming distances of pairwise alignedsequences with pairwise deletion of gaps) across all 121 availablegenomes (Fig 1A) using SDT v12 (Muhire Varsani and Martin2014) Most of the virus genomes in our dataset share 56ndash66genome-wide pairwise identities and only a handful containednearly identical relatives (98 identity) indicating that se-quence diversity among SsHADV-1-like viruses remains largelyunexplored
Pairwise comparison of the Rep and CP protein sequencesrevealed a broader distribution of identity values (Fig 1B and C)Notably the CPs were considerably more divergent that theReps with the highest proportion of pairwise identities being33 (versus 48 for the Rep) This observation is in line withfunctional differences of the two proteins and the fact that viralCPs often encompass host recognition determinants which areunder constant pressure to co-evolve with the cellular receptors(Kolawole et al 2014 Shangjin Cortey and Segales 2009) Basedon the analysis of distribution of the pairwise identities acrossgenomes CPs and Reps we consider a threshold of 78 to be aconservative value for species demarcation Thus all viral ge-nomes showing identities higher than this value should be con-sidered as variants of the existing species Nonetheless theremay be situations where it is difficult to assign species becausea particular new sequence is
1 gt78 similar to sequences from a particular species butislt 78 similar to other variants of that same species
2 gt78 similar to sequences from two or more differentspecies
To resolve the above conflicts we suggest adopting a similarapproach proposed for geminiviruses (Muhire et al 2013Varsani et al 2014a b Brown et al 2015) To resolve conflict 1we suggest that the new sequence be classified within any spe-cies in which it sharesgt78 identity to any one variant for-merly classified as belonging to that species even if it islt78identical to other viruses within that species To resolve conflict2 we suggest that the new sequence be considered as belongingto the species with sequences with which it shares the highestdegree of similarity
3 Rep-based approach for creation of genera
Maximum likelihood phylogenetic analyses based on the Rep of121 genomoviruses revealed several well-supported clades thatcould be considered as genera within the family (Fig 2) Wenote that the clades obtained in the Rep-based phylogeny arenot fully consistent with those obtained in the phylogeneticanalysis of the full genome or the more diverse CP sequences(Figs 3 and 4) This is most explicit in the case of the newly pro-posed genus Gemykolovirus (see below) In the Rep-based treecorresponding sequences form a sister clade to the single repre-sentative of the genus Gemyduguivirus (Fig 2) In contrast in thewhole-genome-based phylogeny gemykoloviruses form a sistergroup to members of the genus Gemycircularvirus (Fig 3) Thereason for this incongruence is likely to be intra-familial recom-bination between different genomovirus genomes resulting inchimeric entities encoding Rep and CP with different evolution-ary histories (Kraberger et al 2015a) Indeed in the CP-based
2 | Virus Evolution 2017 Vol 3 No 1
Tab
le1
Det
ails
of
allm
embe
rso
fth
eG
enom
ovir
idae
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Gem
ycir
cula
rvir
us
Blac
kbir
das
soci
ated
gem
ycir
cula
rvir
us1
KF3
7164
1P9
Tur
dus
mer
ula
Bla
ckbi
rdFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Blac
kbir
das
soci
ated
gem
ycir
cula
rvir
us1
KF3
7164
2P2
2T
urdu
sm
erul
aB
lack
bird
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Blac
kbir
das
soci
ated
gem
ycir
cula
rvir
us1
KF3
7164
3as
41O
vis
arie
sSh
eep
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Bovi
neas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
5352
Fec7
8023
cow
Bos
taur
usC
ow
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Brom
us
asso
ciat
edge
myc
ircu
larv
irus
1K
M51
0192
Bas
CV
-3N
Z-N
ZG
01Se
f-20
12Br
omus
hord
eace
usSo
ftbr
om
eB
ull
gras
sLe
afN
ewZ
eala
nd
Kra
berg
eret
al(
2015
b)
Cas
sava
asso
ciat
edge
myc
ircu
larv
irus
1JQ
4120
56G
14M
anih
otes
cule
nta
Cas
sava
Leaf
Gh
ana
Day
aram
etal
(20
12)
Cas
sava
asso
ciat
edge
myc
ircu
larv
irus
1JQ
4120
57G
5M
anih
otes
cule
nta
Cas
sava
Leaf
Gh
ana
Day
aram
etal
(20
12)
Chi
ckad
eeas
soci
ated
gem
ycir
cula
rvir
us1
KT
3090
2925
4065
908
Poec
ileat
rica
pillu
sB
lack
-cap
ped
chic
kad
eeB
ucc
alan
d
clo
acal
swab
USA
Han
na
etal
(20
15)
Chi
cken
asso
ciat
edge
myc
ircu
larv
iru
s1
KT
8622
4327
Fec7
9971
chic
ken
Gal
lus
gallu
sdo
mes
ticu
sC
hic
ken
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Chi
cken
asso
ciat
edge
myc
ircu
larv
iru
s1
KT
8622
4429
Fec7
9971
Ilam
aLa
ma
glam
aLl
ama
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Chi
cken
asso
ciat
edge
myc
ircu
larv
iru
s1
KT
8622
4630
Fec7
9971
ho
rse
Equu
sfe
rus
caba
llus
Ho
rse
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Chi
cken
asso
ciat
edge
myc
ircu
larv
iru
s2
KT
8622
4227
Fec1
6497
chic
ken
Gal
lus
gallu
sdo
mes
ticu
sC
hic
ken
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Dra
gonfl
yas
soci
ated
gem
ycir
cula
rvir
us1
JX18
5429
FL2-
5X-2
010
Eryt
hem
issi
mpl
icic
ollis
Dra
gon
fly
Abd
om
enU
SAR
osa
rio
etal
(20
12)
Equi
neas
soci
ated
gem
ycir
cula
rvir
us
1K
T86
2248
30Fe
c800
61h
ors
eEq
uus
feru
sca
ballu
sH
ors
eFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Fur
seal
asso
ciat
edge
myc
ircu
larv
irus
1K
F371
638
as50
Arc
toce
phal
usfo
rste
riN
ewZ
eala
nd
fur
seal
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Fur
seal
asso
ciat
edge
myc
ircu
larv
irus
1K
T86
2241
27Fe
c1ch
icke
nG
allu
sga
llus
dom
esti
cus
Ch
icke
nFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Ger
ygon
eas
soci
ated
gem
ycir
cula
rvir
us1
KF3
7163
6P2
4aG
eryg
one
albo
fron
tata
Ch
ath
amIs
lan
dw
arbl
erFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Ger
ygon
eas
soci
ated
gem
ycir
cula
rvir
us2
KF3
7163
7P2
4bG
eryg
one
albo
fron
tata
Ch
ath
amIs
lan
dw
arbl
erFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Ger
ygon
eas
soci
ated
gem
ycir
cula
rvir
us3
KF3
7163
9P2
4cG
eryg
one
albo
fron
tata
Ch
ath
amIs
lan
dw
arbl
erFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Hyp
eric
um
asso
ciat
edge
myc
ircu
larv
irus
1K
F413
620
VN
HJ1
WH
yper
icum
japo
nicu
mH
yper
icu
mLe
afV
ietn
amD
uet
al(
2014
)
Lam
aas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
4529
Fec8
0018
llam
aLa
ma
glam
aLl
ama
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Lam
aas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
4730
Fec8
0018
ho
rse
Equu
sfe
rus
caba
llus
Ho
rse
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Mal
lard
asso
ciat
edge
myc
ircu
larv
iru
s1
KF3
7163
5as
24A
nas
plat
yrhy
ncho
sM
alla
rdd
uck
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Min
iopt
erus
asso
ciat
edge
myc
ircu
larv
irus
1K
J641
719
BtM
f-C
V-2
3G
D20
12M
inio
pter
usfu
ligin
osus
Bat
Phar
ynge
alamp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Mon
goos
eas
soci
ated
gem
ycir
cula
rvir
us1
KP2
6354
747
8dH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Mos
quit
oas
soci
ated
gem
ycir
cula
rvir
us1
HQ
3350
86SD
BV
LG
Cul
exer
ythr
otho
rax
Mo
squ
ito
Mo
squ
ito
sam
ple
sU
SAN
get
al(
2011
)
Odo
nata
asso
ciat
edge
myc
ircu
larv
iru
s1
KM
5983
85O
daG
mV
-1-U
S-26
0BC
-12
Isch
nura
posi
taD
amse
lfly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Odo
nata
asso
ciat
edge
myc
ircu
larv
iru
s1
KM
5983
86O
daG
mV
-1-U
S-26
0SR
1-12
Pant
ala
hym
enae
aD
rago
nfl
yA
bdo
men
USA
Day
aram
etal
(20
15)
Odo
nata
asso
ciat
edge
myc
ircu
larv
iru
s2
KM
5983
87O
daG
mV
-2-U
S-16
42K
W-1
2A
eshn
am
ulti
colo
rD
rago
nfl
yA
bdo
men
USA
Day
aram
etal
(20
15)
Odo
nata
asso
ciat
edge
myc
ircu
larv
iru
s2
KM
5983
88O
daG
mV
-2-U
S-16
34LM
2-12
Libe
llula
satu
rata
Dra
gon
fly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Poac
eae
asso
ciat
edge
myc
ircu
larv
irus
1K
T25
3577
PaG
mV
-1T
OST
O14
-292
0420
14Br
achi
aria
defle
xaSi
gnal
gras
sLe
afT
on
gaM
ale
etal
(20
15)
Poac
eae
asso
ciat
edge
myc
ircu
larv
irus
1K
T25
3578
PaG
mV
-1T
OST
O15
-292
0420
14Br
achi
aria
defle
xaSi
gnal
gras
sLe
afT
on
gaM
ale
etal
(20
15)
Poac
eae
asso
ciat
edge
myc
ircu
larv
irus
1K
T25
3579
PaG
mV
-1T
OST
O18
-292
0420
14Br
achi
aria
defle
xaSi
gnal
gras
sLe
afT
on
gaM
ale
etal
(20
15)
Porc
ine
asso
ciat
edge
myc
ircu
larv
irus
1K
T86
2250
49Fe
c800
61p
igSu
ssc
rofa
dom
esti
caPi
gFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Porc
ine
asso
ciat
edge
myc
ircu
larv
irus
2K
F371
640
as5
Sus
scro
faD
om
esti
cp
igFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
1K
T73
2804
Tba
t45
285
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
1K
T73
2805
Tba
t47
364
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
2K
T73
2792
Tba
t10
3791
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
2K
T73
2793
Tba
tA
1037
91Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
3K
T73
2797
Tba
tA
1038
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
4K
T73
2814
Tba
tH
1038
06Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
5K
T73
2801
Tba
t12
377
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
5K
T73
2802
Tba
tH
1237
7Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
6K
T73
2796
Tba
tH
1036
39Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
6K
T73
2803
Tba
t10
3951
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
7K
T73
2807
Tba
tA
1037
46Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
7K
T73
2808
Tba
tA
1039
09Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
) (co
nti
nu
ed)
A Varsani and M Krupovic | 3
Tab
le1
Co
nti
nu
ed
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
09T
bat
H10
3746
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
10T
bat
H10
3909
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
11T
bat
L10
3746
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
12T
bat
L10
3909
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us8
KT
7328
06T
bat
3157
9Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us9
KT
7327
95T
bat
2138
3Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us10
KT
7327
94T
bat
H10
3958
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Rat
asso
ciat
edge
myc
ircu
larv
irus
1K
R91
2221
Ch
-zjr
at-0
1R
attu
sno
rveg
icus
Rat
Blo
od
Ch
ina
Liet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
GQ
3657
09Ss
HA
DV
-1C
NSc
lero
tini
asc
lero
tior
umSc
lero
tin
iaM
ycel
ials
amp
les
Ch
ina
Yu
etal
(20
10)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
5Ss
HA
DV
-1N
ZH
620
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
6Ss
HA
DV
-1N
ZSR
120
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
7Ss
HA
DV
-1N
ZSR
320
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
8Ss
HA
DV
-1N
ZSR
520
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
82Ss
HA
DV
-1-U
S-54
9LB
-12
Isch
nura
ram
buri
iD
amse
lfly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
83Ss
HA
DV
-1-U
S-54
9DFS
-12
Eryt
hem
issi
mpl
icic
ollis
Dra
gon
fly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
84Ss
HA
DV
-1-U
S-54
9SR
-12
Pant
ala
hym
enae
aD
rago
nfl
yA
bdo
men
USA
Day
aram
etal
(20
15)
Sew
age
deri
ved
gem
ycir
cula
rvir
us1
KJ5
4763
8B
S391
7Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us1
KM
8217
47Sa
Gm
V-1
NZ
-BS3
970-
2012
Sew
age
oxid
atio
npo
ndndash
Sew
age
New
Zea
lan
dK
rabe
rger
etal
(20
15a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us2
KJ5
4764
1B
S411
7Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us3
KJ5
4763
6B
S401
4Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us4
KJ5
4763
7B
S393
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us4
KJ5
4764
0B
S397
2Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us5
KJ5
4763
9B
S397
0Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Shee
pas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
4947
Fec8
0064
shee
pO
vis
arie
sSh
eep
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Shee
pas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
5151
Fec8
0064
shee
pO
vis
arie
sSh
eep
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Soyb
ean
asso
ciat
edge
myc
ircu
larv
irus
1K
T59
8248
SlaG
emV
1-1
Gly
cine
max
Soyb
ean
Leaf
USA
Mar
zan
oan
dD
om
ier
(201
6)
Gem
ydug
uiv
irus
Dra
gonfl
yas
soci
ated
gem
ydug
uivi
rus
1JX
1854
28T
O-D
FS3B
2-20
10Pa
ntal
afla
vesc
ens
Dra
gon
fly
Abd
om
enT
on
gaR
osa
rio
etal
(20
12)
Gem
ygor
viru
sC
anin
eas
soci
ated
gem
ygor
viru
s1
KT
8622
5453
Fec7
do
gC
anis
lupu
sfa
mili
aris
Do
gFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1JN
7046
10V
S470
0006
Mel
esm
eles
Euro
pea
nba
dge
rR
ecta
lsw
abN
eth
erla
nd
sva
nd
enB
ran
det
al(
2012
)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1K
T86
2238
4Fe
c7d
uck
Ana
spl
atyr
hync
hos
Du
ckFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1K
T86
2239
24Fe
c7d
uck
Ana
spl
atyr
hync
hos
Du
ckFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Pter
opus
asso
ciat
edge
myg
orvi
rus
1K
T73
2790
Tba
tA
1039
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myg
orvi
rus
1K
T73
2791
Tba
tH
1039
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Sew
age
deri
ved
gem
ygor
viru
s1
KJ4
1314
434
9H
omo
sapi
ens
Hu
man
Cer
vica
lsam
ple
Sou
thA
fric
a
Sew
age
deri
ved
gem
ygor
viru
s1
KJ5
4763
5B
S396
3Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Star
ling
asso
ciat
edge
myg
orvi
rus
1K
F371
632
P14
Stur
nus
vulg
aris
Euro
pea
nst
arli
ng
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Gem
ykib
ivir
usBa
dger
asso
ciat
edge
myk
ibiv
irus
1K
P263
543
588t
Mel
esm
eles
Euro
pea
nba
dge
rFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Blac
kro
bin
asso
ciat
edge
myk
ibiv
irus
1K
F371
634
P21
Petr
oica
trav
ersi
Ch
ath
amIs
lan
d
blac
kro
bin
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Blac
kbir
das
soci
ated
gem
ykib
ivir
us
1K
F371
633
P22
Tur
dus
mer
ula
Bla
ckbi
rdFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Bovi
neas
soci
ated
gem
ykib
ivir
us1
LK93
1483
HC
BI8
215
Bos
taur
usC
ow
Seru
mG
erm
any
Lam
bert
oet
al(
2014
)
Dra
gonfl
yas
soci
ated
gem
ykib
ivir
us1
JX18
5430
FL1-
2X-2
010
Mia
thyr
iam
arce
llaD
rago
nfl
yA
bdo
men
USA
Ro
sari
oet
al(
2012
)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KJ5
4764
4B
S398
0Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KJ5
4764
5B
S384
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KP9
7469
4D
B2
Hom
osa
pien
sH
um
anPl
asm
aG
erm
any
Zh
ang
etal
(20
16)
Hum
anas
soci
ated
gem
ykib
ivir
us1
LK93
1485
MSS
I22
25H
omo
sapi
ens
Hu
man
Blo
od
Ger
man
yLa
mbe
rto
etal
(20
14)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
5SL
1H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
6SL
2H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
7SL
3H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
(co
nti
nu
ed)
4 | Virus Evolution 2017 Vol 3 No 1
Tab
le1
Co
nti
nu
ed
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
8B
Z1
Hom
osa
pien
sH
um
anFa
eces
Bra
zil
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
9B
Z2
Hom
osa
pien
sH
um
anFa
eces
Bra
zil
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3308
0N
PU
ntre
ated
sew
age
ndashSe
wag
eN
epal
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us3
KP2
6354
654
1cH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us3
KP9
8788
7G
emyC
1cH
omo
sapi
ens
Hu
man
Plas
ma
Fran
ceU
chet
al(
2015
)
Hum
anas
soci
ated
gem
ykib
ivir
us4
KT
3638
39G
eTz1
Hom
osa
pien
sH
um
anC
ereb
rosp
inal
flu
idC
hin
aZ
ho
uet
al(
2015
)
Hum
anas
soci
ated
gem
ykib
ivir
us5
KU
3431
37H
V-G
cV2
Hom
osa
pien
sH
um
anPe
rica
rdia
lflu
idFr
ance
Hal
ary
etal
(20
16)
Mon
goos
eas
soci
ated
gem
ykib
ivir
us1
KP2
6354
516
0bH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Pter
opu
sas
soci
ated
gem
ykib
ivir
us1
KT
7328
13T
bat
A64
418
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
1K
J641
737
BtR
h-C
V-6
Tib
et20
13R
hino
loph
ushi
ppos
ider
osB
atPh
aryn
geal
amp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
1K
P263
544
181a
Her
pest
esic
hneu
mon
Egyp
tian
mo
ngo
ose
Faec
esPo
rtu
gal
Co
nce
icao
-Net
oet
al(
2015
)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
2K
J641
726
BtR
f-C
V-8
NM
2013
Rhi
nolo
phus
ferr
umeq
uinu
mB
atPh
aryn
geal
amp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Sew
age
deri
ved
gem
ykib
ivir
us1
KJ5
4764
3B
S414
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
4027
BS1
4149
chic
ken
Gal
lus
gallu
sdo
mes
ticu
sC
hic
ken
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
5252
BS1
4149
cow
Bos
taur
usC
ow
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
5556
BS1
4149
har
eLe
pus
euro
paeu
sH
are
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us2
KJ5
4764
2B
S391
1Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Gem
ykol
ovir
us
Pter
opu
sas
soci
ated
gem
ykol
ovir
us1
KT
7327
98T
bat
A10
3779
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ykol
ovir
us1
KT
7327
99T
bat
H10
3779
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myk
olov
irus
2K
T73
2800
Tba
tH
1039
21Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Gem
ykro
gvir
usB
ovin
eas
soci
ated
gem
ykro
gvir
us1
LK93
1484
HC
BI9
212
Bos
taur
usC
ow
Seru
mG
erm
any
Lam
bert
oet
al(
2014
)
Car
ibou
asso
ciat
edge
myk
rogv
irus
1K
J938
717
FaG
mC
V-1
3R
angi
fer
tara
ndus
Car
ibou
Faec
esC
anad
aN
get
al(
2014
)
Sew
age
deri
ved
gem
ykro
gvir
us1
KJ5
4763
4B
S391
3Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Gem
ykro
znav
iru
sR
abbi
tas
soci
ated
gem
ykro
znav
irus
1K
F371
631
as35
Ory
ctol
agus
cuni
culu
sR
abbi
tFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Gem
yton
dvir
usO
stri
chas
soci
ated
gem
yton
dvir
us1
KF3
7163
0as
3St
ruth
ioca
