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Sauvage et al., 2011
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A member of a new Picornaviridae genus is shed in pig feces 2
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Virginie Sauvagea, Meriadeg Ar Gouilha, Justine Chevalb, Erika Muthb, Kevin Parienteb, Ana 5
Burguierea, Valérie Caroc, Jean-Claude Manuguerraa, Marc Eloitb,d,e* 6
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aInstitut Pasteur, Laboratory for Urgent Responses to Biological Threats, 25 rue du Docteur 8
Roux, F-75724 Paris Cedex 15, France 9 bPathoquest, 28 rue du Docteur Roux, F-75015 Paris, France 10 cInstitut Pasteur, Genotyping of Pathogens and Public Health Platform, 28 rue du Docteur 11
Roux, F-75015 75724 Paris, France 12 dEcole Nationale Vétérinaire d’Alfort, UMR 1161 Virologie ENVA, INRA, ANSES, 7 avenue 13
Général de Gaulle, F-94704 Maisons Alfort, France 14 eInstitut Pasteur, Department of Virology , 28 rue du Docteur Roux, F-75015 Paris, France 15
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* Corresponding author : Marc Eloit, Department of Virology , 28 rue du Docteur Roux, 17
F-75015 Paris, France 18
tel : 33 1 44 38 92 16 fax : 33 1 40 61 39 40, [email protected] 19
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KEYWORDS: Picornaviridae, SPaV1, swine, piglet, industrial farm 21
RUNNING TITLE: A new Picornaviridae genus discovered in piglets feces 22
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00046-12 JVI Accepts, published online ahead of print on 11 July 2012
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Sauvage et al., 2011
ABSTRACT 34
35
During a study of the fecal microbiome from two healthy piglets using high 36
throughput sequencing (HTS), we identified of a viral genome containing an open reading 37
frame encoding a predicted polyprotein of 2133 amino acids. This novel viral genome 38
displayed the typical organization of picornaviruses containing three structural proteins (VP0, 39
VP3 and VP1) followed by seven non-structural proteins (2A, 2B, 2C, 3A, 3B, 3Cpro and 40
3Dpol). Given its particular relationship with Parechovirus, we propose to name it “Pasivirus” 41
for “Parecho sister-clade virus” with the “Swine Pasivirus 1” (SPaV1) as type species. Fecal 42
samples collected in an industrial farm from healthy sows and piglets from the same herd (25 43
and 75, respectively) of ages ranging from 4 to 28 weeks old were analyzed for the presence 44
of SPaV1 by one-step RT-PCR targeting a 3D region of 151 pb. SPaV1 was detected in fecal 45
samples from 51/75 healthy piglets (68% of animals) and in none of the 25 fecal samples 46
from healthy sows, indicating that SPaV1 circulates through enteric infection of healthy 47
piglets. We propose that SPaV1 represents the first member of a novel Picornaviridae genus 48
related to parechoviruses.49
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INTRODUCTION 50
51
Members of the Picornaviridae family are small, non-enveloped viruses, with a 52
genomic positive single-stranded RNA, responsible for several human and veterinary 53
diseases. As of 2009, the International Committee on Taxonomy of Viruses (ICTV) 54
recognized twelve genera within the Picornaviridae family, namely: Enterovirus, 55
Cardiovirus, Aphtovirus, Hepatovirus, Parechovirus, Erbovirus, Kobuvirus, Teschovirus, 56
Sapelovirus, Senecavirus, Tremovirus and Avihepathovirus (www.picornaviridae.com). 57
However, recent developments of high-throughput sequencing (HTS) identified numerous 58
Picornaviridae species, among which several sequences were proposed as prototype for novel 59
genera. At least eleven novel genera have been proposed to belong to the Picornaviridae 60
family in recent literature: “Cosavirus” (7, 20), “Salivirus” (16, 21, 36, 51) in Human, 61
“Orthoturdivirus” and “Paraturdivirus” in wild birds (58), “Mosavirus” and “Rosavirus” in 62
wild rodents (44), an unnamed genus in ringed seal (SePV-1) (26), an agent responsible for 63
hepatitis in turkey poults (22), two unnamed genera harbored by bats (32) and a virus 64
described in domestic cat (FePV) (33). Very recently, the Picornaviridae study group 65
