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RESEARCH ARTICLE Open Access
Genomic characterization of two novelpathogenic avipoxviruses isolated frompacific shearwaters (Ardenna spp.)Subir Sarker1* , Shubhagata Das2, Jennifer L. Lavers3, Ian Hutton4, Karla Helbig1, Jacob Imbery5, Chris Upton5
and Shane R. Raidal2
Abstract
Background: Over the past 20 years, many marine seabird populations have been gradually declining and thefactors driving this ongoing deterioration are not always well understood. Avipoxvirus infections have been foundin a wide range of bird species worldwide, however, very little is known about the disease ecology of avian poxvirusesin seabirds. Here we present two novel avipoxviruses from pacific shearwaters (Ardenna spp), one from a Flesh-footedShearwater (A. carneipes) (SWPV-1) and the other from a Wedge-tailed Shearwater (A. pacificus) (SWPV-2).
Results: Epidermal pox lesions, liver, and blood samples were examined from A. carneipes and A. pacificus of breedingcolonies in eastern Australia. After histopathological confirmation of the disease, PCR screening was conducted foravipoxvirus, circovirus, reticuloendotheliosis virus, and fungal agents. Two samples that were PCR positive forpoxvirus were further assessed by next generation sequencing, which yielded complete Shearwaterpox virus(SWPV) genomes from A. pacificus and A. carneipes, both showing the highest degree of similarity with Canarypox virus(98% and 67%, respectively). The novel SWPV-1 complete genome from A. carneipes is missing 43 genes compared toCNPV and contains 4 predicted genes which are not found in any other poxvirus, whilst, SWPV-2 complete genomewas deemed to be missing 18 genes compared to CNPV and a further 15 genes significantly fragmented asto probably cause them to be non-functional.
Conclusion: These are the first avipoxvirus complete genome sequences that infect marine seabirds. In thecomparison of SWPV-1 and −2 to existing avipoxvirus sequences, our results indicate that the SWPV completegenome from A. carneipes (SWPV-1) described here is not closely related to any other avipoxvirus genome isolated fromavian or other natural host species, and that it likely should be considered a separate species.
Keywords: Avipoxvirus, Poxvirus, Next generation sequencing, dermatitis, Ardenna, Shearwater
BackgroundThe Avipoxvirus genus includes a divergent group of vi-ruses that cause diseases in more than 278 species of wildand domestic birds in terrestrial and marine environmentsworldwide [1, 2]. Relatively little is known about the ori-gins, worldwide host distribution and genetic diversity ofavipoxviruses [3]. In affected birds, avipoxviruses typicallycause proliferative ‘wart-like’ growths that are most com-monly restricted to the eyes, beak or unfeathered skin of
the body (so-called ‘dry’ pox), but infections can alsodevelop in the upper alimentary and respiratory tracts(‘wet’ or ‘diptheritic’ pox) [2]. The incubation periodand magnitude of avipoxvirus infection is variable, and israrely fatal although secondary bacterial or fungal infec-tions are common and cause increased mortality [2]. Suchconditions in naïve populations can reach a much higherprevalence with substantial fatality [4, 5].Avipoxviruses belong to the subfamily Chordopoxvirinae
(ChPV) of the Poxviridae family, which are relatively largedouble-stranded DNA (dsDNA) viruses that replicate inthe cytoplasm of infected cells [6]. Although poxviruseshave evolved to infect a wide range of host species, to date
* Correspondence: [email protected] of Physiology, Anatomy and Microbiology, School of LifeSciences, La Trobe University, Melbourne, VIC 3086, AustraliaFull list of author information is available at the end of the article
only six avipoxvirus genomes have been published; apathogenic American strain of Fowlpox virus (FPVUS)[7], an attenuated European strain of Fowlpox virus(FP9) [8], a virulent Canarypox virus (CNPV) [9], apathogenic South African strain of Pigeonpox virus(FeP2), a Penguinpox virus (PEPV) [3], and a patho-genic Hungarian strain of Turkeypox virus (TKPV)[10]. Although these genome sequences demonstratethat avipoxviruses have diverged considerably fromthe other chordopoxviruses (ChPVs), approximately 80genes have been found to be conserved amongst allChPVs and to comprise the minimum essential pox-virus genome [11]. These genes tend to be present inthe central core of the linear genome with the remain-der presumed to be immunomodulatory and host spe-cific genes located towards the terminal regions of thegenome [3]. With the exception of TKPV (188 kb),avipoxvirus genomes (266–360 kb) tend to be biggerthan those of other ChPVs due in part to multiplefamilies of genes.Over the past two decades, the status of the world’s
bird populations have deteriorated with seabirds declin-ing faster than any other group of birds [12]. On LordHowe Island in eastern Australia, the Flesh-footedShearwater Ardenna carneipes has been declining formany years and is therefore listed as Vulnerable in thestate of New South Wales [13]. The ongoing threat ofplastic pollution, and toxicity from the elevated concen-tration of trace elements such as mercury could be con-founding drivers of this declining species [14]. Infectiousdiseases, including those caused by avipoxviruses, havealso been identified as an important risk factor in theconservation of small and endangered populations, par-ticularly in island species [15–18]. The impact of theintroduction of avipoxviruses has been severe for theavifauna of various archipelagos [19]. The emergence ofdistinctive avipoxvirus with a high prevalence (88%) inHawaiian Laysan Albatross (Phoebastria immutabilis)enabled one of the first detailed studies of the epidemi-ology and population-level impact of the disease in theseabirds [20]. However, relatively little is known aboutthe general prevalence or effects of poxviruses in seabirdspecies, including for shearwaters (Ardenna or Puffinusspp.). Therefore, the aim of the present study was toidentify and characterize pathogens associated with clin-ical disease in breeding colonies of Flesh-footed Shear-water and Wedge-tailed Shearwater sourced from LordHowe Island in 2015.
ResultsIdentification of fungal pathogensIn the sample from A. pacificus (15–1526, and 15–1527),there were multifocal areas of inflammation and exudationassociated with serocellular surface crust that contained
abundant branching fungal hyphae and aggregations ofbacteria (Fig. 1c). A PCR screening was conducted for thepresence of fungal pathogen using the ITS region toamplify a segment of approximately 550 bp. Two sam-ples (out of 6) were positive for fungal pathogens, anddirect Sanger sequencing of the purified gel bandsresulted in a 550 bp sequence after trimming offprimer sequences (data not shown). These sequenceswere further verified using high-throughput NGS, andgenerated con tigs of 3,430 bp (15–1526; GenBank ac-cession KX857213) and 5,188 bp (15–1527; GenBankaccession KX857212). A BLASTn search for the birdcoinfected with fungal pathogen (15–1526) returnedmultiple hits to various fungal species, all with verysimilar scores; however, the best match (88%) was tothe Phaeosphaeria nodorum (GenBank AccessionEU053989.1, and value ≤ e-153), a major necrotrophicfungal pathogen of wheat [21]. Similar search modelfor the fungal pathogen of bird 15–1527, demon-strated a highest hit (96%) to the Metarhizium aniso-pliae var. anisopliae (GenBank Accession AY884128.1,and value ≤ e-173), an entomopathogenic fungus [22].