mel
usO
stri
chFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Gem
yvon
gvir
usH
um
anas
soci
ated
gem
yvon
gvir
us1
KP9
7469
3D
B1
Hom
osa
pien
sH
um
anPl
asm
aG
erm
any
Zh
ang
etal
(20
16)
A Varsani and M Krupovic | 5
tree gemykoloviruses are firmly nested within the large cladeincluding the majority of gemycircularviruses (Fig 4) Giventhat CP sequences of genomoviruses are considerably more di-vergent than the Rep sequences (Fig 1) it appears reasonable toestablish a higher (ie above the species level) taxonomicframework using the Rep (Fig 2) The latter protein is also
conserved in other eukaryotic ssDNA viruses (which is not thecase for the CP) and can thus be used to assess the place ofgenomoviruses within the larger community of ssDNA viruses
To evaluate the taxonomic structure of the Genomoviridaewe took advantage of the fact that in Rep-based phylogeneticanalyses genomoviruses consistently form a sister group to
Figure 1 Distribution of (A) genome-wide (B) Rep and (C) CP pairwise identities (121 taxa) of genomoviruses calculated using SDT v12 (Muhire Varsani and Martin
2014)
6 | Virus Evolution 2017 Vol 3 No 1
members of the Geminiviridae (Krupovic et al 2016) a compre-hensively characterized family of plant viruses with circularssDNA genomes (Varsani et al 2014b) Thus using the estab-lished taxonomic framework of the Geminiviridae overlaid on theRep-based phylogeny as a guide we could define five cladesand four additional singletons within the Genomoviridae branch(Fig 2) The defined groups displayed equivalent intra-family di-vergence as the established genera within the familyGeminiviridae (Varsani et al 2014b) The nine groups were sup-ported in both nucleotide and protein sequence inferred phylog-enies (Supplementary Fig S2) Consequently in addition to theexisting genus Gemycircularvirus we propose establishing eightnew genera within the family Genomoviridae The details of thenine genera are summarized in Fig 5 and briefly outlinedbelow
31 Gemycircularvirus
This genus has the largest number of new species (nfrac14 43 sev-enty-three genomes Table 1) and includes SsHADV-1 thefounding member of the family Members of the genus display44 diversity Viruses within the forty-three species clusterwith 99 and 96 branch support values in phylogenetic treesconstructed from either Rep or full genome sequences respec-tively (Figs 2 and 3)
32 Gemykibivirus
This is the second most populated genus (nfrac14 16 twenty-nine ge-nomes Table 1) in the family with 43 diversity among its mem-bers The name of the genus is an acronym of words geminivirus-like and myco-like kibi virus (kibi means circular in Amharic)Sequences within the fifteen species cluster with 93 branch sup-port within phylogenetic trees constructed from Rep (Fig 2) andtwo well-supported clades (100 and 96) within trees constructedfrom full genome sequences (Fig 3) suggesting that recombinationhas played an important role in the evolution of this group
33 Gemygorvirus
Members of this genus (nfrac14 5 nine genomes Table 1) display49 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like gor virus (gor means round inHindi) Sequences within the five species cluster with 100 and99 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
34 Gemykolovirus
Members of this genus (nfrac14 2 three genomes Table 1) display37 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like kolo virus (kolo means round inCzech) Sequences within the two species cluster with 100 and89 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
35 Gemykrogvirus
Members of this genus (nfrac14 3 three genomes Table 1) display33 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like krog virus (krog means round inSlovenian) Sequences within the three species cluster with 99and 100 branch support within phylogenetic trees constructedfrom either Rep or full genome sequences respectively (Figs 2and 3)
KP133079 Human associated gemykibivirus 1 KP133080 Human associated gemykibivirus 1 KP133078 Human associated gemykibivirus 1 KP133077 Human associated gemykibivirus 1 KP133075 Human associated gemykibivirus 1 KP133076 Human associated gemykibivirus 1
JX185428 Dragonfly associated gemyduguivirus 1 KP974693 Human associated gemyvongvirus 1
Figure 3 Maximum likelihood phylogenetic tree of the genomes of viruses in the Genomoviridae family The tree was inferred using FastTree (Price Dehal and Arkin
2010) (GTRthornCAT) The numbers at the branches indicate SH-like support values The topology of tree supports the proposed genera demarcation at the genome level
despite there being evidence of recombination within the genomes Branches withlt75 SH-like branch support have been collapsed
8 | Virus Evolution 2017 Vol 3 No 1
35 Gemyvongvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like vong virus (vong means circular in Lao) The sin-gle species Human associated gemyvongvirus 1 (Table 1) within thegenus shares between 56 and 62 genome-wide sequence simi-larity with viruses in other genera and is a divergent taxon inthe phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
36 Gemytondvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like tond virus (tond means round in Maltese) Thesingle species Ostrich associated gemytondvirus 1 (Table 1) withinthe genus shares between 53 and 61 genome-wide sequencesimilarity with viruses in other genera and is a divergent taxonin the phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
37 Gemykroznavirus
The name of the genus is an acronym of words geminivirus-likeand myco-like krozna virus (krozna means circular in Slovenian)The single species Rabbit associated gemykroznavirus 1 (Table 1)
KP974693 Human associated gemyvongvirus 1 KF371631 Rabbit associated gemykroznavirus 1
KP133075 Human associated gemykibivirus 2 KP133080 Human associated gemykibivirus 2 KP133079 Human associated gemykibivirus 2 KP133078 Human associated gemykibivirus 2 KP133076 Human associated gemykibivirus 2 KP133077 Human associated gemykibivirus 2
KJ413144 Human associated gemygorvirus 1 KJ547635 Sewage derived gemygorvirus 1
100
100
100
80
94
86
100
100100
10099
97
9986
8688
95
99
10091
89
100
100
80
96
84
100100
100
87
10083
79
89 95
100
91
9391
100
93
92
94
88
93100
10094
77
10096
90
94
9284
100
10089
9696
99
80
96
95
100
100
94
100
10099
89
94100
100
93100
93
100
9895
100
100
87
02 amino acid subs per site
GemyduguivirusGemycircularvirus
GemykibivirusGemygorvirus
Gemyvongvirus
Gemykolovirus
GemytondvirusGemykroznavirusGemykrogvirus
Figure 4 Maximum likelihood phylogenetic tree of the CP amino acid sequences
inferred using PHYML (Guindon et al 2010) with LGthornGthornI substitution models
and rooted with geminivirus sequences Branches withlt75 SH-like branch sup-
port have been collapsed
0 10 20 30 40 50 60 70 80
Percentage diversity
Number of isolates
Number of species
Gemyvongvirus
Gemytondvirus
Gemykroznavirus
Gemykrogvirus
Gemykolovirus
Gemykibivirus
Gemygorvirus
Gemyduguivirus
Gemycircularvirus
Figure 5 Summary of genera and the associated species and their diversity
(within genera) within the Genomoviridae family
A Varsani and M Krupovic | 9
within the genus shares between 56 and 61 genome-widesequence similarity with other sequences in other genera and isa divergent taxon in the phylogenetic trees constructed fromeither Rep or full genome sequences (Figs 2 and 3)
38 Gemyduguivirus
The name of the genus is an acronym of words geminivirus-likeand myco-like dugui virus (dugui means circular in Mongolian)The single species Dragonfly associated gemyduguivirus 1 (Table 1)within the genus shares between 57 and 62 genome-wide se-quence similarity with viruses in other genera and is a divergenttaxon in the phylogenetic trees constructed from either Rep orfull genome sequences (Figs 2 and 3)
4 Conserved sequence motifs in theGenomoviridae
CRESS DNA viruses replicate through the rolling circle replica-tion (RCR) mechanism which is similar to that used by bacterialplasmids (Khan 1997 Chandler et al 2013 Ruiz-Maso et al2015) RCR is initiated by the Rep encoded by CRESS DNAviruses cleaving the dsDNA between positions 7 and 8 of anonanucleotide sequence located at a putative stem-loopstructure at the origin of replication (Heyraud-Nitschke et al1995 Laufs et al 1995b Timchenko et al 1999 RosarioDuffy and Breitbart 2012) In the case of genomoviruses this
nonanucleotide is variable (lsquoTAWWDWRNrsquo) with lsquoTAATWYATrsquobeing the consensus nonanucleotide for gemycircularviruseswhereas gemykibiruses display the greatest variation inthis motifmdashlsquoWATAWWHANrsquo (Fig 6 Supplementary Data S1)In contrast we note that within the Geminiviridae familyincluding all recently described geminiviruses (Varsani et al2009 Briddon et al 2010 Krenz et al 2012 Loconsole et al 2012Bernardo et al 2013 Heydarnejad et al 2013 Ma et al 2015Bernardo et al 2016) the consensus nonanucleotide motif islsquoTRAKATTRCrsquo
The N terminus of the Rep contains motifs that are impor-tant for initiating RCR and it is not surprising that some of thesemotifs are well conserved across many ssDNA viruses phagesand plasmids that replicate using the RCR mechanism (Ilyinaand Koonin 1992 Vega-Rocha et al 2007a Rosario Duffy andBreitbart 2012 Krupovic 2013) The presence of a single cata-lytic tyrosine residue in the RCR motif III classifies genomovi-rus geminivirus bacilladnavirus circovirus and nanovirus Repsas members of superfamily II (Ilyina and Koonin 1992Krupovic 2013)
In genomoviruses the conserved sequence of the RCRmotif I which is thought to be involved in the recognition ofiterative sequences associated with the origin of replicationis predominantly lsquouuTYxQrsquo (u denotes hydrophobic residuesand x any residue) (Fig 6 Supplementary Data S1) with theexception of the Reps of currently known gemykolovirusesand gemykrogviruses The genomovirus RCR motif II lsquoxHxHxrsquo
Figure 6 Summary of conserved motifs that is nonanucleotide and Rep motifs illustrated using WebLogo3 (Crooks et al 2004) identified in the family Genomoviridae as
a whole and its nine genera separately Note the highly derived Walker A motif (GPHRRRRT) in the sole member of the genus Gemytondvirus
10 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
Tab
le1
Det
ails
of
allm
embe
rso
fth
eG
enom
ovir
idae
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Gem
ycir
cula
rvir
us
Blac
kbir
das
soci
ated
gem
ycir
cula
rvir
us1
KF3
7164
1P9
Tur
dus
mer
ula
Bla
ckbi
rdFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Blac
kbir
das
soci
ated
gem
ycir
cula
rvir
us1
KF3
7164
2P2
2T
urdu
sm
erul
aB
lack
bird
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Blac
kbir
das
soci
ated
gem
ycir
cula
rvir
us1
KF3
7164
3as
41O
vis
arie
sSh
eep
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Bovi
neas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
5352
Fec7
8023
cow
Bos
taur
usC
ow
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Brom
us
asso
ciat
edge
myc
ircu
larv
irus
1K
M51
0192
Bas
CV
-3N
Z-N
ZG
01Se
f-20
12Br
omus
hord
eace
usSo
ftbr
om
eB
ull
gras
sLe
afN
ewZ
eala
nd
Kra
berg
eret
al(
2015
b)
Cas
sava
asso
ciat
edge
myc
ircu
larv
irus
1JQ
4120
56G
14M
anih
otes
cule
nta
Cas
sava
Leaf
Gh
ana
Day
aram
etal
(20
12)
Cas
sava
asso
ciat
edge
myc
ircu
larv
irus
1JQ
4120
57G
5M
anih
otes
cule
nta
Cas
sava
Leaf
Gh
ana
Day
aram
etal
(20
12)
Chi
ckad
eeas
soci
ated
gem
ycir
cula
rvir
us1
KT
3090
2925
4065
908
Poec
ileat
rica
pillu
sB
lack
-cap
ped
chic
kad
eeB
ucc
alan
d
clo
acal
swab
USA
Han
na
etal
(20
15)
Chi
cken
asso
ciat
edge
myc
ircu
larv
iru
s1
KT
8622
4327
Fec7
9971
chic
ken
Gal
lus
gallu
sdo
mes
ticu
sC
hic
ken
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Chi
cken
asso
ciat
edge
myc
ircu
larv
iru
s1
KT
8622
4429
Fec7
9971
Ilam
aLa
ma
glam
aLl
ama
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Chi
cken
asso
ciat
edge
myc
ircu
larv
iru
s1
KT
8622
4630
Fec7
9971
ho
rse
Equu
sfe
rus
caba
llus
Ho
rse
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Chi
cken
asso
ciat
edge
myc
ircu
larv
iru
s2
KT
8622
4227
Fec1
6497
chic
ken
Gal
lus
gallu
sdo
mes
ticu
sC
hic
ken
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Dra
gonfl
yas
soci
ated
gem
ycir
cula
rvir
us1
JX18
5429
FL2-
5X-2
010
Eryt
hem
issi
mpl
icic
ollis
Dra
gon
fly
Abd
om
enU
SAR
osa
rio
etal
(20
12)
Equi
neas
soci
ated
gem
ycir
cula
rvir
us
1K
T86
2248
30Fe
c800
61h
ors
eEq
uus
feru
sca
ballu
sH
ors
eFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Fur
seal
asso
ciat
edge
myc
ircu
larv
irus
1K
F371
638
as50
Arc
toce
phal
usfo
rste
riN
ewZ
eala
nd
fur
seal
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Fur
seal
asso
ciat
edge
myc
ircu
larv
irus
1K
T86
2241
27Fe
c1ch
icke
nG
allu
sga
llus
dom
esti
cus
Ch
icke
nFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Ger
ygon
eas
soci
ated
gem
ycir
cula
rvir
us1
KF3
7163
6P2
4aG
eryg
one
albo
fron
tata
Ch
ath
amIs
lan
dw
arbl
erFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Ger
ygon
eas
soci
ated
gem
ycir
cula
rvir
us2
KF3
7163
7P2
4bG
eryg
one
albo
fron
tata
Ch
ath
amIs
lan
dw
arbl
erFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Ger
ygon
eas
soci
ated
gem
ycir
cula
rvir
us3
KF3
7163
9P2
4cG
eryg
one
albo
fron
tata
Ch
ath
amIs
lan
dw
arbl
erFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Hyp
eric
um
asso
ciat
edge
myc
ircu
larv
irus
1K
F413
620
VN
HJ1
WH
yper
icum
japo
nicu
mH
yper
icu
mLe
afV
ietn
amD
uet
al(
2014
)
Lam
aas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
4529
Fec8
0018
llam
aLa
ma
glam
aLl
ama
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Lam
aas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
4730
Fec8
0018
ho
rse
Equu
sfe
rus
caba
llus
Ho
rse
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Mal
lard
asso
ciat
edge
myc
ircu
larv
iru
s1
KF3
7163
5as
24A
nas
plat
yrhy
ncho
sM
alla
rdd
uck
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Min
iopt
erus
asso
ciat
edge
myc
ircu
larv
irus
1K
J641
719
BtM
f-C
V-2
3G
D20
12M
inio
pter
usfu
ligin
osus
Bat
Phar
ynge
alamp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Mon
goos
eas
soci
ated
gem
ycir
cula
rvir
us1
KP2
6354
747
8dH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Mos
quit
oas
soci
ated
gem
ycir
cula
rvir
us1
HQ
3350
86SD
BV
LG
Cul
exer
ythr
otho
rax
Mo
squ
ito
Mo
squ
ito
sam
ple
sU
SAN
get
al(
2011
)
Odo
nata
asso
ciat
edge
myc
ircu
larv
iru
s1
KM
5983
85O
daG
mV
-1-U
S-26
0BC
-12
Isch
nura
posi
taD
amse
lfly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Odo
nata
asso
ciat
edge
myc
ircu
larv
iru
s1
KM
5983
86O
daG
mV
-1-U
S-26
0SR
1-12
Pant
ala
hym
enae
aD
rago
nfl
yA
bdo
men
USA
Day
aram
etal
(20
15)
Odo
nata
asso
ciat
edge
myc
ircu
larv
iru
s2
KM
5983
87O
daG
mV
-2-U
S-16
42K
W-1
2A
eshn
am
ulti
colo
rD
rago
nfl
yA
bdo
men
USA
Day
aram
etal
(20
15)
Odo
nata
asso
ciat
edge
myc
ircu
larv
iru
s2
KM
5983
88O
daG
mV
-2-U
S-16
34LM
2-12
Libe
llula
satu
rata
Dra
gon
fly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Poac
eae
asso
ciat
edge
myc
ircu
larv
irus
1K
T25
3577
PaG
mV
-1T
OST
O14
-292
0420
14Br
achi
aria
defle
xaSi
gnal
gras
sLe
afT
on
gaM
ale
etal
(20
15)
Poac
eae
asso
ciat
edge
myc
ircu
larv
irus
1K
T25
3578
PaG
mV
-1T
OST
O15
-292
0420
14Br
achi
aria
defle
xaSi
gnal
gras
sLe
afT
on
gaM
ale
etal
(20
15)
Poac
eae
asso
ciat
edge
myc
ircu
larv
irus
1K
T25
3579
PaG
mV
-1T
OST
O18
-292
0420
14Br
achi
aria
defle
xaSi
gnal
gras
sLe
afT
on
gaM
ale
etal
(20
15)
Porc
ine
asso
ciat
edge
myc
ircu
larv
irus
1K
T86
2250
49Fe
c800
61p
igSu
ssc
rofa
dom
esti
caPi
gFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Porc
ine
asso
ciat
edge
myc
ircu
larv
irus
2K
F371
640
as5
Sus
scro
faD
om
esti
cp
igFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
1K
T73
2804
Tba
t45
285
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
1K
T73
2805
Tba
t47
364
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
2K
T73
2792
Tba
t10
3791
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
2K
T73
2793
Tba
tA
1037
91Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
3K
T73
2797
Tba
tA
1038
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
4K
T73
2814
Tba
tH
1038
06Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
5K
T73
2801
Tba
t12
377
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
5K
T73
2802
Tba
tH
1237
7Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
6K
T73
2796
Tba
tH
1036
39Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
6K
T73
2803
Tba
t10
3951
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
7K
T73
2807
Tba
tA
1037
46Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myc
ircu
larv
irus
7K
T73
2808
Tba
tA
1039
09Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
) (co
nti
nu
ed)
A Varsani and M Krupovic | 3
Tab
le1
Co
nti
nu
ed
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
09T
bat
H10
3746
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
10T
bat
H10
3909
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
11T
bat
L10
3746
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
12T
bat
L10
3909
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us8
KT
7328
06T
bat
3157
9Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us9
KT
7327
95T
bat
2138
3Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us10
KT
7327
94T
bat
H10
3958
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Rat
asso
ciat
edge
myc
ircu
larv
irus
1K
R91
2221
Ch
-zjr
at-0
1R
attu
sno
rveg
icus
Rat
Blo
od
Ch
ina
Liet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
GQ
3657
09Ss
HA
DV
-1C
NSc
lero
tini
asc
lero
tior
umSc
lero
tin
iaM
ycel
ials
amp
les
Ch
ina
Yu
etal
(20
10)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
5Ss
HA
DV
-1N
ZH
620
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
6Ss
HA
DV
-1N
ZSR
120
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