suggested that the proposed species “seal picornavirus 1” (SePV1) be named “seal 66
aquamavirus A1” and classified in a new proposed genus called “Aquamavirus” and that the 67
proposed species “Turkey hepatitis virus” be classified in the proposed “Megrivirus” genus. 68
Candidate species have also been reported within the genus Kobuvirus in pig (46), dog (27, 69
34) and rodent (44), and within the genus Sapelovirus in California seal lion (35). 70
To date, viruses belonging to five genera from the Picornaviridae family are 71
responsible for several diseases in domestic pigs. They are the Encephalomyocarditis virus 72
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(genus Cardiovirus), the Porcine enterovirus B (Enterovirus), the “Porcine kobuvirus” 73
(Kobuvirus), the Porcine sapelovirus (Sapelovirus, formerly Porcine enterovirus A) and the 74
Porcine teschovirus (Teschovirus, counting only one species) which is recognized as the 75
etiologic agent of polioencephalomyelitis, the most virulent picornaviral infection of pigs. 76
During a study of the fecal microbiome from two healthy piglets using high 77
throughput sequencing (HTS), we identified a viral genome containing an open reading frame 78
encoding a predicted polyprotein of 2133 amino acids (aa) displaying the typical organization 79
of picornaviruses. According to criteria of ICTV (less than 40%, 40% and 50% aa identities in 80
P1, P2 and P3 regions respectively, for genus demarcation), this virus would represent a novel 81
genus in the family Picornaviridae. Given its particular relationship with Parechovirus, we 82
propose to name it “Pasivirus” for “Parecho sister-clade virus” with the “Swine Pasivirus 1” 83
(SPaV1) as type species. In the present article, we show that SPaV1 causes an acute enteric 84
infection of young pigs and report the putative genomic organization and subsequent 85
phylogenetic analysis of its genome. 86
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MATERIALS AND METHODS 87
88
Fecal samples 89
Two fecal samples of healthy piglets (“index cases”) were submitted to HTS analysis. 90
Subsequently, a prevalence survey was performed by one-step RT-PCR based on the data 91
obtained from these index cases (for details see “detection of SPaV1 by one-step RT-PCR of 92
3D gene” section). This prevalence study included fecal samples from 25 healthy sows (2 93
years old) and from 75 healthy piglets ranging from 4 to 28 weeks old (3 to 4 piglets per age 94
category). All the fecal samples (sows and piglets) were collected from an industrial pig farm 95
located in the center of France in 2011. 96
97
Extraction and amplification of nucleic acids 98
Fecal samples were diluted (0.1 g/mL) in phosphate-buffered saline (Gibco), vigorously 99
homogenized and centrifuged at 12,000g for 25 minutes at 4°C. The supernatants were micro- 100
filtered (0.45 µm, Sartorius, Goettingen, Germany) to remove residual eukaryotic and 101
bacterial cell-sized particles. The fecal filtrates were then treated with 0.5U/µL of DNaseI 102
(Qiagen) for 2 hours at 37°C in order to digest unprotected nucleic acids. The DnaseI was 103
inactivated by 10 mM EDTA at room temperature. A volume of 100 µL of each fecal filtrate 104
was then extracted using a Nucleospin RNA virus kit (Macherey-Nagel), which allows 105
recovery of both DNA and RNA. The nucleic acids were eluted into 50 µL of RNase-free 106
water and cDNA synthesis step was performed with random hexamer primers (Superscript® 107
III RT, Invitrogen, Inc). The two following steps, ligation of cDNA and nucleic acids 108
amplification by the bacteriophage Phi29 polymerase, were performed as previously 109
described (5). 110
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HTS and bioinformatics analysis 111
The HTS and the bioinformatics analysis were performed as previously described (5). Briefly, 112
sequencing was conducted on an Illumina® HiSeq-2000 sequencer (GATC Biotech AG, 113
Konstanz, Germany) with a mean depth per sample of 29 ×106 paired-end reads of 96 nt in 114
length (range 25-37 ×106). The whole porcine genome (SGSC - Sscrofa9.2/susScr2) from 115
http://www.genome.ucsc.edu/ was used as reference sequence for pig sequences mapping 116
conducted by SOAPaligner. 117
118