Identification of virusSamples from six shearwater chicks of two different spe-cies, A. carneipes and A. pacificus, with evidence of grosswell circumscribed, popular, crusting lesions across thefeather skins (Fig. 1a), were biopsied, with blood andliver samples also collected. Histological examinations ofthe skin demonstrated focal to diffuse full thickness ne-crosis of the epidermis and a thick serocellular surfacecrust. A marked heterophilic rich inflammatory cellularresponse and exudation was present alongside abundantmacrophages and perifollicular fibroplasia. In some areasthere was focal proliferation of the adjacent epidermisassociated with ballooning degeneration of keratinocyteswith eosinophilic intracytoplasmic inclusions (Fig. 1b). APCR screening was conducted for the presence of pox-virus, circovirus and reticuloendotheliosis virus, whichare likely to cause this type of skin lesions. Two birds(A. pacificus 15–1526 and A. carneipes 15–1528) werepositive by PCR targeting the 4b gene that encodes acore protein of ChPV, however, there were no evidenceof either circovirus or reticuloendotheliosis for any ofthe samples used in this study. Direct Sanger sequencingof the purified gel bands resulted in a 578 bp sequenceafter trimming off primer sequences (data not shown). ABLASTn search with these sequences returned multiplehits to the 4b core gene from a variety of poxviruses, allwith very similar scores; however, the best match wasto the Canarypox virus 4b core protein gene ((bird15–1526; similarity with AY318871 was 99% andidentity score ≤ e-162), and bird 15–1528; similaritywith LK021654 was 99% and identity score ≤ e-157)).
Sarker et al. BMC Genomics (2017) 18:298 Page 2 of 26
Genome sequence and annotation of virusesThe Shearwaterpox virus complete genomes wereassembled using CLC Genomics workbench 9.5.2 underLa Trobe University Genomics Platform. The assembledcomplete genomes of SWPV-1 and −2 were 326,929 and351,108 nt, respectively. The SWPV-1 and −2 completegenomes were annotated as described in the methodsusing CNPV as a reference genome (Additional file 1:Table S1 and Additional file 2: Table S2). We took a con-servative approach to the annotation in order tominimize the inclusion of ORFs that were unlikely torepresent functional genes. Table 1 lists the 310 and 312genes annotated for SWPV-1 and −2, respectively. Forthe most part, these two new complete genomes are col-linear to CNPV although there are a number of rear-rangements of blocks of 1–6 genes in addition toinsertions and deletions with respect to CNPV (Table 1).Comparison of the predicted proteins of SWPV-2 toorthologs in CNPV reveal the vast majority are >98%identical (aa), with more than 80 being completelyconserved. In contrast, the orthologs of SWPV-1 onlyhave an average aa identity of 67% to CNPV. However,with the lower average identity, greater genetic distance,comes a much greater range of variation in the level ofidentity and a significant number of predicted proteinsare 80 – 90% identical (aa) to CNPV orthologs.
This difference in similarity between the new virusesand CNPV is easily visualized in complete genome dot-plots (Fig. 2a and b). Significantly more indels arepresent in the SWPV-1 vs CNPV dotplot (Fig. 2a). How-ever, when the phylogenetic relationships of these vi-ruses were examined together with the other availablecomplete genomes, SWPV-1 was still part of the CNPVclade (Fig. 3a). From this alignment, CNPV is 99.2%,78.7%, 69.4%, 69.5%, 68.8% and 66.5% identical (nt) toSWPV-2, SWPV-1, FeP2, PEPV, FWPV and TKPV, re-spectively. A greater selection of viruses was included inthe phylogenetic tree by using other fragments of incom-pletely sequenced avipoxvirus genomes. For example,Vultur gryphus poxvirus (VGPV), Flamingopox virus(FGPV) and Hawaiian goose poxvirus (HGPV) are allmore similar to SWPV-2 and CNPV than SWPV-1(Fig. 3b), this confirms that other poxviruses are asclosely related to CNPV as SWPV-2. By also buildingphylogenetic trees with partial nucleotide sequencesfrom the p4b gene (Fig. 4) and DNA polymerase gene(Fig. 5), we discovered that several other viruses arewithin the SWPV-1, SWPV-2 and CNPV clade. Thisincludes a poxvirus isolated from Houbara Bustards(Chlamydotis undulata) in captive-breeding programsin Morocco [23], but named CNPV-morocco, andavipoxviruses isolated from American crow (Corvus