7Ss
HA
DV
-1N
ZSR
320
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
8Ss
HA
DV
-1N
ZSR
520
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
82Ss
HA
DV
-1-U
S-54
9LB
-12
Isch
nura
ram
buri
iD
amse
lfly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
83Ss
HA
DV
-1-U
S-54
9DFS
-12
Eryt
hem
issi
mpl
icic
ollis
Dra
gon
fly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
84Ss
HA
DV
-1-U
S-54
9SR
-12
Pant
ala
hym
enae
aD
rago
nfl
yA
bdo
men
USA
Day
aram
etal
(20
15)
Sew
age
deri
ved
gem
ycir
cula
rvir
us1
KJ5
4763
8B
S391
7Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us1
KM
8217
47Sa
Gm
V-1
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-BS3
970-
2012
Sew
age
oxid
atio
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ndndash
Sew
age
New
Zea
lan
dK
rabe
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etal
(20
15a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us2
KJ5
4764
1B
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7Se
wag
eox
idat
ion
pond
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wag
eN
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eala
nd
Kra
berg
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al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us3
KJ5
4763
6B
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4Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
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eala
nd
Kra
berg
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al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us4
KJ5
4763
7B
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9Se
wag
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idat
ion
pond
ndashSe
wag
eN
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eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us4
KJ5
4764
0B
S397
2Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us5
KJ5
4763
9B
S397
0Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Shee
pas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
4947
Fec8
0064
shee
pO
vis
arie
sSh
eep
Faec
esN
ewZ
eala
nd
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let
al(
2016
)
Shee
pas
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ated
gem
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cula
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us1
KT
8622
5151
Fec8
0064
shee
pO
vis
arie
sSh
eep
Faec
esN
ewZ
eala
nd
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let
al(
2016
)
Soyb
ean
asso
ciat
edge
myc
ircu
larv
irus
1K
T59
8248
SlaG
emV
1-1
Gly
cine
max
Soyb
ean
Leaf
USA
Mar
zan
oan
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om
ier
(201
6)
Gem
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irus
Dra
gonfl
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ated
gem
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rus
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1854
28T
O-D
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10Pa
ntal
afla
vesc
ens
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gon
fly
Abd
om
enT
on
gaR
osa
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(20
12)
Gem
ygor
viru
sC
anin
eas
soci
ated
gem
ygor
viru
s1
KT
8622
5453
Fec7
do
gC
anis
lupu
sfa
mili
aris
Do
gFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1JN
7046
10V
S470
0006
Mel
esm
eles
Euro
pea
nba
dge
rR
ecta
lsw
abN
eth
erla
nd
sva
nd
enB
ran
det
al(
2012
)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1K
T86
2238
4Fe
c7d
uck
Ana
spl
atyr
hync
hos
Du
ckFa
eces
New
Zea
lan
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eele
tal
(20
16)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1K
T86
2239
24Fe
c7d
uck
Ana
spl
atyr
hync
hos
Du
ckFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Pter
opus
asso
ciat
edge
myg
orvi
rus
1K
T73
2790
Tba
tA
1039
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myg
orvi
rus
1K
T73
2791
Tba
tH
1039
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Sew
age
deri
ved
gem
ygor
viru
s1
KJ4
1314
434
9H
omo
sapi
ens
Hu
man
Cer
vica
lsam
ple
Sou
thA
fric
a
Sew
age
deri
ved
gem
ygor
viru
s1
KJ5
4763
5B
S396
3Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Star
ling
asso
ciat
edge
myg
orvi
rus
1K
F371
632
P14
Stur
nus
vulg
aris
Euro
pea
nst
arli
ng
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Gem
ykib
ivir
usBa
dger
asso
ciat
edge
myk
ibiv
irus
1K
P263
543
588t
Mel
esm
eles
Euro
pea
nba
dge
rFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Blac
kro
bin
asso
ciat
edge
myk
ibiv
irus
1K
F371
634
P21
Petr
oica
trav
ersi
Ch
ath
amIs
lan
d
blac
kro
bin
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Blac
kbir
das
soci
ated
gem
ykib
ivir
us
1K
F371
633
P22
Tur
dus
mer
ula
Bla
ckbi
rdFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Bovi
neas
soci
ated
gem
ykib
ivir
us1
LK93
1483
HC
BI8
215
Bos
taur
usC
ow
Seru
mG
erm
any
Lam
bert
oet
al(
2014
)
Dra
gonfl
yas
soci
ated
gem
ykib
ivir
us1
JX18
5430
FL1-
2X-2
010
Mia
thyr
iam
arce
llaD
rago
nfl
yA
bdo
men
USA
Ro
sari
oet
al(
2012
)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KJ5
4764
4B
S398
0Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KJ5
4764
5B
S384
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KP9
7469
4D
B2
Hom
osa
pien
sH
um
anPl
asm
aG
erm
any
Zh
ang
etal
(20
16)
Hum
anas
soci
ated
gem
ykib
ivir
us1
LK93
1485
MSS
I22
25H
omo
sapi
ens
Hu
man
Blo
od
Ger
man
yLa
mbe
rto
etal
(20
14)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
5SL
1H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
6SL
2H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
7SL
3H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
(co
nti
nu
ed)
4 | Virus Evolution 2017 Vol 3 No 1
Tab
le1
Co
nti
nu
ed
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
8B
Z1
Hom
osa
pien
sH
um
anFa
eces
Bra
zil
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
9B
Z2
Hom
osa
pien
sH
um
anFa
eces
Bra
zil
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3308
0N
PU
ntre
ated
sew
age
ndashSe
wag
eN
epal
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us3
KP2
6354
654
1cH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us3
KP9
8788
7G
emyC
1cH
omo
sapi
ens
Hu
man
Plas
ma
Fran
ceU
chet
al(
2015
)
Hum
anas
soci
ated
gem
ykib
ivir
us4
KT
3638
39G
eTz1
Hom
osa
pien
sH
um
anC
ereb
rosp
inal
flu
idC
hin
aZ
ho
uet
al(
2015
)
Hum
anas
soci
ated
gem
ykib
ivir
us5
KU
3431
37H
V-G
cV2
Hom
osa
pien
sH
um
anPe
rica
rdia
lflu
idFr
ance
Hal
ary
etal
(20
16)
Mon
goos
eas
soci
ated
gem
ykib
ivir
us1
KP2
6354
516
0bH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Pter
opu
sas
soci
ated
gem
ykib
ivir
us1
KT
7328
13T
bat
A64
418
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
1K
J641
737
BtR
h-C
V-6
Tib
et20
13R
hino
loph
ushi
ppos
ider
osB
atPh
aryn
geal
amp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
1K
P263
544
181a
Her
pest
esic
hneu
mon
Egyp
tian
mo
ngo
ose
Faec
esPo
rtu
gal
Co
nce
icao
-Net
oet
al(
2015
)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
2K
J641
726
BtR
f-C
V-8
NM
2013
Rhi
nolo
phus
ferr
umeq
uinu
mB
atPh
aryn
geal
amp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Sew
age
deri
ved
gem
ykib
ivir
us1
KJ5
4764
3B
S414
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
4027
BS1
4149
chic
ken
Gal
lus
gallu
sdo
mes
ticu
sC
hic
ken
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
5252
BS1
4149
cow
Bos
taur
usC
ow
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
5556
BS1
4149
har
eLe
pus
euro
paeu
sH
are
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us2
KJ5
4764
2B
S391
1Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Gem
ykol
ovir
us
Pter
opu
sas
soci
ated
gem
ykol
ovir
us1
KT
7327
98T
bat
A10
3779
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ykol
ovir
us1
KT
7327
99T
bat
H10
3779
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myk
olov
irus
2K
T73
2800
Tba
tH
1039
21Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Gem
ykro
gvir
usB
ovin
eas
soci
ated
gem
ykro
gvir
us1
LK93
1484
HC
BI9
212
Bos
taur
usC
ow
Seru
mG
erm
any
Lam
bert
oet
al(
2014
)
Car
ibou
asso
ciat
edge
myk
rogv
irus
1K
J938
717
FaG
mC
V-1
3R
angi
fer
tara
ndus
Car
ibou
Faec
esC
anad
aN
get
al(
2014
)
Sew
age
deri
ved
gem
ykro
gvir
us1
KJ5
4763
4B
S391
3Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Gem
ykro
znav
iru
sR
abbi
tas
soci
ated
gem
ykro
znav
irus
1K
F371
631
as35
Ory
ctol
agus
cuni
culu
sR
abbi
tFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Gem
yton
dvir
usO
stri
chas
soci
ated
gem
yton
dvir
us1
KF3
7163
0as
3St
ruth
ioca
mel
usO
stri
chFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Gem
yvon
gvir
usH
um
anas
soci
ated
gem
yvon
gvir
us1
KP9
7469
3D
B1
Hom
osa
pien
sH
um
anPl
asm
aG
erm
any
Zh
ang
etal
(20
16)
A Varsani and M Krupovic | 5
tree gemykoloviruses are firmly nested within the large cladeincluding the majority of gemycircularviruses (Fig 4) Giventhat CP sequences of genomoviruses are considerably more di-vergent than the Rep sequences (Fig 1) it appears reasonable toestablish a higher (ie above the species level) taxonomicframework using the Rep (Fig 2) The latter protein is also
conserved in other eukaryotic ssDNA viruses (which is not thecase for the CP) and can thus be used to assess the place ofgenomoviruses within the larger community of ssDNA viruses
To evaluate the taxonomic structure of the Genomoviridaewe took advantage of the fact that in Rep-based phylogeneticanalyses genomoviruses consistently form a sister group to
Figure 1 Distribution of (A) genome-wide (B) Rep and (C) CP pairwise identities (121 taxa) of genomoviruses calculated using SDT v12 (Muhire Varsani and Martin
2014)
6 | Virus Evolution 2017 Vol 3 No 1
members of the Geminiviridae (Krupovic et al 2016) a compre-hensively characterized family of plant viruses with circularssDNA genomes (Varsani et al 2014b) Thus using the estab-lished taxonomic framework of the Geminiviridae overlaid on theRep-based phylogeny as a guide we could define five cladesand four additional singletons within the Genomoviridae branch(Fig 2) The defined groups displayed equivalent intra-family di-vergence as the established genera within the familyGeminiviridae (Varsani et al 2014b) The nine groups were sup-ported in both nucleotide and protein sequence inferred phylog-enies (Supplementary Fig S2) Consequently in addition to theexisting genus Gemycircularvirus we propose establishing eightnew genera within the family Genomoviridae The details of thenine genera are summarized in Fig 5 and briefly outlinedbelow
31 Gemycircularvirus
This genus has the largest number of new species (nfrac14 43 sev-enty-three genomes Table 1) and includes SsHADV-1 thefounding member of the family Members of the genus display44 diversity Viruses within the forty-three species clusterwith 99 and 96 branch support values in phylogenetic treesconstructed from either Rep or full genome sequences respec-tively (Figs 2 and 3)
32 Gemykibivirus
This is the second most populated genus (nfrac14 16 twenty-nine ge-nomes Table 1) in the family with 43 diversity among its mem-bers The name of the genus is an acronym of words geminivirus-like and myco-like kibi virus (kibi means circular in Amharic)Sequences within the fifteen species cluster with 93 branch sup-port within phylogenetic trees constructed from Rep (Fig 2) andtwo well-supported clades (100 and 96) within trees constructedfrom full genome sequences (Fig 3) suggesting that recombinationhas played an important role in the evolution of this group
33 Gemygorvirus
Members of this genus (nfrac14 5 nine genomes Table 1) display49 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like gor virus (gor means round inHindi) Sequences within the five species cluster with 100 and99 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
34 Gemykolovirus
Members of this genus (nfrac14 2 three genomes Table 1) display37 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like kolo virus (kolo means round inCzech) Sequences within the two species cluster with 100 and89 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
35 Gemykrogvirus
Members of this genus (nfrac14 3 three genomes Table 1) display33 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like krog virus (krog means round inSlovenian) Sequences within the three species cluster with 99and 100 branch support within phylogenetic trees constructedfrom either Rep or full genome sequences respectively (Figs 2and 3)
KP133079 Human associated gemykibivirus 1 KP133080 Human associated gemykibivirus 1 KP133078 Human associated gemykibivirus 1 KP133077 Human associated gemykibivirus 1 KP133075 Human associated gemykibivirus 1 KP133076 Human associated gemykibivirus 1
JX185428 Dragonfly associated gemyduguivirus 1 KP974693 Human associated gemyvongvirus 1
Figure 3 Maximum likelihood phylogenetic tree of the genomes of viruses in the Genomoviridae family The tree was inferred using FastTree (Price Dehal and Arkin
2010) (GTRthornCAT) The numbers at the branches indicate SH-like support values The topology of tree supports the proposed genera demarcation at the genome level
despite there being evidence of recombination within the genomes Branches withlt75 SH-like branch support have been collapsed
8 | Virus Evolution 2017 Vol 3 No 1
35 Gemyvongvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like vong virus (vong means circular in Lao) The sin-gle species Human associated gemyvongvirus 1 (Table 1) within thegenus shares between 56 and 62 genome-wide sequence simi-larity with viruses in other genera and is a divergent taxon inthe phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
36 Gemytondvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like tond virus (tond means round in Maltese) Thesingle species Ostrich associated gemytondvirus 1 (Table 1) withinthe genus shares between 53 and 61 genome-wide sequencesimilarity with viruses in other genera and is a divergent taxonin the phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
37 Gemykroznavirus
The name of the genus is an acronym of words geminivirus-likeand myco-like krozna virus (krozna means circular in Slovenian)The single species Rabbit associated gemykroznavirus 1 (Table 1)
KP974693 Human associated gemyvongvirus 1 KF371631 Rabbit associated gemykroznavirus 1
KP133075 Human associated gemykibivirus 2 KP133080 Human associated gemykibivirus 2 KP133079 Human associated gemykibivirus 2 KP133078 Human associated gemykibivirus 2 KP133076 Human associated gemykibivirus 2 KP133077 Human associated gemykibivirus 2
KJ413144 Human associated gemygorvirus 1 KJ547635 Sewage derived gemygorvirus 1
100
100
100
80
94
86
100
100100
10099
97
9986
8688
95
99
10091
89
100
100
80
96
84
100100
100
87
10083
79
89 95
100
91
9391
100
93
92
94
88
93100
10094
77
10096
90
94
9284
100
10089
9696
99
80
96
95
100
100
94
100
10099
89
94100
100
93100
93
100
9895
100
100
87
02 amino acid subs per site
GemyduguivirusGemycircularvirus
GemykibivirusGemygorvirus
Gemyvongvirus
Gemykolovirus
GemytondvirusGemykroznavirusGemykrogvirus
Figure 4 Maximum likelihood phylogenetic tree of the CP amino acid sequences
inferred using PHYML (Guindon et al 2010) with LGthornGthornI substitution models
and rooted with geminivirus sequences Branches withlt75 SH-like branch sup-
port have been collapsed
0 10 20 30 40 50 60 70 80
Percentage diversity
Number of isolates
Number of species
Gemyvongvirus
Gemytondvirus
Gemykroznavirus
Gemykrogvirus
Gemykolovirus
Gemykibivirus
Gemygorvirus
Gemyduguivirus
Gemycircularvirus
Figure 5 Summary of genera and the associated species and their diversity
(within genera) within the Genomoviridae family
A Varsani and M Krupovic | 9
within the genus shares between 56 and 61 genome-widesequence similarity with other sequences in other genera and isa divergent taxon in the phylogenetic trees constructed fromeither Rep or full genome sequences (Figs 2 and 3)
38 Gemyduguivirus
The name of the genus is an acronym of words geminivirus-likeand myco-like dugui virus (dugui means circular in Mongolian)The single species Dragonfly associated gemyduguivirus 1 (Table 1)within the genus shares between 57 and 62 genome-wide se-quence similarity with viruses in other genera and is a divergenttaxon in the phylogenetic trees constructed from either Rep orfull genome sequences (Figs 2 and 3)
4 Conserved sequence motifs in theGenomoviridae
CRESS DNA viruses replicate through the rolling circle replica-tion (RCR) mechanism which is similar to that used by bacterialplasmids (Khan 1997 Chandler et al 2013 Ruiz-Maso et al2015) RCR is initiated by the Rep encoded by CRESS DNAviruses cleaving the dsDNA between positions 7 and 8 of anonanucleotide sequence located at a putative stem-loopstructure at the origin of replication (Heyraud-Nitschke et al1995 Laufs et al 1995b Timchenko et al 1999 RosarioDuffy and Breitbart 2012) In the case of genomoviruses this
nonanucleotide is variable (lsquoTAWWDWRNrsquo) with lsquoTAATWYATrsquobeing the consensus nonanucleotide for gemycircularviruseswhereas gemykibiruses display the greatest variation inthis motifmdashlsquoWATAWWHANrsquo (Fig 6 Supplementary Data S1)In contrast we note that within the Geminiviridae familyincluding all recently described geminiviruses (Varsani et al2009 Briddon et al 2010 Krenz et al 2012 Loconsole et al 2012Bernardo et al 2013 Heydarnejad et al 2013 Ma et al 2015Bernardo et al 2016) the consensus nonanucleotide motif islsquoTRAKATTRCrsquo
The N terminus of the Rep contains motifs that are impor-tant for initiating RCR and it is not surprising that some of thesemotifs are well conserved across many ssDNA viruses phagesand plasmids that replicate using the RCR mechanism (Ilyinaand Koonin 1992 Vega-Rocha et al 2007a Rosario Duffy andBreitbart 2012 Krupovic 2013) The presence of a single cata-lytic tyrosine residue in the RCR motif III classifies genomovi-rus geminivirus bacilladnavirus circovirus and nanovirus Repsas members of superfamily II (Ilyina and Koonin 1992Krupovic 2013)
In genomoviruses the conserved sequence of the RCRmotif I which is thought to be involved in the recognition ofiterative sequences associated with the origin of replicationis predominantly lsquouuTYxQrsquo (u denotes hydrophobic residuesand x any residue) (Fig 6 Supplementary Data S1) with theexception of the Reps of currently known gemykolovirusesand gemykrogviruses The genomovirus RCR motif II lsquoxHxHxrsquo
Figure 6 Summary of conserved motifs that is nonanucleotide and Rep motifs illustrated using WebLogo3 (Crooks et al 2004) identified in the family Genomoviridae as
a whole and its nine genera separately Note the highly derived Walker A motif (GPHRRRRT) in the sole member of the genus Gemytondvirus
10 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
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Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
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Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
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et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
Tab
le1
Co
nti
nu
ed
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
09T
bat
H10
3746
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
10T
bat
H10
3909
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
11T
bat
L10
3746
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us7
KT
7328
12T
bat
L10
3909
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us8
KT
7328
06T
bat
3157
9Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us9
KT
7327
95T
bat
2138
3Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opu
sas
soci
ated
gem
ycir
cula
rvir
us10
KT
7327
94T
bat
H10
3958
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Rat
asso
ciat
edge
myc
ircu
larv
irus
1K
R91
2221
Ch
-zjr
at-0
1R
attu
sno
rveg
icus
Rat
Blo
od
Ch
ina
Liet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
GQ
3657
09Ss
HA
DV
-1C
NSc
lero
tini
asc
lero
tior
umSc
lero
tin
iaM
ycel
ials
amp
les
Ch
ina
Yu
etal
(20
10)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
5Ss
HA
DV
-1N
ZH
620
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
6Ss
HA
DV
-1N
ZSR
120
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
7Ss
HA
DV
-1N
ZSR
320
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KF2
6802
8Ss
HA
DV
-1N
ZSR
520
12R
iver
Sedi
men
tsndash
Riv
erSe
dim
ents
New
Zea
lan
dK
rabe
rger
etal
(20
13)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
82Ss
HA
DV
-1-U
S-54
9LB
-12
Isch
nura
ram
buri
iD
amse
lfly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
83Ss
HA
DV
-1-U
S-54
9DFS
-12
Eryt
hem
issi
mpl
icic
ollis
Dra
gon
fly
Abd
om
enU
SAD
ayar
amet
al(
2015
)
Scle
roti
nia
gem
ycir
cula
rvir
us1
KM
5983
84Ss
HA
DV
-1-U
S-54
9SR
-12
Pant
ala
hym
enae
aD
rago
nfl
yA
bdo
men
USA
Day
aram
etal
(20
15)
Sew
age
deri
ved
gem
ycir
cula
rvir
us1
KJ5
4763
8B
S391
7Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us1
KM
8217
47Sa
Gm
V-1
NZ
-BS3
970-
2012
Sew
age
oxid
atio
npo
ndndash
Sew
age
New
Zea
lan
dK
rabe
rger
etal
(20
15a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us2
KJ5
4764
1B
S411
7Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us3
KJ5
4763
6B
S401
4Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us4
KJ5
4763
7B
S393
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us4
KJ5
4764
0B
S397
2Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ycir
cula
rvir
us5
KJ5
4763
9B
S397
0Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Shee
pas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
4947
Fec8
0064
shee
pO
vis
arie
sSh
eep
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Shee
pas
soci
ated
gem
ycir
cula
rvir
us1
KT
8622
5151
Fec8
0064
shee
pO
vis
arie
sSh
eep
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Soyb
ean
asso
ciat
edge
myc
ircu
larv
irus
1K
T59
8248
SlaG
emV
1-1
Gly
cine
max
Soyb
ean
Leaf
USA
Mar
zan
oan
dD
om
ier
(201
6)
Gem
ydug
uiv
irus
Dra
gonfl
yas
soci
ated
gem
ydug
uivi
rus
1JX
1854
28T
O-D
FS3B
2-20
10Pa
ntal
afla
vesc
ens
Dra
gon
fly
Abd
om
enT
on
gaR
osa
rio
etal
(20
12)
Gem
ygor
viru
sC
anin
eas
soci
ated
gem
ygor
viru
s1
KT
8622
5453
Fec7
do
gC
anis
lupu
sfa
mili
aris
Do
gFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1JN
7046
10V
S470
0006
Mel
esm
eles
Euro
pea
nba
dge
rR
ecta
lsw
abN
eth
erla
nd
sva
nd
enB
ran
det
al(
2012
)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1K
T86
2238
4Fe
c7d
uck
Ana
spl
atyr
hync
hos
Du
ckFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Mal
lard
asso
ciat
edge
myg
orvi
rus
1K
T86
2239
24Fe
c7d
uck
Ana
spl
atyr
hync
hos
Du
ckFa
eces
New
Zea
lan
dSt
eele
tal
(20
16)
Pter
opus
asso
ciat
edge
myg
orvi
rus
1K
T73
2790
Tba
tA
1039
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Pter
opus
asso
ciat
edge
myg
orvi
rus
1K
T73
2791
Tba
tH
1039
52Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Sew
age
deri
ved
gem
ygor
viru
s1
KJ4
1314
434
9H
omo
sapi
ens
Hu
man
Cer
vica
lsam
ple
Sou
thA
fric
a
Sew
age
deri
ved
gem
ygor
viru
s1
KJ5
4763
5B
S396
3Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Star
ling
asso
ciat
edge
myg
orvi
rus
1K
F371
632
P14
Stur
nus
vulg
aris
Euro
pea
nst
arli
ng
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Gem
ykib
ivir
usBa
dger
asso
ciat
edge
myk
ibiv
irus
1K
P263
543
588t
Mel
esm
eles
Euro
pea
nba
dge
rFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Blac
kro
bin
asso
ciat
edge
myk
ibiv
irus
1K
F371
634
P21
Petr
oica
trav
ersi
Ch
ath
amIs
lan
d
blac
kro
bin
Faec
esN
ewZ
eala
nd
Siko
rski
etal
(20
13)
Blac
kbir
das
soci
ated
gem
ykib
ivir
us
1K
F371
633
P22
Tur
dus
mer
ula
Bla
ckbi
rdFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Bovi
neas
soci
ated
gem
ykib
ivir
us1
LK93
1483
HC
BI8
215
Bos
taur
usC
ow
Seru
mG
erm
any
Lam
bert
oet
al(
2014
)
Dra
gonfl
yas
soci
ated
gem
ykib
ivir
us1
JX18
5430
FL1-
2X-2
010
Mia
thyr
iam
arce
llaD
rago
nfl
yA
bdo
men
USA
Ro
sari
oet
al(
2012
)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KJ5
4764
4B
S398
0Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KJ5
4764
5B
S384
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Hum
anas
soci
ated
gem
ykib
ivir
us1
KP9
7469
4D
B2
Hom
osa
pien
sH
um
anPl
asm
aG
erm
any
Zh
ang
etal
(20
16)
Hum
anas
soci
ated
gem
ykib
ivir
us1
LK93
1485
MSS
I22
25H
omo
sapi
ens
Hu
man
Blo
od
Ger
man
yLa
mbe
rto
etal
(20
14)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
5SL
1H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
6SL
2H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
7SL
3H
omo
sapi
ens
Hu
man
Cer
ebro
spin
alfl
uid
SriL
anka
Phan
etal
(20
15)
(co
nti
nu
ed)
4 | Virus Evolution 2017 Vol 3 No 1
Tab
le1
Co
nti
nu
ed
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
8B
Z1
Hom
osa
pien
sH
um
anFa
eces
Bra
zil
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
9B
Z2
Hom
osa
pien
sH
um
anFa
eces
Bra
zil
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3308
0N
PU
ntre
ated
sew
age
ndashSe
wag
eN
epal
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us3
KP2
6354
654
1cH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us3
KP9
8788
7G
emyC
1cH
omo
sapi
ens
Hu
man
Plas
ma
Fran
ceU
chet
al(
2015
)
Hum
anas
soci
ated
gem
ykib
ivir
us4
KT
3638
39G
eTz1
Hom
osa
pien
sH
um
anC
ereb
rosp
inal
flu
idC
hin
aZ
ho
uet
al(
2015
)
Hum
anas
soci
ated
gem
ykib
ivir
us5
KU
3431
37H
V-G
cV2
Hom
osa
pien
sH
um
anPe
rica
rdia
lflu
idFr
ance
Hal
ary
etal
(20
16)
Mon
goos
eas
soci
ated
gem
ykib
ivir
us1
KP2
6354
516
0bH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Pter
opu
sas
soci
ated
gem
ykib
ivir
us1
KT
7328
13T
bat
A64
418
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
1K
J641
737
BtR
h-C
V-6
Tib
et20
13R
hino
loph
ushi
ppos
ider
osB
atPh
aryn
geal
amp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
1K
P263
544
181a
Her
pest
esic
hneu
mon
Egyp
tian
mo
ngo
ose
Faec
esPo
rtu
gal
Co
nce
icao
-Net
oet
al(
2015
)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
2K
J641
726
BtR
f-C
V-8
NM
2013
Rhi
nolo
phus
ferr
umeq
uinu
mB
atPh
aryn
geal
amp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Sew
age
deri
ved
gem
ykib
ivir
us1
KJ5
4764
3B
S414
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
4027
BS1
4149
chic
ken
Gal
lus
gallu
sdo
mes
ticu
sC
hic
ken
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
5252
BS1
4149
cow
Bos
taur
usC
ow
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
5556
BS1
4149
har
eLe
pus
euro
paeu
sH
are
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us2
KJ5
4764
2B
S391
1Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Gem
ykol
ovir
us
Pter
opu
sas
soci
ated
gem
ykol
ovir
us1
KT
7327
98T
bat
A10
3779
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ykol
ovir
us1
KT
7327
99T
bat
H10
3779
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myk
olov
irus
2K
T73
2800
Tba
tH
1039
21Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Gem
ykro
gvir
usB
ovin
eas
soci
ated
gem
ykro
gvir
us1
LK93
1484
HC
BI9
212
Bos
taur
usC
ow
Seru
mG
erm
any
Lam
bert
oet
al(
2014
)
Car
ibou
asso
ciat
edge
myk
rogv
irus
1K
J938
717
FaG
mC
V-1
3R
angi
fer
tara
ndus
Car
ibou
Faec
esC
anad
aN
get
al(
2014
)
Sew
age
deri
ved
gem
ykro
gvir
us1
KJ5
4763
4B
S391
3Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Gem
ykro
znav
iru
sR
abbi
tas
soci
ated
gem
ykro
znav
irus
1K
F371
631
as35
Ory
ctol
agus
cuni
culu
sR
abbi
tFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Gem
yton
dvir
usO
stri
chas
soci
ated
gem
yton
dvir
us1
KF3
7163
0as
3St
ruth
ioca
mel
usO
stri
chFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Gem
yvon
gvir
usH
um
anas
soci
ated
gem
yvon
gvir
us1
KP9
7469
3D
B1
Hom
osa
pien
sH
um
anPl
asm
aG
erm
any
Zh
ang
etal
(20
16)
A Varsani and M Krupovic | 5
tree gemykoloviruses are firmly nested within the large cladeincluding the majority of gemycircularviruses (Fig 4) Giventhat CP sequences of genomoviruses are considerably more di-vergent than the Rep sequences (Fig 1) it appears reasonable toestablish a higher (ie above the species level) taxonomicframework using the Rep (Fig 2) The latter protein is also
conserved in other eukaryotic ssDNA viruses (which is not thecase for the CP) and can thus be used to assess the place ofgenomoviruses within the larger community of ssDNA viruses
To evaluate the taxonomic structure of the Genomoviridaewe took advantage of the fact that in Rep-based phylogeneticanalyses genomoviruses consistently form a sister group to
Figure 1 Distribution of (A) genome-wide (B) Rep and (C) CP pairwise identities (121 taxa) of genomoviruses calculated using SDT v12 (Muhire Varsani and Martin
2014)
6 | Virus Evolution 2017 Vol 3 No 1
members of the Geminiviridae (Krupovic et al 2016) a compre-hensively characterized family of plant viruses with circularssDNA genomes (Varsani et al 2014b) Thus using the estab-lished taxonomic framework of the Geminiviridae overlaid on theRep-based phylogeny as a guide we could define five cladesand four additional singletons within the Genomoviridae branch(Fig 2) The defined groups displayed equivalent intra-family di-vergence as the established genera within the familyGeminiviridae (Varsani et al 2014b) The nine groups were sup-ported in both nucleotide and protein sequence inferred phylog-enies (Supplementary Fig S2) Consequently in addition to theexisting genus Gemycircularvirus we propose establishing eightnew genera within the family Genomoviridae The details of thenine genera are summarized in Fig 5 and briefly outlinedbelow
31 Gemycircularvirus
This genus has the largest number of new species (nfrac14 43 sev-enty-three genomes Table 1) and includes SsHADV-1 thefounding member of the family Members of the genus display44 diversity Viruses within the forty-three species clusterwith 99 and 96 branch support values in phylogenetic treesconstructed from either Rep or full genome sequences respec-tively (Figs 2 and 3)
32 Gemykibivirus
This is the second most populated genus (nfrac14 16 twenty-nine ge-nomes Table 1) in the family with 43 diversity among its mem-bers The name of the genus is an acronym of words geminivirus-like and myco-like kibi virus (kibi means circular in Amharic)Sequences within the fifteen species cluster with 93 branch sup-port within phylogenetic trees constructed from Rep (Fig 2) andtwo well-supported clades (100 and 96) within trees constructedfrom full genome sequences (Fig 3) suggesting that recombinationhas played an important role in the evolution of this group
33 Gemygorvirus
Members of this genus (nfrac14 5 nine genomes Table 1) display49 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like gor virus (gor means round inHindi) Sequences within the five species cluster with 100 and99 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
34 Gemykolovirus
Members of this genus (nfrac14 2 three genomes Table 1) display37 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like kolo virus (kolo means round inCzech) Sequences within the two species cluster with 100 and89 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
35 Gemykrogvirus
Members of this genus (nfrac14 3 three genomes Table 1) display33 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like krog virus (krog means round inSlovenian) Sequences within the three species cluster with 99and 100 branch support within phylogenetic trees constructedfrom either Rep or full genome sequences respectively (Figs 2and 3)
KP133079 Human associated gemykibivirus 1 KP133080 Human associated gemykibivirus 1 KP133078 Human associated gemykibivirus 1 KP133077 Human associated gemykibivirus 1 KP133075 Human associated gemykibivirus 1 KP133076 Human associated gemykibivirus 1
JX185428 Dragonfly associated gemyduguivirus 1 KP974693 Human associated gemyvongvirus 1
Figure 3 Maximum likelihood phylogenetic tree of the genomes of viruses in the Genomoviridae family The tree was inferred using FastTree (Price Dehal and Arkin
2010) (GTRthornCAT) The numbers at the branches indicate SH-like support values The topology of tree supports the proposed genera demarcation at the genome level
despite there being evidence of recombination within the genomes Branches withlt75 SH-like branch support have been collapsed
8 | Virus Evolution 2017 Vol 3 No 1
35 Gemyvongvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like vong virus (vong means circular in Lao) The sin-gle species Human associated gemyvongvirus 1 (Table 1) within thegenus shares between 56 and 62 genome-wide sequence simi-larity with viruses in other genera and is a divergent taxon inthe phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
36 Gemytondvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like tond virus (tond means round in Maltese) Thesingle species Ostrich associated gemytondvirus 1 (Table 1) withinthe genus shares between 53 and 61 genome-wide sequencesimilarity with viruses in other genera and is a divergent taxonin the phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
37 Gemykroznavirus
The name of the genus is an acronym of words geminivirus-likeand myco-like krozna virus (krozna means circular in Slovenian)The single species Rabbit associated gemykroznavirus 1 (Table 1)
KP974693 Human associated gemyvongvirus 1 KF371631 Rabbit associated gemykroznavirus 1
KP133075 Human associated gemykibivirus 2 KP133080 Human associated gemykibivirus 2 KP133079 Human associated gemykibivirus 2 KP133078 Human associated gemykibivirus 2 KP133076 Human associated gemykibivirus 2 KP133077 Human associated gemykibivirus 2
KJ413144 Human associated gemygorvirus 1 KJ547635 Sewage derived gemygorvirus 1
100
100
100
80
94
86
100
100100
10099
97
9986
8688
95
99
10091
89
100
100
80
96
84
100100
100
87
10083
79
89 95
100
91
9391
100
93
92
94
88
93100
10094
77
10096
90
94
9284
100
10089
9696
99
80
96
95
100
100
94
100
10099
89
94100
100
93100
93
100
9895
100
100
87
02 amino acid subs per site
GemyduguivirusGemycircularvirus
GemykibivirusGemygorvirus
Gemyvongvirus
Gemykolovirus
GemytondvirusGemykroznavirusGemykrogvirus
Figure 4 Maximum likelihood phylogenetic tree of the CP amino acid sequences
inferred using PHYML (Guindon et al 2010) with LGthornGthornI substitution models
and rooted with geminivirus sequences Branches withlt75 SH-like branch sup-
port have been collapsed
0 10 20 30 40 50 60 70 80
Percentage diversity
Number of isolates
Number of species
Gemyvongvirus
Gemytondvirus
Gemykroznavirus
Gemykrogvirus
Gemykolovirus
Gemykibivirus
Gemygorvirus
Gemyduguivirus
Gemycircularvirus
Figure 5 Summary of genera and the associated species and their diversity
(within genera) within the Genomoviridae family
A Varsani and M Krupovic | 9
within the genus shares between 56 and 61 genome-widesequence similarity with other sequences in other genera and isa divergent taxon in the phylogenetic trees constructed fromeither Rep or full genome sequences (Figs 2 and 3)
38 Gemyduguivirus
The name of the genus is an acronym of words geminivirus-likeand myco-like dugui virus (dugui means circular in Mongolian)The single species Dragonfly associated gemyduguivirus 1 (Table 1)within the genus shares between 57 and 62 genome-wide se-quence similarity with viruses in other genera and is a divergenttaxon in the phylogenetic trees constructed from either Rep orfull genome sequences (Figs 2 and 3)
4 Conserved sequence motifs in theGenomoviridae