Fig. 1 Pathological evidence of characteristic pox and fungal lesions. a Grossly well circumscribed, popular, crusting pox lesions across the featherless skins(white arrows). b Feather skin demonstrating diffuse proliferation of the epidermis and follicular infundibula with keratinocytes containing eosinophilicintracytoplasmic inclusions (Bollinger bodies) and serocellular surface crust (double head arrow). c Inflammatory exudates associated with serocellularsurface crust that contained abundant branching fungal hyphae and aggregations of bacteria
Sarker et al. BMC Genomics (2017) 18:298 Page 3 of 26
Table 1 Shearwaterpox virus (SWPV) genome annotations and comparative analysis of ORFs relative to CNPV genomes
SWPV1synteny
SWPV2synteny
CNPVsynteny
CNPV BLAST hits SWPV1 %identity
SWPV2 %identity
SWPV1 AAsize
SWPV2 AAsize
Reference AAsize
notes
CNPV001 CNPV001hypothetical protein
72
SWPV2-001 CNPV002 CNPV002hypothetical protein
92.941 171 171
SWPV1-001 SWPV2-002 CNPV003 CNPV003 C-typelectin-like protein
32.044 85.99 181 208 204
SWPV1-002 CNPV004 CNPV004 ankyrinrepeat protein
56.458 468 514
SWPV1-003 SWPV2-003 CNPV005 CNPV005 conservedhypothetical protein
87.387 99.55 220 222 222
SWPV2-004 CNPV006 CNPV006hypothetical protein
88.71 134 182 SWPV2: C-terminusfragment, not likelytranslated
CNPV007 CNPV007 ankyrinrepeat protein
674
SWPV1-004 SWPV2-005 CNPV008 CNPV008 C-typelectin-like protein
50 98.225 174 169 169
SWPV2-006 CNPV009 CNPV009 ankyrinrepeat protein
99.564 688 688
CNPV010 CNPV010 ankyrinrepeat protein
734
SWPV2-007 CNPV011 CNPV011 ankyrinrepeat protein
99.147 586 586
SWPV2-008 CNPV012 CNPV012hypothetical protein
100 189 189
SWPV2-009 CNPV013 CNPV013hypothetical protein
98.81 168 168
SWPV2-010 CNPV014 CNPV014immunoglobulin-likedomain protein
99.184 490 490
SWPV2-011 CNPV015 CNPV015 ankyrinrepeat protein
97.538 528 528
SWPV1-005 CNPV035 C-typelectin-like protein
35.556 138 134
SWPV1-006 CNPV318 ankyrinrepeat protein
58.932 487 514
SWPV1-007 SWPV2-012 CNPV016 CNPV016 C-typelectin-like protein
52.128 98.81 117 168 168
SWPV1-008 SWPV2-013 CNPV017 CNPV017 ankyrinrepeat protein
64.471 97.912 425 479 486
SWPV1-009 CNPV295 ankyrinrepeat protein
56.41 277 396
SWPV2-014 CNPV018 CNPV018 IL-10-likeprotein
90.805 190 191
SWPV2-015 CNPV019 CNPV019 ankyrinrepeat protein
99.083 436 436
SWPV1-010 SWPV2-016 CNPV020 CNPV020 ankyrinrepeat protein
56.311 99.761 412 419 419
SWPV1-011 CNPV320 Ig-like domain protein
31.656 483 469
SWPV1-012 SWPV2-017 CNPV021 CNPV021 ankyrinrepeat protein
SWPV1-291 SWPV2-298 CNPV312 CNPV312 conservedhypothetical protein
53.704 98.795 168 166 166
SWPV1-292 SWPV2-299 CNPV313 CNPV313 Ig-like do-main protein
69.43 98.165 213 218 218
SWPV1-293 SWPV2-300 CNPV314 CNPV314 ankyrinrepeat protein
71.552 99.829 580 629 584
SWPV1-294 CNPV011 ankyrinrepeat protein
32 513 586
SWPV1-295 SWPV2-301 CNPV315 CNPV315 G protein-coupled receptor-likeprotein
59.17 99.365 315 315 315
SWPV1-296 CNPV014 Ig-like do-main protein
59.624 230 490
SWPV1-297 CNPV014 Ig-like do-main protein
59.641 240 490
SWPV1-298 CNPV015 ankyrinrepeat protein
45.455 74 528
SWPV1-299 CNPV150 ankyrinrepeat protein
36.364 84 351
SWPV1-300 SWPV2-302 CNPV316 CNPV316 ankyrinrepeat protein
35.294 99.632 162 544 544
SWPV2-303 CNPV317 CNPV317hypothetical protein
100 55 55
SWPV2-304 CNPV318 CNPV318 ankyrinrepeat protein
98.054 514 514
SWPV2-305 CNPV319 CNPV319 ankyrinrepeat protein
97.638 637 739 SWPV2: C-terminusfragment, not likelytranslated
SWPV1-301 PIPV253 EFc-likeprotein
69 124 124
SWPV1-302 CNPV015 ankyrinrepeat protein
45.276 520 528
SWPV1-303 CNPV223 ankyrinrepeat protein
40 480 847
SWPV1-304 SWPV2-306 CNPV320 CNPV320 Ig-like do-main protein
76.858 99.787 468 469 469
SWPV2-307 CNPV321 CNPV321 EFc-likeprotein
99.194 124 124
SWPV2-308 CNPV322 CNPV322 ankyrinrepeat protein
98.408 689 690
SWPV1-305 CNPV035 C-typelectin-like protein
35.556 138 134
Sarker et al. BMC Genomics (2017) 18:298 Page 18 of 26
brachyrhynchos) and American robin (Turdus migrator-ius) [24], which is almost identical to CPNV-1 withinthis relatively small fragment of the genome.
Features of SWPV-2As noted above, and displayed in the Dotplot (Fig. 2b),SWPV-2 is very similar to CNPV with almost 98% ntidentity. However, a 1% difference still gives approxi-mately 10 mutations in an average sized gene any ofwhich could have drastic effects if an early STOP codonis introduced to the gene sequence. Similarly, smallchanges to promoter regions can significantly alter geneexpressions that are impossible to predict in these vi-ruses. With this annotation strategy, 18 CNPV genes
were deemed to be missing from the SWPV-2 completegenome and a further 15 genes significantly fragmentedas to probably cause them to be non-functional (Table 1).No novel genes were predicted in SWPV-2, and no re-arrangement of genes compared to CNPV was observed.