CRESS DNA viruses replicate through the rolling circle replica-tion (RCR) mechanism which is similar to that used by bacterialplasmids (Khan 1997 Chandler et al 2013 Ruiz-Maso et al2015) RCR is initiated by the Rep encoded by CRESS DNAviruses cleaving the dsDNA between positions 7 and 8 of anonanucleotide sequence located at a putative stem-loopstructure at the origin of replication (Heyraud-Nitschke et al1995 Laufs et al 1995b Timchenko et al 1999 RosarioDuffy and Breitbart 2012) In the case of genomoviruses this
nonanucleotide is variable (lsquoTAWWDWRNrsquo) with lsquoTAATWYATrsquobeing the consensus nonanucleotide for gemycircularviruseswhereas gemykibiruses display the greatest variation inthis motifmdashlsquoWATAWWHANrsquo (Fig 6 Supplementary Data S1)In contrast we note that within the Geminiviridae familyincluding all recently described geminiviruses (Varsani et al2009 Briddon et al 2010 Krenz et al 2012 Loconsole et al 2012Bernardo et al 2013 Heydarnejad et al 2013 Ma et al 2015Bernardo et al 2016) the consensus nonanucleotide motif islsquoTRAKATTRCrsquo
The N terminus of the Rep contains motifs that are impor-tant for initiating RCR and it is not surprising that some of thesemotifs are well conserved across many ssDNA viruses phagesand plasmids that replicate using the RCR mechanism (Ilyinaand Koonin 1992 Vega-Rocha et al 2007a Rosario Duffy andBreitbart 2012 Krupovic 2013) The presence of a single cata-lytic tyrosine residue in the RCR motif III classifies genomovi-rus geminivirus bacilladnavirus circovirus and nanovirus Repsas members of superfamily II (Ilyina and Koonin 1992Krupovic 2013)
In genomoviruses the conserved sequence of the RCRmotif I which is thought to be involved in the recognition ofiterative sequences associated with the origin of replicationis predominantly lsquouuTYxQrsquo (u denotes hydrophobic residuesand x any residue) (Fig 6 Supplementary Data S1) with theexception of the Reps of currently known gemykolovirusesand gemykrogviruses The genomovirus RCR motif II lsquoxHxHxrsquo
Figure 6 Summary of conserved motifs that is nonanucleotide and Rep motifs illustrated using WebLogo3 (Crooks et al 2004) identified in the family Genomoviridae as
a whole and its nine genera separately Note the highly derived Walker A motif (GPHRRRRT) in the sole member of the genus Gemytondvirus
10 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
Tab
le1
Co
nti
nu
ed
Gen
us
Spec
ies
Acc
essi
on
Se
qu
ence
IDIs
ola
tio
nso
urc
eC
om
mo
nn
ame
Sam
ple
typ
eC
ou
ntr
yR
efer
ence
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
8B
Z1
Hom
osa
pien
sH
um
anFa
eces
Bra
zil
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3307
9B
Z2
Hom
osa
pien
sH
um
anFa
eces
Bra
zil
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us2
KP1
3308
0N
PU
ntre
ated
sew
age
ndashSe
wag
eN
epal
Phan
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us3
KP2
6354
654
1cH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Hum
anas
soci
ated
gem
ykib
ivir
us3
KP9
8788
7G
emyC
1cH
omo
sapi
ens
Hu
man
Plas
ma
Fran
ceU
chet
al(
2015
)
Hum
anas
soci
ated
gem
ykib
ivir
us4
KT
3638
39G
eTz1
Hom
osa
pien
sH
um
anC
ereb
rosp
inal
flu
idC
hin
aZ
ho
uet
al(
2015
)
Hum
anas
soci
ated
gem
ykib
ivir
us5
KU
3431
37H
V-G
cV2
Hom
osa
pien
sH
um
anPe
rica
rdia
lflu
idFr
ance
Hal
ary
etal
(20
16)
Mon
goos
eas
soci
ated
gem
ykib
ivir
us1
KP2
6354
516
0bH
erpe
stes
ichn
eum
onEg
ypti
anm
on
goo
seFa
eces
Port
uga
lC
on
ceic
ao-N
eto
etal
(20
15)
Pter
opu
sas
soci
ated
gem
ykib
ivir
us1
KT
7328
13T
bat
A64
418
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
1K
J641
737
BtR
h-C
V-6
Tib
et20
13R
hino
loph
ushi
ppos
ider
osB
atPh
aryn
geal
amp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
1K
P263
544
181a
Her
pest
esic
hneu
mon
Egyp
tian
mo
ngo
ose
Faec
esPo
rtu
gal
Co
nce
icao
-Net
oet
al(
2015
)
Rhi
nolo
phus
asso
ciat
edge
myk
ibiv
irus
2K
J641
726
BtR
f-C
V-8
NM
2013
Rhi
nolo
phus
ferr
umeq
uinu
mB
atPh
aryn
geal
amp
rect
alsw
abs
Ch
ina
Wu
etal
(20
16)
Sew
age
deri
ved
gem
ykib
ivir
us1
KJ5
4764
3B
S414
9Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
4027
BS1
4149
chic
ken
Gal
lus
gallu
sdo
mes
ticu
sC
hic
ken
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
5252
BS1
4149
cow
Bos
taur
usC
ow
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us1
KT
8622
5556
BS1
4149
har
eLe
pus
euro
paeu
sH
are
Faec
esN
ewZ
eala
nd
Stee
let
al(
2016
)
Sew
age
deri
ved
gem
ykib
ivir
us2
KJ5
4764
2B
S391
1Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Gem
ykol
ovir
us
Pter
opu
sas
soci
ated
gem
ykol
ovir
us1
KT
7327
98T
bat
A10
3779
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opu
sas
soci
ated
gem
ykol
ovir
us1
KT
7327
99T
bat
H10
3779
Pter
opus
tong
anus
Bat
Faec
esT
on
gaM
ale
etal
(20
16)
Pter
opus
asso
ciat
edge
myk
olov
irus
2K
T73
2800
Tba
tH
1039
21Pt
erop
usto
ngan
usB
atFa
eces
To
nga
Mal
eet
al(
2016
)
Gem
ykro
gvir
usB
ovin
eas
soci
ated
gem
ykro
gvir
us1
LK93
1484
HC
BI9
212
Bos
taur
usC
ow
Seru
mG
erm
any
Lam
bert
oet
al(
2014
)
Car
ibou
asso
ciat
edge
myk
rogv
irus
1K
J938
717
FaG
mC
V-1
3R
angi
fer
tara
ndus
Car
ibou
Faec
esC
anad
aN
get
al(
2014
)
Sew
age
deri
ved
gem
ykro
gvir
us1
KJ5
4763
4B
S391
3Se
wag
eox
idat
ion
pond
ndashSe
wag
eN
ewZ
eala
nd
Kra
berg
eret
al(
2015
a)
Gem
ykro
znav
iru
sR
abbi
tas
soci
ated
gem
ykro
znav
irus
1K
F371
631
as35
Ory
ctol
agus
cuni
culu
sR
abbi
tFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Gem
yton
dvir
usO
stri
chas
soci
ated
gem
yton
dvir
us1
KF3
7163
0as
3St
ruth
ioca
mel
usO
stri
chFa
eces
New
Zea
lan
dSi
kors
kiet
al(
2013
)
Gem
yvon
gvir
usH
um
anas
soci
ated
gem
yvon
gvir
us1
KP9
7469
3D
B1
Hom
osa
pien
sH
um
anPl
asm
aG
erm
any
Zh
ang
etal
(20
16)
A Varsani and M Krupovic | 5
tree gemykoloviruses are firmly nested within the large cladeincluding the majority of gemycircularviruses (Fig 4) Giventhat CP sequences of genomoviruses are considerably more di-vergent than the Rep sequences (Fig 1) it appears reasonable toestablish a higher (ie above the species level) taxonomicframework using the Rep (Fig 2) The latter protein is also
conserved in other eukaryotic ssDNA viruses (which is not thecase for the CP) and can thus be used to assess the place ofgenomoviruses within the larger community of ssDNA viruses
To evaluate the taxonomic structure of the Genomoviridaewe took advantage of the fact that in Rep-based phylogeneticanalyses genomoviruses consistently form a sister group to
Figure 1 Distribution of (A) genome-wide (B) Rep and (C) CP pairwise identities (121 taxa) of genomoviruses calculated using SDT v12 (Muhire Varsani and Martin
2014)
6 | Virus Evolution 2017 Vol 3 No 1
members of the Geminiviridae (Krupovic et al 2016) a compre-hensively characterized family of plant viruses with circularssDNA genomes (Varsani et al 2014b) Thus using the estab-lished taxonomic framework of the Geminiviridae overlaid on theRep-based phylogeny as a guide we could define five cladesand four additional singletons within the Genomoviridae branch(Fig 2) The defined groups displayed equivalent intra-family di-vergence as the established genera within the familyGeminiviridae (Varsani et al 2014b) The nine groups were sup-ported in both nucleotide and protein sequence inferred phylog-enies (Supplementary Fig S2) Consequently in addition to theexisting genus Gemycircularvirus we propose establishing eightnew genera within the family Genomoviridae The details of thenine genera are summarized in Fig 5 and briefly outlinedbelow
31 Gemycircularvirus
This genus has the largest number of new species (nfrac14 43 sev-enty-three genomes Table 1) and includes SsHADV-1 thefounding member of the family Members of the genus display44 diversity Viruses within the forty-three species clusterwith 99 and 96 branch support values in phylogenetic treesconstructed from either Rep or full genome sequences respec-tively (Figs 2 and 3)
32 Gemykibivirus
This is the second most populated genus (nfrac14 16 twenty-nine ge-nomes Table 1) in the family with 43 diversity among its mem-bers The name of the genus is an acronym of words geminivirus-like and myco-like kibi virus (kibi means circular in Amharic)Sequences within the fifteen species cluster with 93 branch sup-port within phylogenetic trees constructed from Rep (Fig 2) andtwo well-supported clades (100 and 96) within trees constructedfrom full genome sequences (Fig 3) suggesting that recombinationhas played an important role in the evolution of this group
33 Gemygorvirus
Members of this genus (nfrac14 5 nine genomes Table 1) display49 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like gor virus (gor means round inHindi) Sequences within the five species cluster with 100 and99 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
34 Gemykolovirus
Members of this genus (nfrac14 2 three genomes Table 1) display37 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like kolo virus (kolo means round inCzech) Sequences within the two species cluster with 100 and89 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
35 Gemykrogvirus
Members of this genus (nfrac14 3 three genomes Table 1) display33 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like krog virus (krog means round inSlovenian) Sequences within the three species cluster with 99and 100 branch support within phylogenetic trees constructedfrom either Rep or full genome sequences respectively (Figs 2and 3)
KP133079 Human associated gemykibivirus 1 KP133080 Human associated gemykibivirus 1 KP133078 Human associated gemykibivirus 1 KP133077 Human associated gemykibivirus 1 KP133075 Human associated gemykibivirus 1 KP133076 Human associated gemykibivirus 1
JX185428 Dragonfly associated gemyduguivirus 1 KP974693 Human associated gemyvongvirus 1
Figure 3 Maximum likelihood phylogenetic tree of the genomes of viruses in the Genomoviridae family The tree was inferred using FastTree (Price Dehal and Arkin
2010) (GTRthornCAT) The numbers at the branches indicate SH-like support values The topology of tree supports the proposed genera demarcation at the genome level
despite there being evidence of recombination within the genomes Branches withlt75 SH-like branch support have been collapsed
8 | Virus Evolution 2017 Vol 3 No 1
35 Gemyvongvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like vong virus (vong means circular in Lao) The sin-gle species Human associated gemyvongvirus 1 (Table 1) within thegenus shares between 56 and 62 genome-wide sequence simi-larity with viruses in other genera and is a divergent taxon inthe phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
36 Gemytondvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like tond virus (tond means round in Maltese) Thesingle species Ostrich associated gemytondvirus 1 (Table 1) withinthe genus shares between 53 and 61 genome-wide sequencesimilarity with viruses in other genera and is a divergent taxonin the phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
37 Gemykroznavirus
The name of the genus is an acronym of words geminivirus-likeand myco-like krozna virus (krozna means circular in Slovenian)The single species Rabbit associated gemykroznavirus 1 (Table 1)
KP974693 Human associated gemyvongvirus 1 KF371631 Rabbit associated gemykroznavirus 1
KP133075 Human associated gemykibivirus 2 KP133080 Human associated gemykibivirus 2 KP133079 Human associated gemykibivirus 2 KP133078 Human associated gemykibivirus 2 KP133076 Human associated gemykibivirus 2 KP133077 Human associated gemykibivirus 2
KJ413144 Human associated gemygorvirus 1 KJ547635 Sewage derived gemygorvirus 1
100
100
100
80
94
86
100
100100
10099
97
9986
8688
95
99
10091
89
100
100
80
96
84
100100
100
87
10083
79
89 95
100
91
9391
100
93
92
94
88
93100
10094
77
10096
90
94
9284
100
10089
9696
99
80
96
95
100
100
94
100
10099
89
94100
100
93100
93
100
9895
100
100
87
02 amino acid subs per site
GemyduguivirusGemycircularvirus
GemykibivirusGemygorvirus
Gemyvongvirus
Gemykolovirus
GemytondvirusGemykroznavirusGemykrogvirus
Figure 4 Maximum likelihood phylogenetic tree of the CP amino acid sequences
inferred using PHYML (Guindon et al 2010) with LGthornGthornI substitution models
and rooted with geminivirus sequences Branches withlt75 SH-like branch sup-
port have been collapsed
0 10 20 30 40 50 60 70 80
Percentage diversity
Number of isolates
Number of species
Gemyvongvirus
Gemytondvirus
Gemykroznavirus
Gemykrogvirus
Gemykolovirus
Gemykibivirus
Gemygorvirus
Gemyduguivirus
Gemycircularvirus
Figure 5 Summary of genera and the associated species and their diversity
(within genera) within the Genomoviridae family
A Varsani and M Krupovic | 9
within the genus shares between 56 and 61 genome-widesequence similarity with other sequences in other genera and isa divergent taxon in the phylogenetic trees constructed fromeither Rep or full genome sequences (Figs 2 and 3)
38 Gemyduguivirus
The name of the genus is an acronym of words geminivirus-likeand myco-like dugui virus (dugui means circular in Mongolian)The single species Dragonfly associated gemyduguivirus 1 (Table 1)within the genus shares between 57 and 62 genome-wide se-quence similarity with viruses in other genera and is a divergenttaxon in the phylogenetic trees constructed from either Rep orfull genome sequences (Figs 2 and 3)
4 Conserved sequence motifs in theGenomoviridae
CRESS DNA viruses replicate through the rolling circle replica-tion (RCR) mechanism which is similar to that used by bacterialplasmids (Khan 1997 Chandler et al 2013 Ruiz-Maso et al2015) RCR is initiated by the Rep encoded by CRESS DNAviruses cleaving the dsDNA between positions 7 and 8 of anonanucleotide sequence located at a putative stem-loopstructure at the origin of replication (Heyraud-Nitschke et al1995 Laufs et al 1995b Timchenko et al 1999 RosarioDuffy and Breitbart 2012) In the case of genomoviruses this
nonanucleotide is variable (lsquoTAWWDWRNrsquo) with lsquoTAATWYATrsquobeing the consensus nonanucleotide for gemycircularviruseswhereas gemykibiruses display the greatest variation inthis motifmdashlsquoWATAWWHANrsquo (Fig 6 Supplementary Data S1)In contrast we note that within the Geminiviridae familyincluding all recently described geminiviruses (Varsani et al2009 Briddon et al 2010 Krenz et al 2012 Loconsole et al 2012Bernardo et al 2013 Heydarnejad et al 2013 Ma et al 2015Bernardo et al 2016) the consensus nonanucleotide motif islsquoTRAKATTRCrsquo
The N terminus of the Rep contains motifs that are impor-tant for initiating RCR and it is not surprising that some of thesemotifs are well conserved across many ssDNA viruses phagesand plasmids that replicate using the RCR mechanism (Ilyinaand Koonin 1992 Vega-Rocha et al 2007a Rosario Duffy andBreitbart 2012 Krupovic 2013) The presence of a single cata-lytic tyrosine residue in the RCR motif III classifies genomovi-rus geminivirus bacilladnavirus circovirus and nanovirus Repsas members of superfamily II (Ilyina and Koonin 1992Krupovic 2013)
In genomoviruses the conserved sequence of the RCRmotif I which is thought to be involved in the recognition ofiterative sequences associated with the origin of replicationis predominantly lsquouuTYxQrsquo (u denotes hydrophobic residuesand x any residue) (Fig 6 Supplementary Data S1) with theexception of the Reps of currently known gemykolovirusesand gemykrogviruses The genomovirus RCR motif II lsquoxHxHxrsquo
Figure 6 Summary of conserved motifs that is nonanucleotide and Rep motifs illustrated using WebLogo3 (Crooks et al 2004) identified in the family Genomoviridae as
a whole and its nine genera separately Note the highly derived Walker A motif (GPHRRRRT) in the sole member of the genus Gemytondvirus
10 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
tree gemykoloviruses are firmly nested within the large cladeincluding the majority of gemycircularviruses (Fig 4) Giventhat CP sequences of genomoviruses are considerably more di-vergent than the Rep sequences (Fig 1) it appears reasonable toestablish a higher (ie above the species level) taxonomicframework using the Rep (Fig 2) The latter protein is also
conserved in other eukaryotic ssDNA viruses (which is not thecase for the CP) and can thus be used to assess the place ofgenomoviruses within the larger community of ssDNA viruses
To evaluate the taxonomic structure of the Genomoviridaewe took advantage of the fact that in Rep-based phylogeneticanalyses genomoviruses consistently form a sister group to
Figure 1 Distribution of (A) genome-wide (B) Rep and (C) CP pairwise identities (121 taxa) of genomoviruses calculated using SDT v12 (Muhire Varsani and Martin
2014)
6 | Virus Evolution 2017 Vol 3 No 1
members of the Geminiviridae (Krupovic et al 2016) a compre-hensively characterized family of plant viruses with circularssDNA genomes (Varsani et al 2014b) Thus using the estab-lished taxonomic framework of the Geminiviridae overlaid on theRep-based phylogeny as a guide we could define five cladesand four additional singletons within the Genomoviridae branch(Fig 2) The defined groups displayed equivalent intra-family di-vergence as the established genera within the familyGeminiviridae (Varsani et al 2014b) The nine groups were sup-ported in both nucleotide and protein sequence inferred phylog-enies (Supplementary Fig S2) Consequently in addition to theexisting genus Gemycircularvirus we propose establishing eightnew genera within the family Genomoviridae The details of thenine genera are summarized in Fig 5 and briefly outlinedbelow
31 Gemycircularvirus
This genus has the largest number of new species (nfrac14 43 sev-enty-three genomes Table 1) and includes SsHADV-1 thefounding member of the family Members of the genus display44 diversity Viruses within the forty-three species clusterwith 99 and 96 branch support values in phylogenetic treesconstructed from either Rep or full genome sequences respec-tively (Figs 2 and 3)
32 Gemykibivirus
This is the second most populated genus (nfrac14 16 twenty-nine ge-nomes Table 1) in the family with 43 diversity among its mem-bers The name of the genus is an acronym of words geminivirus-like and myco-like kibi virus (kibi means circular in Amharic)Sequences within the fifteen species cluster with 93 branch sup-port within phylogenetic trees constructed from Rep (Fig 2) andtwo well-supported clades (100 and 96) within trees constructedfrom full genome sequences (Fig 3) suggesting that recombinationhas played an important role in the evolution of this group
33 Gemygorvirus
Members of this genus (nfrac14 5 nine genomes Table 1) display49 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like gor virus (gor means round inHindi) Sequences within the five species cluster with 100 and99 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
34 Gemykolovirus
Members of this genus (nfrac14 2 three genomes Table 1) display37 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like kolo virus (kolo means round inCzech) Sequences within the two species cluster with 100 and89 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
35 Gemykrogvirus
Members of this genus (nfrac14 3 three genomes Table 1) display33 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like krog virus (krog means round inSlovenian) Sequences within the three species cluster with 99and 100 branch support within phylogenetic trees constructedfrom either Rep or full genome sequences respectively (Figs 2and 3)
KP133079 Human associated gemykibivirus 1 KP133080 Human associated gemykibivirus 1 KP133078 Human associated gemykibivirus 1 KP133077 Human associated gemykibivirus 1 KP133075 Human associated gemykibivirus 1 KP133076 Human associated gemykibivirus 1
JX185428 Dragonfly associated gemyduguivirus 1 KP974693 Human associated gemyvongvirus 1
Figure 3 Maximum likelihood phylogenetic tree of the genomes of viruses in the Genomoviridae family The tree was inferred using FastTree (Price Dehal and Arkin
2010) (GTRthornCAT) The numbers at the branches indicate SH-like support values The topology of tree supports the proposed genera demarcation at the genome level
despite there being evidence of recombination within the genomes Branches withlt75 SH-like branch support have been collapsed
8 | Virus Evolution 2017 Vol 3 No 1
35 Gemyvongvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like vong virus (vong means circular in Lao) The sin-gle species Human associated gemyvongvirus 1 (Table 1) within thegenus shares between 56 and 62 genome-wide sequence simi-larity with viruses in other genera and is a divergent taxon inthe phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
36 Gemytondvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like tond virus (tond means round in Maltese) Thesingle species Ostrich associated gemytondvirus 1 (Table 1) withinthe genus shares between 53 and 61 genome-wide sequencesimilarity with viruses in other genera and is a divergent taxonin the phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
37 Gemykroznavirus
The name of the genus is an acronym of words geminivirus-likeand myco-like krozna virus (krozna means circular in Slovenian)The single species Rabbit associated gemykroznavirus 1 (Table 1)
KP974693 Human associated gemyvongvirus 1 KF371631 Rabbit associated gemykroznavirus 1
KP133075 Human associated gemykibivirus 2 KP133080 Human associated gemykibivirus 2 KP133079 Human associated gemykibivirus 2 KP133078 Human associated gemykibivirus 2 KP133076 Human associated gemykibivirus 2 KP133077 Human associated gemykibivirus 2
KJ413144 Human associated gemygorvirus 1 KJ547635 Sewage derived gemygorvirus 1
100
100
100
80
94
86
100
100100
10099
97
9986
8688
95
99
10091
89
100
100
80
96
84
100100
100
87
10083
79
89 95
100
91
9391
100
93
92
94
88
93100
10094
77
10096
90
94
9284
100
10089
9696
99
80
96
95
100
100
94
100
10099
89
94100
100
93100
93
100
9895
100
100
87
02 amino acid subs per site
GemyduguivirusGemycircularvirus
GemykibivirusGemygorvirus
Gemyvongvirus
Gemykolovirus
GemytondvirusGemykroznavirusGemykrogvirus
Figure 4 Maximum likelihood phylogenetic tree of the CP amino acid sequences
inferred using PHYML (Guindon et al 2010) with LGthornGthornI substitution models
and rooted with geminivirus sequences Branches withlt75 SH-like branch sup-
port have been collapsed
0 10 20 30 40 50 60 70 80
Percentage diversity
Number of isolates
Number of species
Gemyvongvirus
Gemytondvirus
Gemykroznavirus
Gemykrogvirus
Gemykolovirus
Gemykibivirus
Gemygorvirus
Gemyduguivirus
Gemycircularvirus
Figure 5 Summary of genera and the associated species and their diversity
(within genera) within the Genomoviridae family
A Varsani and M Krupovic | 9
within the genus shares between 56 and 61 genome-widesequence similarity with other sequences in other genera and isa divergent taxon in the phylogenetic trees constructed fromeither Rep or full genome sequences (Figs 2 and 3)
38 Gemyduguivirus
The name of the genus is an acronym of words geminivirus-likeand myco-like dugui virus (dugui means circular in Mongolian)The single species Dragonfly associated gemyduguivirus 1 (Table 1)within the genus shares between 57 and 62 genome-wide se-quence similarity with viruses in other genera and is a divergenttaxon in the phylogenetic trees constructed from either Rep orfull genome sequences (Figs 2 and 3)
4 Conserved sequence motifs in theGenomoviridae
CRESS DNA viruses replicate through the rolling circle replica-tion (RCR) mechanism which is similar to that used by bacterialplasmids (Khan 1997 Chandler et al 2013 Ruiz-Maso et al2015) RCR is initiated by the Rep encoded by CRESS DNAviruses cleaving the dsDNA between positions 7 and 8 of anonanucleotide sequence located at a putative stem-loopstructure at the origin of replication (Heyraud-Nitschke et al1995 Laufs et al 1995b Timchenko et al 1999 RosarioDuffy and Breitbart 2012) In the case of genomoviruses this
nonanucleotide is variable (lsquoTAWWDWRNrsquo) with lsquoTAATWYATrsquobeing the consensus nonanucleotide for gemycircularviruseswhereas gemykibiruses display the greatest variation inthis motifmdashlsquoWATAWWHANrsquo (Fig 6 Supplementary Data S1)In contrast we note that within the Geminiviridae familyincluding all recently described geminiviruses (Varsani et al2009 Briddon et al 2010 Krenz et al 2012 Loconsole et al 2012Bernardo et al 2013 Heydarnejad et al 2013 Ma et al 2015Bernardo et al 2016) the consensus nonanucleotide motif islsquoTRAKATTRCrsquo
The N terminus of the Rep contains motifs that are impor-tant for initiating RCR and it is not surprising that some of thesemotifs are well conserved across many ssDNA viruses phagesand plasmids that replicate using the RCR mechanism (Ilyinaand Koonin 1992 Vega-Rocha et al 2007a Rosario Duffy andBreitbart 2012 Krupovic 2013) The presence of a single cata-lytic tyrosine residue in the RCR motif III classifies genomovi-rus geminivirus bacilladnavirus circovirus and nanovirus Repsas members of superfamily II (Ilyina and Koonin 1992Krupovic 2013)
In genomoviruses the conserved sequence of the RCRmotif I which is thought to be involved in the recognition ofiterative sequences associated with the origin of replicationis predominantly lsquouuTYxQrsquo (u denotes hydrophobic residuesand x any residue) (Fig 6 Supplementary Data S1) with theexception of the Reps of currently known gemykolovirusesand gemykrogviruses The genomovirus RCR motif II lsquoxHxHxrsquo
Figure 6 Summary of conserved motifs that is nonanucleotide and Rep motifs illustrated using WebLogo3 (Crooks et al 2004) identified in the family Genomoviridae as
a whole and its nine genera separately Note the highly derived Walker A motif (GPHRRRRT) in the sole member of the genus Gemytondvirus
10 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
members of the Geminiviridae (Krupovic et al 2016) a compre-hensively characterized family of plant viruses with circularssDNA genomes (Varsani et al 2014b) Thus using the estab-lished taxonomic framework of the Geminiviridae overlaid on theRep-based phylogeny as a guide we could define five cladesand four additional singletons within the Genomoviridae branch(Fig 2) The defined groups displayed equivalent intra-family di-vergence as the established genera within the familyGeminiviridae (Varsani et al 2014b) The nine groups were sup-ported in both nucleotide and protein sequence inferred phylog-enies (Supplementary Fig S2) Consequently in addition to theexisting genus Gemycircularvirus we propose establishing eightnew genera within the family Genomoviridae The details of thenine genera are summarized in Fig 5 and briefly outlinedbelow
31 Gemycircularvirus
This genus has the largest number of new species (nfrac14 43 sev-enty-three genomes Table 1) and includes SsHADV-1 thefounding member of the family Members of the genus display44 diversity Viruses within the forty-three species clusterwith 99 and 96 branch support values in phylogenetic treesconstructed from either Rep or full genome sequences respec-tively (Figs 2 and 3)
32 Gemykibivirus
This is the second most populated genus (nfrac14 16 twenty-nine ge-nomes Table 1) in the family with 43 diversity among its mem-bers The name of the genus is an acronym of words geminivirus-like and myco-like kibi virus (kibi means circular in Amharic)Sequences within the fifteen species cluster with 93 branch sup-port within phylogenetic trees constructed from Rep (Fig 2) andtwo well-supported clades (100 and 96) within trees constructedfrom full genome sequences (Fig 3) suggesting that recombinationhas played an important role in the evolution of this group
33 Gemygorvirus
Members of this genus (nfrac14 5 nine genomes Table 1) display49 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like gor virus (gor means round inHindi) Sequences within the five species cluster with 100 and99 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
34 Gemykolovirus
Members of this genus (nfrac14 2 three genomes Table 1) display37 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like kolo virus (kolo means round inCzech) Sequences within the two species cluster with 100 and89 branch support within phylogenetic trees constructed fromeither Rep or full genome sequences respectively (Figs 2 and 3)
35 Gemykrogvirus
Members of this genus (nfrac14 3 three genomes Table 1) display33 diversity The name of the genus is an acronym of wordsgeminivirus-like and myco-like krog virus (krog means round inSlovenian) Sequences within the three species cluster with 99and 100 branch support within phylogenetic trees constructedfrom either Rep or full genome sequences respectively (Figs 2and 3)
KP133079 Human associated gemykibivirus 1 KP133080 Human associated gemykibivirus 1 KP133078 Human associated gemykibivirus 1 KP133077 Human associated gemykibivirus 1 KP133075 Human associated gemykibivirus 1 KP133076 Human associated gemykibivirus 1
JX185428 Dragonfly associated gemyduguivirus 1 KP974693 Human associated gemyvongvirus 1
Figure 3 Maximum likelihood phylogenetic tree of the genomes of viruses in the Genomoviridae family The tree was inferred using FastTree (Price Dehal and Arkin
2010) (GTRthornCAT) The numbers at the branches indicate SH-like support values The topology of tree supports the proposed genera demarcation at the genome level
despite there being evidence of recombination within the genomes Branches withlt75 SH-like branch support have been collapsed
8 | Virus Evolution 2017 Vol 3 No 1
35 Gemyvongvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like vong virus (vong means circular in Lao) The sin-gle species Human associated gemyvongvirus 1 (Table 1) within thegenus shares between 56 and 62 genome-wide sequence simi-larity with viruses in other genera and is a divergent taxon inthe phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
36 Gemytondvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like tond virus (tond means round in Maltese) Thesingle species Ostrich associated gemytondvirus 1 (Table 1) withinthe genus shares between 53 and 61 genome-wide sequencesimilarity with viruses in other genera and is a divergent taxonin the phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
37 Gemykroznavirus
The name of the genus is an acronym of words geminivirus-likeand myco-like krozna virus (krozna means circular in Slovenian)The single species Rabbit associated gemykroznavirus 1 (Table 1)
KP974693 Human associated gemyvongvirus 1 KF371631 Rabbit associated gemykroznavirus 1
KP133075 Human associated gemykibivirus 2 KP133080 Human associated gemykibivirus 2 KP133079 Human associated gemykibivirus 2 KP133078 Human associated gemykibivirus 2 KP133076 Human associated gemykibivirus 2 KP133077 Human associated gemykibivirus 2
KJ413144 Human associated gemygorvirus 1 KJ547635 Sewage derived gemygorvirus 1
100
100
100
80
94
86
100
100100
10099
97
9986
8688
95
99
10091
89
100
100
80
96
84
100100
100
87
10083
79
89 95
100
91
9391
100
93
92
94
88
93100
10094
77
10096
90
94
9284
100
10089
9696
99
80
96
95
100
100
94
100
10099
89
94100
100
93100
93
100
9895
100
100
87
02 amino acid subs per site
GemyduguivirusGemycircularvirus
GemykibivirusGemygorvirus
Gemyvongvirus
Gemykolovirus
GemytondvirusGemykroznavirusGemykrogvirus
Figure 4 Maximum likelihood phylogenetic tree of the CP amino acid sequences
inferred using PHYML (Guindon et al 2010) with LGthornGthornI substitution models
and rooted with geminivirus sequences Branches withlt75 SH-like branch sup-
port have been collapsed
0 10 20 30 40 50 60 70 80
Percentage diversity
Number of isolates
Number of species
Gemyvongvirus
Gemytondvirus
Gemykroznavirus
Gemykrogvirus
Gemykolovirus
Gemykibivirus
Gemygorvirus
Gemyduguivirus
Gemycircularvirus
Figure 5 Summary of genera and the associated species and their diversity
(within genera) within the Genomoviridae family
A Varsani and M Krupovic | 9
within the genus shares between 56 and 61 genome-widesequence similarity with other sequences in other genera and isa divergent taxon in the phylogenetic trees constructed fromeither Rep or full genome sequences (Figs 2 and 3)
38 Gemyduguivirus
The name of the genus is an acronym of words geminivirus-likeand myco-like dugui virus (dugui means circular in Mongolian)The single species Dragonfly associated gemyduguivirus 1 (Table 1)within the genus shares between 57 and 62 genome-wide se-quence similarity with viruses in other genera and is a divergenttaxon in the phylogenetic trees constructed from either Rep orfull genome sequences (Figs 2 and 3)
4 Conserved sequence motifs in theGenomoviridae
CRESS DNA viruses replicate through the rolling circle replica-tion (RCR) mechanism which is similar to that used by bacterialplasmids (Khan 1997 Chandler et al 2013 Ruiz-Maso et al2015) RCR is initiated by the Rep encoded by CRESS DNAviruses cleaving the dsDNA between positions 7 and 8 of anonanucleotide sequence located at a putative stem-loopstructure at the origin of replication (Heyraud-Nitschke et al1995 Laufs et al 1995b Timchenko et al 1999 RosarioDuffy and Breitbart 2012) In the case of genomoviruses this
nonanucleotide is variable (lsquoTAWWDWRNrsquo) with lsquoTAATWYATrsquobeing the consensus nonanucleotide for gemycircularviruseswhereas gemykibiruses display the greatest variation inthis motifmdashlsquoWATAWWHANrsquo (Fig 6 Supplementary Data S1)In contrast we note that within the Geminiviridae familyincluding all recently described geminiviruses (Varsani et al2009 Briddon et al 2010 Krenz et al 2012 Loconsole et al 2012Bernardo et al 2013 Heydarnejad et al 2013 Ma et al 2015Bernardo et al 2016) the consensus nonanucleotide motif islsquoTRAKATTRCrsquo
The N terminus of the Rep contains motifs that are impor-tant for initiating RCR and it is not surprising that some of thesemotifs are well conserved across many ssDNA viruses phagesand plasmids that replicate using the RCR mechanism (Ilyinaand Koonin 1992 Vega-Rocha et al 2007a Rosario Duffy andBreitbart 2012 Krupovic 2013) The presence of a single cata-lytic tyrosine residue in the RCR motif III classifies genomovi-rus geminivirus bacilladnavirus circovirus and nanovirus Repsas members of superfamily II (Ilyina and Koonin 1992Krupovic 2013)
In genomoviruses the conserved sequence of the RCRmotif I which is thought to be involved in the recognition ofiterative sequences associated with the origin of replicationis predominantly lsquouuTYxQrsquo (u denotes hydrophobic residuesand x any residue) (Fig 6 Supplementary Data S1) with theexception of the Reps of currently known gemykolovirusesand gemykrogviruses The genomovirus RCR motif II lsquoxHxHxrsquo
Figure 6 Summary of conserved motifs that is nonanucleotide and Rep motifs illustrated using WebLogo3 (Crooks et al 2004) identified in the family Genomoviridae as
a whole and its nine genera separately Note the highly derived Walker A motif (GPHRRRRT) in the sole member of the genus Gemytondvirus
10 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
KP133079 Human associated gemykibivirus 1 KP133080 Human associated gemykibivirus 1 KP133078 Human associated gemykibivirus 1 KP133077 Human associated gemykibivirus 1 KP133075 Human associated gemykibivirus 1 KP133076 Human associated gemykibivirus 1
JX185428 Dragonfly associated gemyduguivirus 1 KP974693 Human associated gemyvongvirus 1
Figure 3 Maximum likelihood phylogenetic tree of the genomes of viruses in the Genomoviridae family The tree was inferred using FastTree (Price Dehal and Arkin
2010) (GTRthornCAT) The numbers at the branches indicate SH-like support values The topology of tree supports the proposed genera demarcation at the genome level
despite there being evidence of recombination within the genomes Branches withlt75 SH-like branch support have been collapsed