Features of SWPV-1As expected from the much lower percent nt identity,SWPV-1 was found to be considerably more different toCNPV than SWPV-2 when compared at the level ofgenes present or absent. (Table 1). 43 CNPV genes areabsent from SWPV-1 and a further 6 are significantlyfragmented. There are 4 predicted genes in SWPV-1 thatare not present in any other poxvirus, nor do they match
Table 1 Shearwaterpox virus (SWPV) genome annotations and comparative analysis of ORFs relative to CNPV genomes (Continued)
SWPV1-306 CNPV008 C-typelectin-like protein
50 174 169
SWPV1-307 SWPV2-309 CNPV323 CNPV323 conservedhypothetical protein
75.61 93.651 84 186 182
SWPV1-308 SWPV2-310 CNPV324 CNPV324 conservedhypothetical protein
87.387 99.55 220 222 222
SWPV1-309 CNPV325 CNPV325 ankyrinrepeat protein
56.458 468 514
SWPV1-310 SWPV2-311 CNPV326 CNPV326 C-typelectin-like protein
32.044 85.99 181 208 204
SWPV2-312 CNPV327 CNPV327hypothetical protein
92.941 171 171
CNPV328 CNPV328hypothetical protein
72
Fig. 2 Dotplots of Shearwaterpox viruses (SWPV-1 and 2) vs CNPV genomes. Horizontal sequence: SWPV-1 (a) and SWPV-2 (b), vertical sequenceCNPV. Red and blue boxes represent genes transcribed to the right and left of the genome, respectively
Sarker et al. BMC Genomics (2017) 18:298 Page 19 of 26
any sequences in the NR protein database usingBLASTP. However, they are all relatively short ORFsand it is possible that they are not functional genes.Additionally, SWPV-1 encodes nine polypeptides that donot match CNPV proteins, but do match proteins fromother avipoxviruses (penguinpox, turkeypox, pigeonpoxand fowlpox). This could be due to recombinationamong ancestral viruses, but could also result from the
loss of the corresponding ortholog in CNPV leaving an-other virus to provide the “best match”.As might be expected given the greater distance be-
tween SWPV-1 and CPNV than between SWPV-2 andCNPV, there are more instances of minor rearrange-ments that created a loss of synteny (Table 1). However,since most of these involve the families of repeatedgenes, it is also possible that divergence of these
Fig. 3 Phylogenetic relationship between Shearwaterpox viruses (SWPV-1 and 2) and other avipoxviruses. a Phylogenetic tree of 173 kbp coreregion (large gaps removed) from available complete avipoxvirus genomes. b Phylogenetic tree highlighting viruses closely related to CNPV. Thesequences were aligned with ClustalO and MEGA7 was used to create a maximum likelihood tree based on the Tamura-Nei method and testedby bootstrapping with 1000 replicates. The abbreviations and GenBank accession details for poxviruses strains were used: Canarypox virus (CNPV;AY318871), Pigeonpox virus (FeP2; KJ801920), Penguinpox virus (PEPV; KJ859677) Fowlpox virus (FWPV; AF198100), Shearwaterpox virus 1 (SWPV-1;KX857216), Shearwaterpox virus 2 (SWPV-2; KX857215), Turkeypox virus (TKPV; NC_028238), Vultur Gryphus poxvirus (VGPV; AY246559), Flamingopoxvirus (FGPV; HQ875129 and KM974726), Hawaiian goose poxvirus (HGPV; AY255628)
Fig. 4 Maximum likelihood phylogenetic tree from partial DNA sequences of p4b gene of avipoxviruses. Novel Shearwaterpox viruses (SWPV-1and SWPV-2) are highlighted by gray background
Sarker et al. BMC Genomics (2017) 18:298 Page 20 of 26
sequences has led to the inability to distinguish betweenthe orthologous and paralogous genes.
Evidence of recombination among avipoxvirusesWhen we reviewed a graph of nt identity between the2 new complete genomes and CNPV using BBB (notshown), there were several relatively short syntenic re-gions where 1) SWPV-1 matched CNPV significantlybetter than the majority of the genome, and 2) SWPV-2 matched CNPV significantly worse than the majorityof the genome. To examine these regions in more de-tail, the Visual Summary feature of BBB was used todisplay individual SNPs for these genome comparisons(Fig. 6a and b). This analysis revealed that SWPV-1
and SWPV-2 were unique in these regions and con-firmed that the genome sequences of SWPV-1andSWPV-2 were not contaminated during their assem-bly. However, when these regions were used as querysequences in BLASTN searches of all poxvirus se-quences the best match remained CNPV suggestingthat these sequences originated from avipoxvirus ge-nomes that are not represented in the publicdatabases.
DiscussionThis paper describes the detection and characterizationof two novel avipoxvirus complete genome sequences ina naturally occurring infections of avian pox in a naïve
Fig. 5 Maximum likelihood phylogenetic tree from partial DNA sequences of DNA polymerase gene of avipoxviruses. Novel Shearwaterpoxviruses (SWPV-1 and SWPV-2) are highlighted by gray background
Fig. 6 Region of recombination in Shearwaterpox viruses (SWPV) detected in A. carneipes and A. pacificus. Nucleotide differences to CNPV areshown in blue (SNPs), green/red (indels). Figure 6a. Region of recombination in SWPV-2. On the middle track, SWPV-2 has very few differences toCNPV except for highly divergent block in the middle of this region. Figure 6b. Region of recombination in SWPV-1. On the bottom track, SWPV-1is very different to CNPV except for highly similar block between nt 193,000 and 195,500
Sarker et al. BMC Genomics (2017) 18:298 Page 21 of 26
population of shearwaters. The DNA sequences ofSWPV-1 and SWPV-2 are significantly different thaneach other but nevertheless had closest similarity withCanarypox virus (67% and 98%, respectively). Further-more, the genetic distance and novel genome structureof SWPV-1 from A. carneipes considered to be miss-ing 43 genes likened to CNPV and contained 4 pre-dicted genes which are not found in any otherpoxvirus and is overall sufficiently genetically differentto be considered a separate virus species. Whilst, theSWPV-2 complete genome was missing 18 genes com-pared to CNPV, with a further 15 genes significantlyfragmented as to probably cause them to be non-functional. Furthermore, the phylogenetic distributionof SWPV-1 indicates that shearwaters and perhapsother long-lived, vagile marine birds could be import-ant hosts for avipoxvirus dispersal around the globe.The natural hosts of these avipoxviruses maybe thispopulation of shearwaters, other migratory birds thatuse Lord Howe Island for breeding or resident avianhost reservoir species. Species such as the Lord HoweWhite-eye (Zosterops tephropleura) and Lord HoweGolden Whistler (Pachcephala petoralis contempta)are candidate passerine birds that might provide suchfunction.Examining the phylogenetic relationship between the
Shearwaterpox viruses and other avipoxviruses, it is evi-dent that the SWPV-2 is most closely related to Canary-pox virus. The SWPV-1 and SWPV-2 complete genomesboth contain several genes that are more closely relatedto CNPV throughout their entire genome. As shown inFig. 3 it is reasonable to postulate that these viruses orig-inated from a common ancestor that diverged from aCNPV-like progenitor related to fowlpox, penguinpoxand pigeonpox viruses. Finer resolution of the phylogen-etic relationship using partial nucleotide sequences ofp4b and DNA polymerase genes of avipoxviruses re-vealed that SWPV isolated from seabirds also clusteredin global clade B consisting of avipoxviruses originatingfrom Canary Morocco, Canarypox and poxviruses fromAmerican crow and American robin. Given their geneticdiversity, it is perhaps not surprising that Shearwaterspecies can be exposed to multiple avipoxviral infections.Studies such as those by Barnett et al. [25] suggest thatthe species specificity of poxviruses is variable. Somegenera, such as Suipoxvirus are highly restricted to indi-vidual vertebrate hosts, swinepox for instance, whereasothers, such as avipoxviruses demonstrate some evi-dence of cross-species infection within a predator–preysystem [24]. This suggests that the avipoxviruses can in-fect a diverse range of bird species if they are within aclose enough proximity to each other [26]. Thus far,there were no clear patterns regarding species-specificityin the Shearwaterpox viruses described here.