8 | Virus Evolution 2017 Vol 3 No 1
35 Gemyvongvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like vong virus (vong means circular in Lao) The sin-gle species Human associated gemyvongvirus 1 (Table 1) within thegenus shares between 56 and 62 genome-wide sequence simi-larity with viruses in other genera and is a divergent taxon inthe phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
36 Gemytondvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like tond virus (tond means round in Maltese) Thesingle species Ostrich associated gemytondvirus 1 (Table 1) withinthe genus shares between 53 and 61 genome-wide sequencesimilarity with viruses in other genera and is a divergent taxonin the phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
37 Gemykroznavirus
The name of the genus is an acronym of words geminivirus-likeand myco-like krozna virus (krozna means circular in Slovenian)The single species Rabbit associated gemykroznavirus 1 (Table 1)
KP974693 Human associated gemyvongvirus 1 KF371631 Rabbit associated gemykroznavirus 1
KP133075 Human associated gemykibivirus 2 KP133080 Human associated gemykibivirus 2 KP133079 Human associated gemykibivirus 2 KP133078 Human associated gemykibivirus 2 KP133076 Human associated gemykibivirus 2 KP133077 Human associated gemykibivirus 2
KJ413144 Human associated gemygorvirus 1 KJ547635 Sewage derived gemygorvirus 1
100
100
100
80
94
86
100
100100
10099
97
9986
8688
95
99
10091
89
100
100
80
96
84
100100
100
87
10083
79
89 95
100
91
9391
100
93
92
94
88
93100
10094
77
10096
90
94
9284
100
10089
9696
99
80
96
95
100
100
94
100
10099
89
94100
100
93100
93
100
9895
100
100
87
02 amino acid subs per site
GemyduguivirusGemycircularvirus
GemykibivirusGemygorvirus
Gemyvongvirus
Gemykolovirus
GemytondvirusGemykroznavirusGemykrogvirus
Figure 4 Maximum likelihood phylogenetic tree of the CP amino acid sequences
inferred using PHYML (Guindon et al 2010) with LGthornGthornI substitution models
and rooted with geminivirus sequences Branches withlt75 SH-like branch sup-
port have been collapsed
0 10 20 30 40 50 60 70 80
Percentage diversity
Number of isolates
Number of species
Gemyvongvirus
Gemytondvirus
Gemykroznavirus
Gemykrogvirus
Gemykolovirus
Gemykibivirus
Gemygorvirus
Gemyduguivirus
Gemycircularvirus
Figure 5 Summary of genera and the associated species and their diversity
(within genera) within the Genomoviridae family
A Varsani and M Krupovic | 9
within the genus shares between 56 and 61 genome-widesequence similarity with other sequences in other genera and isa divergent taxon in the phylogenetic trees constructed fromeither Rep or full genome sequences (Figs 2 and 3)
38 Gemyduguivirus
The name of the genus is an acronym of words geminivirus-likeand myco-like dugui virus (dugui means circular in Mongolian)The single species Dragonfly associated gemyduguivirus 1 (Table 1)within the genus shares between 57 and 62 genome-wide se-quence similarity with viruses in other genera and is a divergenttaxon in the phylogenetic trees constructed from either Rep orfull genome sequences (Figs 2 and 3)
4 Conserved sequence motifs in theGenomoviridae
CRESS DNA viruses replicate through the rolling circle replica-tion (RCR) mechanism which is similar to that used by bacterialplasmids (Khan 1997 Chandler et al 2013 Ruiz-Maso et al2015) RCR is initiated by the Rep encoded by CRESS DNAviruses cleaving the dsDNA between positions 7 and 8 of anonanucleotide sequence located at a putative stem-loopstructure at the origin of replication (Heyraud-Nitschke et al1995 Laufs et al 1995b Timchenko et al 1999 RosarioDuffy and Breitbart 2012) In the case of genomoviruses this
nonanucleotide is variable (lsquoTAWWDWRNrsquo) with lsquoTAATWYATrsquobeing the consensus nonanucleotide for gemycircularviruseswhereas gemykibiruses display the greatest variation inthis motifmdashlsquoWATAWWHANrsquo (Fig 6 Supplementary Data S1)In contrast we note that within the Geminiviridae familyincluding all recently described geminiviruses (Varsani et al2009 Briddon et al 2010 Krenz et al 2012 Loconsole et al 2012Bernardo et al 2013 Heydarnejad et al 2013 Ma et al 2015Bernardo et al 2016) the consensus nonanucleotide motif islsquoTRAKATTRCrsquo
The N terminus of the Rep contains motifs that are impor-tant for initiating RCR and it is not surprising that some of thesemotifs are well conserved across many ssDNA viruses phagesand plasmids that replicate using the RCR mechanism (Ilyinaand Koonin 1992 Vega-Rocha et al 2007a Rosario Duffy andBreitbart 2012 Krupovic 2013) The presence of a single cata-lytic tyrosine residue in the RCR motif III classifies genomovi-rus geminivirus bacilladnavirus circovirus and nanovirus Repsas members of superfamily II (Ilyina and Koonin 1992Krupovic 2013)
In genomoviruses the conserved sequence of the RCRmotif I which is thought to be involved in the recognition ofiterative sequences associated with the origin of replicationis predominantly lsquouuTYxQrsquo (u denotes hydrophobic residuesand x any residue) (Fig 6 Supplementary Data S1) with theexception of the Reps of currently known gemykolovirusesand gemykrogviruses The genomovirus RCR motif II lsquoxHxHxrsquo
Figure 6 Summary of conserved motifs that is nonanucleotide and Rep motifs illustrated using WebLogo3 (Crooks et al 2004) identified in the family Genomoviridae as
a whole and its nine genera separately Note the highly derived Walker A motif (GPHRRRRT) in the sole member of the genus Gemytondvirus
10 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
35 Gemyvongvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like vong virus (vong means circular in Lao) The sin-gle species Human associated gemyvongvirus 1 (Table 1) within thegenus shares between 56 and 62 genome-wide sequence simi-larity with viruses in other genera and is a divergent taxon inthe phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
36 Gemytondvirus
The name of the genus is an acronym of words geminivirus-likeand myco-like tond virus (tond means round in Maltese) Thesingle species Ostrich associated gemytondvirus 1 (Table 1) withinthe genus shares between 53 and 61 genome-wide sequencesimilarity with viruses in other genera and is a divergent taxonin the phylogenetic trees constructed from either Rep or full ge-nome sequences (Figs 2 and 3)
37 Gemykroznavirus
The name of the genus is an acronym of words geminivirus-likeand myco-like krozna virus (krozna means circular in Slovenian)The single species Rabbit associated gemykroznavirus 1 (Table 1)
KP974693 Human associated gemyvongvirus 1 KF371631 Rabbit associated gemykroznavirus 1
KP133075 Human associated gemykibivirus 2 KP133080 Human associated gemykibivirus 2 KP133079 Human associated gemykibivirus 2 KP133078 Human associated gemykibivirus 2 KP133076 Human associated gemykibivirus 2 KP133077 Human associated gemykibivirus 2
KJ413144 Human associated gemygorvirus 1 KJ547635 Sewage derived gemygorvirus 1
100
100
100
80
94
86
100
100100
10099
97
9986
8688
95
99
10091
89
100
100
80
96
84
100100
100
87
10083
79
89 95
100
91
9391
100
93
92
94
88
93100
10094
77
10096
90
94
9284
100
10089
9696
99
80
96
95
100
100
94
100
10099
89
94100
100
93100
93
100
9895
100
100
87
02 amino acid subs per site
GemyduguivirusGemycircularvirus
GemykibivirusGemygorvirus
Gemyvongvirus
Gemykolovirus
GemytondvirusGemykroznavirusGemykrogvirus
Figure 4 Maximum likelihood phylogenetic tree of the CP amino acid sequences
inferred using PHYML (Guindon et al 2010) with LGthornGthornI substitution models
and rooted with geminivirus sequences Branches withlt75 SH-like branch sup-
port have been collapsed
0 10 20 30 40 50 60 70 80
Percentage diversity
Number of isolates
Number of species
Gemyvongvirus
Gemytondvirus
Gemykroznavirus
Gemykrogvirus
Gemykolovirus
Gemykibivirus
Gemygorvirus
Gemyduguivirus
Gemycircularvirus
Figure 5 Summary of genera and the associated species and their diversity
(within genera) within the Genomoviridae family
A Varsani and M Krupovic | 9
within the genus shares between 56 and 61 genome-widesequence similarity with other sequences in other genera and isa divergent taxon in the phylogenetic trees constructed fromeither Rep or full genome sequences (Figs 2 and 3)
38 Gemyduguivirus
The name of the genus is an acronym of words geminivirus-likeand myco-like dugui virus (dugui means circular in Mongolian)The single species Dragonfly associated gemyduguivirus 1 (Table 1)within the genus shares between 57 and 62 genome-wide se-quence similarity with viruses in other genera and is a divergenttaxon in the phylogenetic trees constructed from either Rep orfull genome sequences (Figs 2 and 3)
4 Conserved sequence motifs in theGenomoviridae
CRESS DNA viruses replicate through the rolling circle replica-tion (RCR) mechanism which is similar to that used by bacterialplasmids (Khan 1997 Chandler et al 2013 Ruiz-Maso et al2015) RCR is initiated by the Rep encoded by CRESS DNAviruses cleaving the dsDNA between positions 7 and 8 of anonanucleotide sequence located at a putative stem-loopstructure at the origin of replication (Heyraud-Nitschke et al1995 Laufs et al 1995b Timchenko et al 1999 RosarioDuffy and Breitbart 2012) In the case of genomoviruses this
nonanucleotide is variable (lsquoTAWWDWRNrsquo) with lsquoTAATWYATrsquobeing the consensus nonanucleotide for gemycircularviruseswhereas gemykibiruses display the greatest variation inthis motifmdashlsquoWATAWWHANrsquo (Fig 6 Supplementary Data S1)In contrast we note that within the Geminiviridae familyincluding all recently described geminiviruses (Varsani et al2009 Briddon et al 2010 Krenz et al 2012 Loconsole et al 2012Bernardo et al 2013 Heydarnejad et al 2013 Ma et al 2015Bernardo et al 2016) the consensus nonanucleotide motif islsquoTRAKATTRCrsquo
The N terminus of the Rep contains motifs that are impor-tant for initiating RCR and it is not surprising that some of thesemotifs are well conserved across many ssDNA viruses phagesand plasmids that replicate using the RCR mechanism (Ilyinaand Koonin 1992 Vega-Rocha et al 2007a Rosario Duffy andBreitbart 2012 Krupovic 2013) The presence of a single cata-lytic tyrosine residue in the RCR motif III classifies genomovi-rus geminivirus bacilladnavirus circovirus and nanovirus Repsas members of superfamily II (Ilyina and Koonin 1992Krupovic 2013)
In genomoviruses the conserved sequence of the RCRmotif I which is thought to be involved in the recognition ofiterative sequences associated with the origin of replicationis predominantly lsquouuTYxQrsquo (u denotes hydrophobic residuesand x any residue) (Fig 6 Supplementary Data S1) with theexception of the Reps of currently known gemykolovirusesand gemykrogviruses The genomovirus RCR motif II lsquoxHxHxrsquo
Figure 6 Summary of conserved motifs that is nonanucleotide and Rep motifs illustrated using WebLogo3 (Crooks et al 2004) identified in the family Genomoviridae as
a whole and its nine genera separately Note the highly derived Walker A motif (GPHRRRRT) in the sole member of the genus Gemytondvirus
10 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
within the genus shares between 56 and 61 genome-widesequence similarity with other sequences in other genera and isa divergent taxon in the phylogenetic trees constructed fromeither Rep or full genome sequences (Figs 2 and 3)
38 Gemyduguivirus
The name of the genus is an acronym of words geminivirus-likeand myco-like dugui virus (dugui means circular in Mongolian)The single species Dragonfly associated gemyduguivirus 1 (Table 1)within the genus shares between 57 and 62 genome-wide se-quence similarity with viruses in other genera and is a divergenttaxon in the phylogenetic trees constructed from either Rep orfull genome sequences (Figs 2 and 3)
4 Conserved sequence motifs in theGenomoviridae
CRESS DNA viruses replicate through the rolling circle replica-tion (RCR) mechanism which is similar to that used by bacterialplasmids (Khan 1997 Chandler et al 2013 Ruiz-Maso et al2015) RCR is initiated by the Rep encoded by CRESS DNAviruses cleaving the dsDNA between positions 7 and 8 of anonanucleotide sequence located at a putative stem-loopstructure at the origin of replication (Heyraud-Nitschke et al1995 Laufs et al 1995b Timchenko et al 1999 RosarioDuffy and Breitbart 2012) In the case of genomoviruses this
nonanucleotide is variable (lsquoTAWWDWRNrsquo) with lsquoTAATWYATrsquobeing the consensus nonanucleotide for gemycircularviruseswhereas gemykibiruses display the greatest variation inthis motifmdashlsquoWATAWWHANrsquo (Fig 6 Supplementary Data S1)In contrast we note that within the Geminiviridae familyincluding all recently described geminiviruses (Varsani et al2009 Briddon et al 2010 Krenz et al 2012 Loconsole et al 2012Bernardo et al 2013 Heydarnejad et al 2013 Ma et al 2015Bernardo et al 2016) the consensus nonanucleotide motif islsquoTRAKATTRCrsquo
The N terminus of the Rep contains motifs that are impor-tant for initiating RCR and it is not surprising that some of thesemotifs are well conserved across many ssDNA viruses phagesand plasmids that replicate using the RCR mechanism (Ilyinaand Koonin 1992 Vega-Rocha et al 2007a Rosario Duffy andBreitbart 2012 Krupovic 2013) The presence of a single cata-lytic tyrosine residue in the RCR motif III classifies genomovi-rus geminivirus bacilladnavirus circovirus and nanovirus Repsas members of superfamily II (Ilyina and Koonin 1992Krupovic 2013)
In genomoviruses the conserved sequence of the RCRmotif I which is thought to be involved in the recognition ofiterative sequences associated with the origin of replicationis predominantly lsquouuTYxQrsquo (u denotes hydrophobic residuesand x any residue) (Fig 6 Supplementary Data S1) with theexception of the Reps of currently known gemykolovirusesand gemykrogviruses The genomovirus RCR motif II lsquoxHxHxrsquo
Figure 6 Summary of conserved motifs that is nonanucleotide and Rep motifs illustrated using WebLogo3 (Crooks et al 2004) identified in the family Genomoviridae as
a whole and its nine genera separately Note the highly derived Walker A motif (GPHRRRRT) in the sole member of the genus Gemytondvirus
10 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
(Fig 6 Supplementary Data S1) resembles that found in gemini-viruses and early work has shown that histidines in this motifcoordinate divalent metal ions Mg2thornor Mn2thorn which areimportant cofactors for endonuclease activity at the origin ofreplication (Koonin and Ilyina 1992 Laufs et al 1995b)Genomoviruses have an RCR motif III of lsquoYxxKrsquo and based onother Rep studies this motif is involved in the dsDNA cleavageand subsequent covalent attachment of Rep through thecatalytic tyrosine residue to the 50 end of the cleaved product(Laufs et al 1995a b Orozco and Hanley-Bowdoin 1998Timchenko et al 1999 Steinfeldt Finsterbusch and Mankertz2006 Rosario Duffy and Breitbart 2012) The conserved lysineresidue in the RCR motif III (Fig 6 Supplementary Data S1) isproposed to mediate binding and positioning during catalysis(Vega-Rocha et al 2007a b) A fourth conserved motif the gemi-nivirus Rep sequence (GRS) is only found in geminiviruses andgenomoviruses (Fig 6) In geminiviruses it enables appropriatespatial arrangements of RCR motifs II and III (Nash et al 2011)Site-directed mutagenesis of the GRS domain in tomato goldenmosaic virus yielded non-infectious clones demonstrating thatthe GRS is essential for geminivirus replication (Nash et al 2011)and it is likely this is also the case for genomoviruses
Rep is a multifunctional protein with both endonucleaseand helicase activities Rep helicase activity is mediated by con-served motifs known as Walker A Walker B and motif C locatedin a C-terminal NTP-binding domain (Fig 6 SupplementaryData S1) (Gorbalenya Koonin and Wolf 1990 Koonin 1993Choudhury et al 2006 Clerot and Bernardi 2006) The helicasedomain found in Rep proteins of eukaryotic ssDNA viruses be-longs to the helicase superfamily 3 (Gorbalenya Koonin andWolf 1990 Koonin 1993) The conserved Walker A motif ofgenomoviruses is lsquoGxxxxGKTrsquo with the exception of gemytond-virus which contains a highly derived variant of this motif(GPHRRRRT Fig 6) Previous studies have shown that duringsynthesis of progeny strands Rep helicase activity unwinds thedsDNA intermediate in the 30ndash50 direction using nucleotide tri-phosphates as an energy source (Choudhury et al 2006 Clerotand Bernardi 2006) Walker A motif forms part of the lsquoP-looprsquostructure in the NTP-binding domain that facilitates ATP recog-nition and binding with a conserved lysine residue (Desbiez et al1995 Timchenko et al 1999 Choudhury et al 2006 Clerot andBernardi 2006 Rosario Duffy and Breitbart 2012 George et al2014) The Walker B of genomoviruses is predominantly lsquouuDDursquo(Fig 6 Supplementary Data S1) whereas the motif C is lsquouxxNrsquo(u denotes hydrophobic residues and x any residue Fig 6Supplementary Data S1) The hydrophobic residues in Walker Bmotif contribute to ATP binding and are essential for ATP hydro-lysis whereas the one in motif C (Fig 6 Supplementary Data S1)interacts with the gamma phosphate of ATP and the nucleo-philic water molecule via a conserved asparagine residue(Choudhury et al 2006 George et al 2014)
Genomoviruses from different genera display distinct signa-tures within the nonanucleotide as well as conserved nucleaseand helicase motifs which are generally consistent with theproposed taxa (Fig 6 Supplementary Data S1)
5 Concluding remarks
The Reps of genomoviruses are most closely related to those ofgeminiviruses and hence here we used a geminivirustaxonomy-informed approach to classify 121 genomovirusesinto Rep sequence-based genera Within the Genomoviridae fam-ily we establish eight new genera in addition to the one createdpreviously (Krupovic et al 2016) Detailed analysis of sequence
motifs conserved within the genomoviral genomes further sup-ports the validity of the proposed genera We also define a spe-cies demarcation criterion of 78 genome-wide identity that issequences that sharegt78 pairwise identity with othergenomovirus sequences belong to the same species and thosethat sharelt78 can be considered as new species It is worthnoting that despite the fact that geminiviruses have been stud-ied for over two decades the sequence diversity of all knowngeminiviruses is similar to that of the recently discoveredgenomoviruses (46 vs 47 respectively) This observationstrongly suggests that the extent of sequence diversity withinthis expansive virus group remains largely unexplored
Although the guidelines presented here are tailored for theclassification of viral genomes in the family Genomoviridae asimilar sequence-based framework can be easily adapted forother virus clusters identified though metagenomics studiesand lacking a pre-existing taxonomic framework in particularfor novel CRESS DNA viruses We do acknowledge that this ap-proach deviates from a previous norm that used a set of criteriaincluding biological properties such as host range pathologyvectors etc coupled with sequence data However given thatthe rate at which genome sequences of uncultivated viruses arebeing identified from various sources we need to establishmore robust classification approaches that can easily be imple-mented on the bases of sequence data alone Indeed this neces-sity is acknowledged by the ICTV which encouragessubmissions of taxonomic proposals for classification of virusesthat are known exclusively from their genome sequences(Simmonds et al 2017) This new tide in virus taxonomy is ex-pected to catalyze the comprehension of the diversity ecologyand evolution of the global virome
Supplementary data
Supplementary data are available at Virus Evolution online
Disclaimer
This article is based on the taxonomic proposal 2016001a-agFUv5Genomoviridae which has been considered and ap-proved by the Executive Committee (EC) of the ICTV AV and MKare elected members of the ICTV EC
Conflict of interest None declared
ReferencesAdams M J et al (2016) lsquoRatification Vote on Taxonomic
Proposals to the International Committee on Taxonomy ofVirusesrsquo Archives of Virology 161 2921ndash49
Bernardo P et al (2013) lsquoIdentification and Characterisation of aHighly Divergent Geminivirus Evolutionary and TaxonomicImplicationsrsquo Virus Research 177 35ndash45
et al (2016) lsquoMolecular Characterization and Prevalence ofTwo Capulaviruses Alfalfa Leaf Curl Virus From France andEuphorbia Caput-Medusae Latent Virus From South AfricarsquoVirology 493 142ndash53
Briddon R W et al (2010) lsquoTurnip Curly Top Virus a HighlyDivergent Geminivirus Infecting Turnip in Iranrsquo Virus Research152 169ndash75
Brown J K et al (2015) lsquoRevision of Begomovirus TaxonomyBased on Pairwise Sequence Comparisonsrsquo Archives of Virology160 1593ndash619
A Varsani and M Krupovic | 11
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
Cadar D et al (2013) lsquoPhylogeny Spatio-TemporalPhylodynamics and Evolutionary Scenario of Torque teno susvirus 1 (TTSuV1) and 2 (TTSuV2) in Wild Boars Fast Dispersaland High Genetic Diversityrsquo Veterinary Microbiology 166 200ndash13
Chandler M et al (2013) lsquoBreaking and Joining Single-StrandedDNA the HUH Endonuclease Superfamilyrsquo Nature ReviewsMicrobiology 11 525ndash38
Choudhury N R et al (2006) lsquoThe Oligomeric Rep Protein ofMungbean Yellow Mosaic India Virus (MYMIV) Is a LikelyReplicative Helicasersquo Nucleic Acids Research 34 6362ndash77
Clerot D and Bernardi F (2006) lsquoDNA Helicase Activity IsAssociated with the Replication Initiator Protein Rep ofTomato Yellow Leaf Curl Geminivirusrsquo Journal of Virology 8011322ndash30
Conceicao-Neto N et al (2015) lsquoFecal Virome Analysis of ThreeCarnivores Reveals a Novel Nodavirus and MultipleGemycircularvirusesrsquo Virology Journal 12 79
Crooks G E et al (2004) lsquoWebLogo a Sequence Logo GeneratorrsquoGenome Research 14 1188ndash90
Dayaram A et al (2012) lsquoMolecular Characterisation of a NovelCassava Associated Circular ssDNA Virusrsquo Virus Research 166130ndash5
et al (2015) lsquoIdentification of Diverse Circular Single-Stranded DNA Viruses in Adult Dragonflies and Damselflies(Insecta Odonata) of Arizona and Oklahoma USArsquo InfectionGenetics and Evolution 30 278ndash87
et al (2016) lsquoDiverse Circular Replication-AssociatedProtein Encoding Viruses Circulating in InvertebratesWithin a Lake Ecosystemrsquo Infection Genetics and Evolution 39304ndash16
Desbiez C et al (1995) lsquoRep Protein of Tomato Yellow Leaf CurlGeminivirus Has an ATPase Activity Required for Viral DNAReplicationrsquo Proceedings of the National Academy of Sciences of theUnited States of America 92 5640ndash4
Du Z et al (2014) lsquoIdentification and MolecularCharacterization of a Single-Stranded Circular DNA Virus withSimilarities to Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1rsquo Archives of Virology 159 1527ndash31
Duffy S and Holmes E C (2008) lsquoPhylogenetic Evidence forRapid Rates of Molecular Evolution in the Single-StrandedDNA Begomovirus Tomato Yellow Leaf Curl Virusrsquo Journal ofVirology 82 957ndash65
and (2009) lsquoValidation of High Rates of NucleotideSubstitution in Geminiviruses Phylogenetic Evidence FromEast African Cassava Mosaic Virusesrsquo Journal of GeneralVirology 90 1539ndash47
Dutilh B E et al (2014) lsquoA Highly Abundant BacteriophageDiscovered in the Unknown Sequences of Human FaecalMetagenomesrsquo Nature Communications 5 4498
Firth C et al (2009) lsquoInsights into the Evolutionary History of anEmerging Livestock Pathogen Porcine Circovirus 2rsquo Journal ofVirology 83 12813ndash21
George B et al (2014) lsquoMutational Analysis of the HelicaseDomain of a Replication Initiator Protein Reveals Critical Rolesof Lys 272 of the Brsquo Motif and Lys 289 of the Beta-Hairpin Loopin Geminivirus Replicationrsquo Journal of General Virology 951591ndash602
Gorbalenya A E Koonin E V and Wolf Y I (1990) lsquoA NewSuperfamily of Putative NTP-Binding Domains Encoded byGenomes of Small DNA and RNA Virusesrsquo FEBS Letters 262145ndash8
Grigoras I et al (2010) lsquoHigh Variability and Rapid Evolution of aNanovirusrsquo Journal of Virology 84 9105ndash17
Guindon S et al (2010) lsquoNew Algorithms and Methods toEstimate Maximum-Likelihood Phylogenies Assessing thePerformance of PhyML 30rsquo Systems Biology 59 307ndash21
Halary S et al (2016) lsquoNovel Single-Stranded DNA CircularViruses in Pericardial Fluid of Patient with RecurrentPericarditisrsquo Emerging infectious diseases 22 1839ndash41
Hanna Z R et al (2015) lsquoIsolation of a Complete Circular VirusGenome Sequence from an Alaskan Black-Capped Chickadee(Poecile atricapillus) Gastrointestinal Tract Samplersquo GenomeAnnouncements 3 e01081_15
Harkins G W et al (2009) lsquoExperimental Evidence Indicatingthat Mastreviruses Probably Did Not Co-Diverge with TheirHostsrsquo Virology Journal 6 104
et al (2014) lsquoTowards Inferring the Global Movement ofBeak and Feather Disease Virusrsquo Virology 450ndash451 24ndash33
Heydarnejad J et al (2013) lsquoFulfilling Kochrsquos Postulates for BeetCurly Top Iran Virus and Proposal for Consideration of NewGenus in the Family Geminiviridaersquo Archives of Virology 158435ndash43
Heyraud-Nitschke F et al (1995) lsquoDetermination of the OriginCleavage and Joining Domain of Geminivirus Rep ProteinsrsquoNucleic Acids Research 23 910ndash6
Ilyina T V and Koonin E V (1992) lsquoConserved SequenceMotifs in the Initiator Proteins for Rolling Circle DNAReplication Encoded by Diverse Replicons from EubacteriaEucaryotes and Archaebacteriarsquo Nucleic Acids Research 203279ndash85
Khan S A (1997) lsquoRolling-Circle Replication of Bacterial PlasmidsrsquoMicrobiology and Molecular Biology Reviews 61 442ndash55
Kolawole A O et al (2014) lsquoFlexibility in Surface-Exposed Loopsin a Virus Capsid Mediates Escape From AntibodyNeutralizationrsquo Journal of Virology 88 4543ndash57
Koonin E V (1993) lsquoA Common Set of Conserved Motifs in a VastVariety of Putative Nucleic Acid-Dependent ATPases IncludingMCM Proteins Involved in the Initiation of Eukaryotic DNAReplicationrsquo Nucleic Acids Research 21 2541ndash7
and Ilyina T V (1992) lsquoGeminivirus Replication ProteinsAre Related to Prokaryotic Plasmid Rolling Circle DNAReplication Initiator Proteinsrsquo Journal of General Virology 732763ndash6
Kraberger S et al (2013) lsquoDiscovery of Sclerotinia sclerotiorumHypovirulence-Associated Virus-1 in Urban River Sedimentsof Heathcote and Styx Rivers in Christchurch City NewZealandrsquo Genome Announcements 1 e00559_13
et al (2015a) lsquoCharacterisation of a Diverse Range ofCircular Replication-Associated Protein Encoding DNA VirusesRecovered From a Sewage Treatment Oxidation PondrsquoInfection Genetics and Evolution 31 73ndash86
et al (2015b) lsquoIdentification of Novel Bromus- andTrifolium-Associated Circular DNA Virusesrsquo Archives ofVirology 160 1303ndash11
Krenz B et al (2012) lsquoComplete Genome Sequence of aNew Circular DNA Virus From Grapevinersquo Journal of Virology86 7715
Krupovic M (2013) lsquoNetworks of Evolutionary InteractionsUnderlying the Polyphyletic Origin of ssDNA Virusesrsquo CurrentOpinion in Virology 3 578ndash86
et al (2016) lsquoGenomoviridae a New Family of WidespreadSingle-Stranded DNA Virusesrsquo Archives of Virology 1612633ndash43
Labonte J M and Suttle C A (2013) lsquoPreviously Unknown andHighly Divergent ssDNA Viruses Populate the Oceansrsquo ISMEJournal 7 2169ndash77
12 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
Lamberto I et al (2014) lsquoMycovirus-Like DNA Virus SequencesFrom Cattle Serum and Human Brain and Serum SamplesFrom Multiple Sclerosis Patientsrsquo Genome Announcements 2e00848_14
Laufs J et al (1995a) lsquoIdentification of the Nicking Tyrosine ofGeminivirus Rep Proteinrsquo FEBS Letters 377 258ndash62
et al (1995b) lsquoIn Vitro Cleavage and Joining at the ViralOrigin of Replication by the Replication Initiator Protein ofTomato Yellow Leaf Curl Virusrsquo Proceedings of the NationalAcademy of Sciences of the United States of America 923879ndash83
Li W et al (2015) lsquoA Novel Gemycircularvirus From ExperimentalRatsrsquo Virus Genes 51 302ndash5
Liu S et al (2016) lsquoFungal DNA Virus Infects a MycophagousInsect and Utilizes It as a Transmission Vectorrsquo Proceedings ofthe National Academy of Sciences of the United States of AmericaDOI 101073pnas1608013113
Loconsole G et al (2012) lsquoIdentification of a Single-StrandedDNA Virus Associated with Citrus Chlorotic Dwarf Disease aNew Member in the Family Geminiviridaersquo Virology 432162ndash72
Ma Y et al (2015) lsquoIdentification and MolecularCharacterization of a Novel Monopartite GeminivirusAssociated with Mulberry Mosaic Dwarf Diseasersquo Journal ofGeneral Virology 96 2421ndash34
Male M F et al (2015) lsquoGenome Sequences of Poaceae-Associated Gemycircularviruses from the Pacific Ocean Island ofTongarsquo Genome Announcements 3 e01144_15
et al (2016) lsquoCycloviruses Gemycircularviruses and OtherNovel Replication-Associated Protein Encoding CircularViruses in Pacific flying fox (Pteropus tonganus) Faecesrsquo InfectionGenetics and Evolution 39 279ndash92
Martin D P et al (2011) lsquoRecombination in Eukaryotic SingleStranded DNA Virusesrsquo Viruses 3 1699ndash738
Marzano S Y and Domier L L (2016) lsquoNovel MycovirusesDiscovered from Metatranscriptomics Survey of SoybeanPhyllosphere Phytobiomesrsquo Virus Research 213 332ndash42
Muhire B et al (2013) lsquoA Genome-Wide Pairwise-Identity-BasedProposal for the Classification of Viruses in the GenusMastrevirus (family Geminiviridae)rsquo Archives of Virology 1581411ndash24
Muhire B M Varsani A and Martin D P (2014) lsquoSDT a VirusClassification Tool Based on Pairwise Sequence Alignment andIdentity Calculationrsquo PLoS One 9 e108277
Nash T E et al (2011) lsquoFunctional Analysis of a Novel MotifConserved Across Geminivirus Rep Proteinsrsquo Journal ofVirology 85 1182ndash92
Ng T F et al (2011) lsquoBroad Surveys of DNA Viral DiversityObtained Through Viral Metagenomics of Mosquitoesrsquo PLoSOne 6 e20579
et al (2014) lsquoPreservation of Viral Genomes in 700-y-oldCaribou Feces from a Subarctic Ice Patchrsquo Proceedings of theNational Academy of Sciences of the United States of America 11116842ndash7
Nguyen V G et al (2012) lsquoPopulation Dynamics and ORF3 GeneEvolution of Porcine Circovirus Type 2 Circulating in KorearsquoArchives of Virology 157 799ndash810
Orozco B M and Hanley-Bowdoin L (1998) lsquoConservedSequence and Structural Motifs Contribute to the DNA Bindingand Cleavage Activities of a Geminivirus Replication ProteinrsquoJournal of Biological Chemistry 273 24448ndash56
Phan T G et al (2015) lsquoSmall Circular Single Stranded DNAViral Genomes in Unexplained Cases of Human EncephalitisDiarrhea and in Untreated Sewagersquo Virology 482 98ndash104
Price M N Dehal P S and Arkin A P (2010) lsquoFastTree 2mdashApproximately Maximum-Likelihood Trees for LargeAlignmentsrsquo PLoS One 5 e9490
Rosario K et al (2012) lsquoDiverse Circular ssDNA VirusesDiscovered in Dragonflies (Odonata Epiprocta)rsquo Journal ofGeneral Virology 93 2668ndash81
Duffy S and Breitbart M (2012) lsquoA Field Guide toEukaryotic Circular Single-Stranded DNA Viruses InsightsGained From Metagenomicsrsquo Archives of Virology 157 1851ndash71
Roux S et al (2012) lsquoEvolution and Diversity of the MicroviridaeViral Family Through a Collection of 81 New CompleteGenomes Assembled from Virome Readsrsquo PLoS One 7 e40418
et al (2013) lsquoChimeric Viruses Blur the Borders Betweenthe Major Groups of Eukaryotic Single-Stranded DNA VirusesrsquoNature Communications 4 2700
Ruiz-Maso J A et al (2015) lsquoPlasmid Rolling-Circle ReplicationrsquoMicrobiology Spectrum 3 PLAS-0035-2014
Shangjin C Cortey M and Segales J (2009) lsquoPhylogeny andEvolution of the NS1 and VP1VP2 Gene Sequences fromPorcine Parvovirusrsquo Virus Research 140 209ndash15
Sikorski A et al (2013) lsquoNovel Myco-Like DNA VirusesDiscovered in the Faecal Matter of Various Animalsrsquo VirusResearch 177 209ndash16
Simmonds P et al (2017) lsquoVirus Taxonomy in the Age ofMetagenomicsrsquo Nature Reviews Microbiology (in press) DOI101038nrmicro2016177
Steel O et al (2016) lsquoCircular Replication-Associated ProteinEncoding DNA Viruses Identified in the Faecal Matter ofVarious Animals in New Zealandrsquo Infection Genetics andEvolution 43 151ndash64
Steinfeldt T Finsterbusch T and Mankertz A (2006)lsquoDemonstration of NickingJoining Activity at the Origin ofDNA Replication Associated with the Rep and Reprsquo Proteins ofPorcine Circovirus Type 1rsquo Journal of Virology 80 6225ndash34
Streck A F et al (2011) lsquoHigh Rate of Viral Evolution in theCapsid Protein of Porcine Parvovirusrsquo Journal of GeneralVirology 92 2628ndash36
Timchenko T et al (1999) lsquoA Single Rep Protein InitiatesReplication of Multiple Genome Components of Faba BeanNecrotic Yellows Virus a Single-Stranded DNA Virus ofPlantsrsquo Journal of Virology 73 10173ndash82
Uch R et al (2015) lsquoDivergent Gemycircularvirus in HIV-PositiveBlood Francersquo Emerging Infectious Diseases 21 2096ndash8
van den Brand J M et al (2012) lsquoMetagenomic Analysis of theViral Flora of Pine Marten and European Badger Fecesrsquo Journalof Virology 86 2360ndash5
Varsani A et al (2009) lsquoA Highly Divergent South AfricanGeminivirus Species Illuminates the Ancient EvolutionaryHistory of This Familyrsquo Virology Journal 6 36
et al (2014a) lsquoRevisiting the Classification of CurtovirusesBased on Genome-Wide Pairwise Identityrsquo Archives of Virology159 1873ndash82
et al (2014b) lsquoEstablishment of Three New Genera in theFamily Geminiviridae Becurtovirus Eragrovirus andTurncurtovirusrsquo Archives of Virology 159 2193ndash203
Vega-Rocha S et al (2007a) lsquoSolution Structure Divalent Metaland DNA Binding of the Endonuclease Domain from theReplication Initiation Protein from Porcine Circovirus 2rsquoJournal of Molecular Biology 367 473ndash87
et al (2007b) lsquoSolution Structure of the EndonucleaseDomain from the Master Replication Initiator Protein of theNanovirus Faba Bean Necrotic Yellows Virus and Comparisonwith the Corresponding Geminivirus and CircovirusStructuresrsquo Biochemistry 46 6201ndash12
A Varsani and M Krupovic | 13
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197
14 | Virus Evolution 2017 Vol 3 No 1
Wu Z et al (2016) lsquoDeciphering the Bat Virome Catalog to BetterUnderstand the Ecological Diversity of Bat Viruses and the BatOrigin of Emerging Infectious Diseasesrsquo ISME Journal 10609ndash20
Yau S et al (2011) lsquoVirophage Control of Antarctic Algal Host-Virus Dynamicsrsquo Proceedings of the National Academy of Sciencesof the United States of America 108 6163ndash8
Yu X et al (2010) lsquoA Geminivirus-Related DNA Mycovirus thatConfers Hypovirulence to a Plant Pathogenic FungusrsquoProceedings of the National Academy of Sciences of the United Statesof America 107 8387ndash92
et al (2013) lsquoExtracellular Transmission of a DNAMycovirus and Its Use as a Natural Fungicidersquo Proceedings of theNational Academy of Sciences of the United States of America 1101452ndash7
Yutin N et al (2015) lsquoA Novel Group of Diverse Polinton-LikeViruses Discovered by Metagenome Analysisrsquo BMC Biology 13 95
Zhang W et al (2016) lsquoViral Nucleic Acids in Human PlasmaPoolsrsquo Transfusion 56 2248ndash55
Zhou C et al (2015) lsquoA Novel Gemycircularvirus in anUnexplained Case of Child Encephalitisrsquo Virology Journal12 197