While overt and systemic lesions and fatal disease canoccur, avian pox tends to be a self-limiting localized in-fection of apterial skin with full recovery possible.Many bird species experience life-long immunity if theimmune system is not weakened and or the birds arenot infected by different strains [27, 28]. As shown inour example, secondary infections can occur and thesemay contribute to morbidity and mortality [29–31].Similar to the example in shearwaters, Shivaprasad etal. [30] reported evidence of poxvirus infection and sec-ondary fungal pathogens in canaries (Serinus canaria).Stressful conditions, poor nutrition, overt environmen-tal contamination and other underlying causes of im-munosuppression and ill health may contribute to thepathogenesis of such lesions. This was the primary rea-son we tested for avian circovirus and other potentialpathogens.Avian pox has not been previously reported in shear-
waters (Ardenna spp.) from Lord Howe Island, norhas it been documented for any other bird species inthis region. So it is difficult to attribute the causalityof this unique event in these species. The value ofcomplete genome characterization and analysis ishighlighted since a phylogenetic relationship based onsingle gene studies such as the polymerase gene mayhave falsely implicated Canarypox virus as a potentialexotic introduced emerging disease from domesticatedbirds. Although we cannot trace the actual source ofinfection in the shearwater chicks, it is more likelythat the infection in the birds resulted from parentalfeeding or arthropod mediated transmission fromother island bird species [32]. While, the reservoirhost of these novel Shearwaterpox viruses is unknown,mosquitoes are suspected to play a part in transmis-sion within the island. Avipoxvirus infection appearsto be relatively rare in seabirds, but it has been re-ported in several species when they occur on human-inhabited islands that harbor mosquito vectors [33].According to the Lord Howe Island Board, ship rats,mice, cats, humans and other invasive pest speciessuch as owls are implicated in the extinction of at leastfive endemic birds, two reptiles, 49 flowering plants,12 vegetation communities and numerous threatenedinvertebrates [34]. These rodents and invasive pestshave also been highlighted for the potential reservoirof poxvirus infections [3, 35]. Transmission of avipox-virus by prey–predator and other migratory seabirdslikely plays a prominent role; however, the mode ofavipoxvirus transmission on Lord Howe Island is notcompletely understood. Studies by Gyuranecz et al.[24], for example, postulated that raptors may acquirepoxvirus infection from their avian prey. This suggeststhat the poxvirus in shearwaters is likely to be trans-mitted from other island species such as other
Sarker et al. BMC Genomics (2017) 18:298 Page 22 of 26
migratory seabirds and/or prey–predator, although, itis difficult to be certain without further studies.Interestingly, these new shearwaterpox virus complete
genomes also provide evidence that supports the hy-pothesis that recombination may play an important rolein the evolution of avipoxviruses. A number of genes inSWPV-1 appear to be rearranged compared to CNPVand blocks of unusual similarity scores were seen in bothSWPVs. Software that is designed to look for gross re-combination between two viruses, such as two strains ofHIV, fails to detect this level of recombination and it isleft to the investigator to observe such small events byeye after visualizing the distribution of SNPs between vi-ruses. Such relatively small exchanges of DNA may stillexert important influences on virus evolution, and hasbeen predicted to have been a driver in the evolution ofsmallpox [36].
ConclusionsThese are the first avipoxvirus complete genome se-quences that infect marine bird species. The novelcomplete genome sequences of SWPV-1 and −2 havegreatly enhanced the genomic information for the Avi-poxvirus genus, which will contribute to our understand-ing of the avipoxvirus more generally, and track theevolution of poxvirus infection in such a non-modelavian species. Together with the sequence similaritiesobserved between SWPV and other avipoxviruses, thisstudy concluded that the SWPV complete genome fromA. carneipes (SWPV-1) described here is not closely re-lated to any other avipoxvirus complete genome isolatedfrom avian or other natural host species, and that itlikely should be considered a separate species. Furtherinvestigations of Shearwaterpox viruses genetic andpathogenesis will provide a unique approach to betterassess the risk associated to poxvirus transmissionwithin and between marine bird species.
MethodsSource of samplingA total of six samples were collected from two differentspecies of shearwater, five were from Flesh-footedShearwater (ID: 15-1527-31), and other one was fromWedge-tailed Shearwater (ID: 15–1526). Of size birds,two were recoded to have evidence of gross well cir-cumscribed lesions in the beak (Fig. 1a) and ankle, andothers had feather defects (fault lines across the vanesof feathers). Samples were collected from fledglings(approximately 80–90 days of age) of both species onLord Howe Island, New South Wales (32.53S, 159.08E)located approximately 500 km off the east coast ofAustralia during April-May 2015. Samples were col-lected with the permission of the Lord Howe IslandBoard (permit no. LHIB 02/14) under the approval of
the University of Tasmania and Charles Sturt UniversityAnimal Ethics Committees (permit no. A0010874,A0011586, and 09/046). Samples from one individual ofeach shearwater species were collected including skinlesions, liver and skin biopsies, as well as blood foridentifying the causative agents. Depending on the sam-ples, either 25 mg of skin tissue were cut out andchopped into small pieces or 50–100 μL of blood wereaseptically transferred into clean 1.5 mL microcentri-fuge tube (Eppendorf ), and genomic DNA was isolatedusing the Qiagen blood and tissue mini kit (Qiagen,Germany). The extracted DNA has been stored at −20 °C for further testing. Histopathological examination ofthe skin was performed.
Archived viral and fungal pathogen testingInitially, the extracted DNA was screened for detectingnovel circoviruses [37, 38] and reticuloendotheliosisvirus [39]. For poxvirus screening, the primers PoxP1(5′-CAGCAGGTGCTAAACAACAA-3′) and PoxP2(5′-CGGTAGCTTAACGCCGAATA-3′) were synthe-sized from published literature and used to amplify asegment of approximately 578 bp from the 4b core pro-tein gene for all ChPV species [40]. Optimized PCR re-actions mixture contained 3 μL of extracted genomicDNA, 25 pmol of each primer (GeneWorks, Australia),1.5 mM MgCl2, 1.25 mM of each dNTP, 1xGoTaq®Green Flexi Reaction Buffer, 1 U of Go Taq DNA poly-merase (Promega Corporation, USA) and DEPC dis-tilled H2O (Invitrogen, USA) was added to a finalvolume of 25 μL. The PCR amplification was carriedout in an iCycler thermal cycler (Bio-Rad) under thefollowing conditions: denaturation at 94 °C for 2 minfollowed by 35 cycles of 94 °C for 1 min, 60 °C for1 min and 72 °C for 1 min, and a final extension step of2 min at 72 °C.The internal transcribed spacer (ITS) region was
chosen for screening and identification of fungal patho-gens [41]. A set of fungus-specific primers ITS1 (5′-TCCGTAGGTGAACCTGCGG -3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC -3′) were designed andused to amplify a segment of approximately 550 bp fromthe fungal ITS gene [42]. The PCR was standardized toamplify ITS genes, and the 25-μL reaction mixture con-tained 3 μL of extracted genomic DNA, 25 pmol of eachprimer (GeneWorks, Australia), 1.5 mM MgCl2,1.25 mM of each dNTP, 1xGoTaq® Green Flexi ReactionBuffer, 1 U of Go Taq DNA polymerase (PromegaCorporation, USA). The PCR reaction involved initialdenaturation at 95 °C for 5 min, followed by 30 cycles ofdenaturation at 94 °C for 30 s, annealing at 58 °C for30 s, and extension at 72 °C for 1 min, and with a finalstep of one cycle extension at 72 °C for 10 min.
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Amplified PCR products, together with a standardmolecular mass marker (Sigma), were separated byelectrophoresis in 2.0% agarose gel and stained withGelRed (Biotium, CA). Selected bands were excised andpurified using the Wizard® SV Gel and PCR Clean-UpSystem (Promega, USA) according to the manufac-turer’s instructions. Purified amplicons were se-quenced with PCR primers by the Australian GenomeResearch Facility Ltd (Sydney) using an AB 3730xl unit(Applied Biosystems). For each amplicon, sequenceswere obtained at least twice in each direction for eachisolate. The sequences were trimmed for primers andaligned to construct contigs (minimum overlap of35 bp, minimum match percentage of 95%) using Gen-eious Pro (version 10.0.2).
High throughput sequencingNext-generation sequencing (NGS) was used to se-quence the poxvirus genomes. Virion enrichment wasperformed by centrifugation for 2 min at 800 × g to re-move tissue debris, and the supernatants were subse-quently filtered through 5 μm centrifuge filters(Millipore) [43]. The filtrates were nuclease treated toremove unprotected nucleic acids using 8 μL RNaseCocktail Enzyme Mix (Invitrogen). Viral nucleic acidswere subsequently extracted using QIAamp DNA mini(Qiagen). The genomic libraries were prepared with aninsert size of 150 paired-end. DNA sequencing (NGS)was performed on a HiSeq4000 sequencing platform(Illumina) by Novogene, China.
BioinformaticsAssembly of the viral genome was conducted accord-ing to the established pipeline [44] in CLC Genomicsworkbench 9.5.2 under La Trobe University GenomicsPlatform. Briefly, the preliminary quality evaluationfor each raw read was generated using quality control(QC) report. The raw data were preprocessed to re-move ambiguous base calls, and bases or entire readsof poor quality using default parameters. The datasetswere trimmed to pass the quality control based onPHRED score or per base sequence quality score.Trimmed sequence reads were mapped against closelyavailable host genome (Albatross) to remove possibleremaining host DNA contamination, and post-filteredreads were mapped against reference Canarypox viruscomplete genome sequence. Consensus sequenceswere used to generate the complete poxvirus genome.Avipoxvirus complete genome sequences were alignedusing MAFFT [45]. Then the poxvirus specific bio-informatics analyses were performed using the ViralBioinformatics Resource Centre (virology.uvic.ca) [46],and the further analyses were conducted using thefollowing tools: Viral Orthologous Clusters Database
for sequence management (VOCs) [11]; Base-By-Basefor genome/gene/protein alignments [47, 48]; ViralGenome Organizer for genome organization compari-sons (VGO) [11], and Genome Annotation TransferUtility for annotation (GATU) [49].Open reading frames (ORFs) longer than 60 amino
acids with minimal overlapping (overlaps cannot ex-ceed 25% of one of the genes) to other ORFs were cap-tured using the CLC Genomics Workbench (CLC)ORF analysis tool as well as GATU [49], and otherprotein coding sequence and annotation software de-scribed in Geneious (version 10.0.2, Biomatters, NewZealand). These ORFs were subsequently extractedinto a FASTA file, and similarity searches includingnucleotide (BLASTN) and protein (BLASTP) wereperformed on annotated ORFs as potential genes ifthey shared significant sequence similarity to knownviral or cellular genes (BLAST E value ≤ e-5) or con-tained a putative conserved domain as predicted byBLASTp [50]. The final SWPV annotation was furtherexamined with other poxvirus ortholog alignments todetermine the correct methionine start site, correctstop codons, signs of truncation, and validity ofoverlaps.
Phylogenetic analysisPhylogenetic analyses were performed using full poxvirusgenome sequences for Shearwater species determined inthis study with related avipoxvirus genome sequencesavailable in GenBank database. A selection of partial se-quences from seven completely sequenced avipoxvirusgenomes and fragments of incompletely sequenced avipox-virus genomes from Vultur Gryphus poxvirus (VGPV), fla-mingopox virus (FGPV) and Hawaiian goose poxvirus(HGPV) were also used for phylogenetic analysis. To inves-tigate closer evolutionary relationship among avipox-viruses, partial nucleotide sequences of p4b and DNApolymerase genes were selected. The avipoxvirus se-quences were aligned using ClustalO, and then manuallyedited in Base-by-Base. MEGA7 was used to create a max-imum likelihood tree based on the Tamura-Nei methodand tested by bootstrapping with 1000 replicates. An add-itional analysis was performed using complete genome nu-cleotide sequences of Canarypox virus (CNPV; AY318871),Pigeonpox virus (FeP2; KJ801920), Fowlpox virus (FPV;AF198100), Turkeypox virus (TKPV; NC_028238), Shear-waterpox virus strain-1 (SWPV-1; KX857216), and Shear-waterpox virus strain-2 (SWPV-2; KX857215), which werealigned with MAFTT in Base-By-Base for genome/gene/protein alignments [48]. The program jModelTest 2.1.3favoured a general-time-reversible model with gammadistribution rate variation and a proportion of invariablesites (GTR + I +G4) for the ML analysis [51].
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AcknowledgmentsThe authors are extremely grateful to La Trobe University School of LifeScience Publication Booster Award for their financial support to SS.Additional funding for this project was generously provided by theDetached Foundation, Trading Consultants Ltd, and L. Bryce. Assistance inthe field was provided by the Lord Howe Island community and numerousdedicated volunteers, particularly A. Fidler, P. Lewis, A. Lombal, K. Richards,and V. Wellington. The authors thank Chad Smithson for help with genomeassembly.
FundingFunding for this project was generously provided by the DetachedFoundation, Trading Consultants Ltd, and L. Bryce. However, none of thesehave grant numbers assigned since all are donations from privatephilanthropists. Additional financial support was provided to SS through theLa Trobe University School of Life Science Publication Booster Award. CUand JI were funded by the Natural Sciences and Engineering ResearchCouncil of Canada (NSERC) Discovery Grant. The funding bodies had no rolein the design of the study and collection, analysis, and interpretation of dataand in writing the manuscript.
Availability of data and materialsThe complete genome sequences of the Shearwaterpox virus 1 from a Flesh-footed Shearwater (Ardenna carneipes) and Shearwaterpox virus 2 from aWedge-tailed Shearwater (Ardenna pacificus) have been deposited in theNCBI database under GenBank accession numbers: [SWPV-1, GenBank:KX857216] and [SWPV-2, GenBank: KX857215].
Authors’ contributionsConceived and designed the experiments: SS, SRR. Performed theexperiments: SS, SRR. Analyzed the data: SS, CU, JI, SRR. Contributedreagents/materials/analysis tools: SS, SD, JLL, IH, KH, CU, JI, SRR. SS, JLL, CU, JI,SRR wrote the initial manuscript. All authors read, edited and approved thefinal manuscript.
Competing interestsThe authors declare that they have no competing interests.
Consent for publicationNot applicable.
Ethics approvalSamples were collected with the permission of the Lord Howe Island Board(permit no. LHIB 02/14) under the approval of the University of Tasmaniaand Charles Sturt University Animal Ethics Committees (permit no. A13836,A0010874, A0011586, and 09/046).
Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.
Author details1Department of Physiology, Anatomy and Microbiology, School of LifeSciences, La Trobe University, Melbourne, VIC 3086, Australia. 2School ofAnimal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW2678, Australia. 3Institute for Marine and Antarctic Studies, University ofTasmania, Hobart, TAS 7004, Australia. 4Lord Howe Island Museum, LordHowe Island, NSW 2898, Australia. 5Department of Biochemistry andMicrobiology, University of Victoria, Victoria, BC, Canada.
3. Offerman K, Carulei O, van der Walt AP, Douglass N, Williamson A-L. Thecomplete genome sequences of poxviruses isolated from a penguin and apigeon in South Africa and comparison to other sequenced avipoxviruses.BMC Genomics. 2014;15(1):1–17.
4. Atkinson CT, LaPointe DA. Introduced avian diseases, climate change, andthe future of Hawaiian Honeycreepers. J Avian Med Surg. 2009;23(1):53–63.
5. Alley MR, Hale KA, Cash W, Ha HJ, Howe L. Concurrent avian malaria andavipox virus infection in translocated South Island saddlebacks (Philesturnuscarunculatus carunculatus). N Z Vet J. 2010;58(4):218–23.
6. Gubser C, Hué S, Kellam P, Smith GL. Poxvirus genomes: a phylogeneticanalysis. J Gen Virol. 2004;85(1):105–17.
7. Afonso CL, Tulman ER, Lu Z, Zsak L, Kutish GF, Rock DL. The genome offowlpox virus. J Virol. 2000;74(8):3815–31.
8. Laidlaw SM, Skinner MA. Comparison of the genome sequence of FP9, anattenuated, tissue culture-adapted European strain of Fowlpox virus, with thoseof virulent American and European viruses. J Gen Virol. 2004;85(Pt 2):305–22.
9. Tulman ER, Afonso CL, Lu Z, Zsak L, Kutish GF, Rock DL. The Genome ofCanarypox Virus. J Virol. 2004;78(1):353–66.
10. Banyai K, Palya V, Denes B, Glavits R, Ivanics E, Horvath B, Farkas SL,Marton S, Balint A, Gyuranecz M, et al. Unique genomic organization of anovel Avipoxvirus detected in turkey (Meleagris gallopavo). Infect GenetEvol. 2015;35:221–9.
12. Croxall JP, Butchart SHM, Lascelles BEN, Stattersfield AJ, Sullivan BEN, SymesA, Taylor P. Seabird conservation status, threats and priority actions: a globalassessment. Bird Conserv Int. 2012;22(1):1–34.
13. Reid T, Hindell M, Lavers JL, Wilcox C. Re-examining mortality sources andpopulation trends in a declining seabird: using Bayesian methods toincorporate existing information and new data. PLoS One. 2013;8(4):e58230.
14. Bond AL, Lavers JL. Trace element concentrations in feathers of flesh-footedshearwaters (Puffinus carneipes) from across their breeding range. ArchEnviron Contam Toxicol. 2011;61(2):318–26.
15. Illera JC, Emerson BC, Richardson DS. Genetic characterization, distributionand prevalence of avian pox and avian malaria in the Berthelot’s pipit(Anthus berthelotii) in Macaronesia. Parasitol Res. 2008;103(6):1435–43.
16. Lecis R, Secci F, Antuofermo E, Nuvoli S, Scagliarini A, Pittau M, Alberti A.Multiple gene typing and phylogeny of avipoxvirus associated withcutaneous lesions in a stone curlew. Vet Res Commun. 2017;4:1–7.
17. Woolaver LG, Nichols RK, Morton ES, Stutchbury BJM. Population geneticsand relatedness in a critically endangered island raptor, Ridgway’s HawkButeo ridgwayi. Conserv Genet. 2013;14(3):559–71.
18. Thiel T, Whiteman NK, Tirape A, Baquero MI, Cedeno V, Walsh T, UzcateguiGJ, Parker PG. Characterization of canarypox-like viruses infecting endemicbirds in the Galapagos Islands. J Wildl Dis. 2005;41(2):342–53.
19. van Riper C, van Riper SG, Hansen WR. Epizootiology and Effect of AvianPox on Hawaiian Forest Birds. Auk. 2002;119(4):929–42.
20. Young LC, VanderWerf EA. Prevalence of avian pox virus and effect on thefledging success of Laysan Albatross. J Field Ornithol. 2008;79(1):93–8.
21. Hane JK, Lowe RG, Solomon PS, Tan KC, Schoch CL, Spatafora JW, CrousPW, Kodira C, Birren BW, Galagan JE, et al. Dothideomycete plant
Sarker et al. BMC Genomics (2017) 18:298 Page 25 of 26
interactions illuminated by genome sequencing and EST analysis of thewheat pathogen Stagonospora nodorum. Plant Cell. 2007;19(11):3347–68.
22. Ghikas DV, Kouvelis VN, Typas MA. The complete mitochondrial genome ofthe entomopathogenic fungus Metarhizium anisopliae var. anisopliae: geneorder and trn gene clusters reveal a common evolutionary course for allSordariomycetes, while intergenic regions show variation. Arch Microbiol.2006;185(5):393–401.
23. Le Loc’h G, Ducatez MF, Camus-Bouclainville C, Guérin J-L, Bertagnoli S. Diversityof avipoxviruses in captive-bred Houbara bustard. Vet Res. 2014;45(1):98.
24. Gyuranecz M, Foster JT, Dán Á, Ip HS, Egstad KF, Parker PG, Higashiguchi JM,Skinner MA, Höfle U, Kreizinger Z, et al. Worldwide PhylogeneticRelationship of Avian Poxviruses. J Virol. 2013;87(9):4938–51.
25. Barnett J, Dastjerdi A, Davison N, Deaville R, Everest D, Peake J, Finnegan C,Jepson P, Steinbach F. Identification of Novel Cetacean Poxviruses inCetaceans Stranded in South West England. PLoS One. 2015;10(6):e0124315.
26. Haller SL, Peng C, McFadden G, Rothenburg S. Poxviruses and the Evolutionof Host Range and Virulence. Infect Genet Evol. 2014;21:15–40.
28. Winterfield RW, Reed W. Avian pox: infection and immunity with quail,psittacine, fowl, and pigeon pox viruses. Poult Sci. 1985;64(1):65–70.
29. Johnson BJ, Castro AE. Canary pox causing high mortality in an aviary. J AmVet Med Assoc. 1986;189(10):1345–7.
30. Shivaprasad HL, Kim T, Tripathy D, Woolcock PR, Uzal F. Unusual pathologyof canary poxvirus infection associated with high mortality in young andadult breeder canaries (Serinus canaria). Avian Pathol. 2009;38(4):311–6.
31. Reza K, Nasrin A, Mahmoud S. Clinical and pathological findings ofconcurrent poxvirus lesions and aspergillosis infection in canaries. Asian PacJ Trop Biomed. 2013;3(3):182–5.
32. Shearn-Bochsler V, Green DE, Converse KA, Docherty DE, Thiel T, Geisz HN,Fraser WR, Patterson-Fraser DL. Cutaneous and diphtheritic avian poxvirusinfection in a nestling Southern Giant Petrel (Macronectes giganteus) fromAntarctica. Polar Biol. 2008;31(5):569–73.
33. VanderWerf EA, Young LC. Juvenile survival, recruitment, population size,and effects of avian pox virus in Laysan Albatross (Phoebastria immutabilis)on Oahu, Hawaii, USA. The Condor. 2016;118(4):804–14.
34. Lord Howe Island Board. http://www.lhib.nsw.gov.au/environment/biodiversity/research. Accessed 20 Sep 2016.
35. Tantawi HH, Zaghloul TM, Zakaria M. Poxvirus infection in a rat (Rattusnorvegicus) in Kuwait. Int J Zoonoses. 1983;10(1):28–32.
36. Smithson C, Purdy A, Verster AJ, Upton C. Prediction of Steps in theEvolution of Variola Virus Host Range. PLoS One. 2014;9(3):e91520.
37. Sarker S, Lloyd C, Forwood J, Raidal SR. Forensic genetic evidence of beakand feather disease virus infection in a Powerful Owl. Ninox strenua Emu.2016;116(1):71–4.
38. Sarker S, Moylan KG, Ghorashi SA, Forwood JK, Peters A, Raidal SR. Evidenceof a deep viral host switch event with beak and feather disease virusinfection in rainbow bee-eaters (Merops ornatus). Sci Rep. 2015;5:14511.
39. Biswas SK, Jana C, Chand K, Rehman W, Mondal B. Detection of fowlpoxvirus integrated with reticuloendotheliosis virus sequences from anoutbreak in backyard chickens in India. Vet Ital. 2011;47(2):147–53.
40. Huw Lee L, Hwa Lee K. Application of the polymerase chain reaction for thediagnosis of fowl poxvirus infection. J Virol Methods. 1997;63(1–2):113–9.
41. Kumar M, Shukla PK. Use of PCR Targeting of Internal Transcribed SpacerRegions and Single-Stranded Conformation Polymorphism Analysis ofSequence Variation in Different Regions of rRNA Genes in Fungi for RapidDiagnosis of Mycotic Keratitis. J Clin Microbiol. 2005;43(2):662–8.
42. Lindsley MD, Hurst SF, Iqbal NJ, Morrison CJ. Rapid Identification ofDimorphic and Yeast-Like Fungal Pathogens Using Specific DNA Probes. JClin Microbiol. 2001;39(10):3505–11.
43. Jensen RH, Mollerup S, Mourier T, Hansen TA, Fridholm H, Nielsen LP,Willerslev E, Hansen AJ, Vinner L. Target-dependent enrichment of virionsdetermines the reduction of high-throughput sequencing in virus discovery.PLoS One. 2015;10(4):e0122636.
44. Zhao K, Wohlhueter RM, Li Y. Finishing monkeypox genomes from shortreads: assembly analysis and a neural network method. BMC Genomics.2016;17 Suppl 5:497.
45. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: A novel method for rapidmultiple sequence alignment based on fast Fourier transform. Nucleic AcidsRes. 2002;30(14):3059–66.
46. Ehlers A, Osborne J, Slack S, Roper RL, Upton C. Poxvirus OrthologousClusters (POCs). Bioinformatics. 2002;18(11):1544–5.
47. Brodie R, Smith AJ, Roper RL, Tcherepanov V, Upton C. Base-By-Base: Singlenucleotide-level analysis of whole viral genome alignments. BMCBioinformatics. 2004;5(1):1–9.
48. Hillary W, Lin S-H, Upton C. Base-By-Base version 2: single nucleotide-levelanalysis of whole viral genome alignments. Microb Inf Exp. 2011;1:2–2.
49. Tcherepanov V, Ehlers A, Upton C. Genome Annotation Transfer Utility(GATU): rapid annotation of viral genomes using a closely related referencegenome. BMC Genomics. 2006;7(1):1–10.
50. Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J,Sayers EW. GenBank. Nucleic Acids Res. 2013;41(Database issue):D36–42.
51. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models,new heuristics and parallel computing. Nat Methods. 2012;9(8):772–2
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