Viral genome sequencing and analysis 119
Twelve specific primer pairs (Table 1) were designed from contigs obtained by HTS to 120
amplify and determine the nucleotide sequence of SPaV1. All PCR amplifications were 121
performed by using the Taq Core kit (MPBio, Illkrich, France) following the manufacturer's 122
instructions. PCR products were sequenced directly using the Big Dye Terminator v1.1 cycle 123
sequencing kit (Applied Biosystems). Sequence chromatograms from both strands were 124
obtained on automated sequence analyzer ABI3730 XL (Applied Biosystems). Attempts to 125
acquire the end of the 3D polymerase and 3’ UTR were made by three methods: i) a Ligation-126
Anchored PCR (LA-PCR) method (3), ii) a 3’ step-out rapid amplification of cDNA ends (3’ 127
step-out RACE) according to the published protocol of Matz D et al. (39) and iii) a novel 128
method using a combination of single strand DNA circularization and rolling circle 129
amplification (RCA) (55). Briefly, LA-PCR involves the ligation of an oligonucleotide by T4 130
RNA Ligase (Ambion) to the 3’ end of RNA before synthesis of cDNA. This method allows 131
the reverse transcription of nonpolyadenylated RNA virus genome. For the 3’ step-out RACE, 132
the 3’ UTR of the genome was amplified with an oligo-dT primer and a specific forward 133
primer (5’-ATATGACTGTTCTTGAGGAGGAG-3’). The method based on template 134
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circularization and RCA, used cDNA as template and a specific extension primer (EP) 5’ end 135
phosphorylated. ssDNA was generated with Phusion High-Fidelity DNA Polymerase (New 136
England Biolabs) and self-ligated by using the CircLigase enzyme (Epicentre 137
Biotechnologies). This step was followed by RCA using Phi29 DNA polymerase, yielding 138
linear concatemeric DNA which served as template for inverse-PCR. This PCR involved two 139
set of primers (P2-P3 and P1-P3) listed in Table 2. The detailed protocol is available upon 140
request. 141
The putative proteolytic cleavage sites were predicted by submitting the polyprotein sequence 142
to the analysis performed by the NetPicoRNA prediction server 143
(http://www.cbs.dtu.dk/services/NetPicoRNA/). The protein sequences were aligned using 144
Jalview 11.0 (56). The whole polyprotein of SPaV1 was used as reference in a sliding window 145
analysis implemented in the RAT software (11) (Figure S1, supplemental data). The complete 146
coding sequence of SPaV1 has been deposited in Genbank database under the accession 147
number JQ316470. 148
149
Phylogenetic analysis 150
All complete available amino acid sequences of the polyproteins of Picornaviridae were 151
aligned in a matrix counting up to 92. Complete reference sequences were used when 152
applicable but the matrix was not restricted to reference sequences and the taxa diversity was 153
optimized by including only reference sequence for well-described genera or lower taxonomic 154
levels. Taxa recently described were also included as they may carry valuable information on 155
the diversity of the corresponding group. A sequence of a picornavirus isolated from fish 156
(bluegill virus Montana lake) (2) was used for alignment and tree rooting (GenBank accession 157
number JX134222). A screening of putative recombination breakpoint was performed using 158
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RDP3 package prior to phylogenetic analyzes (38). This aligned matrix was then sliced 159
following each protein’s orf encoded in the genome taxa and redundant gene sequences were 160
excluded from the analysis. Protein matrices were constituted in accordance with conserved 161
amino acid motifs reported to be characteristic of proteins start and end for each reference 162
sequence. Other taxa were aligned to these reference sequences by several iterations of multi-163
alignment performed under the Muscle algorithm implemented in Seaview software version 164
4.2.11 (15). Sea-Al software version 2.0a11 was also used to edit the matrices 165
(http://tree.bio.ed.ac.uk/software/seal/). Reading frame was respected for subsequent analyzes 166
and phylogenetic tests. Matrices were converted back to their nucleotidic sequences before 167
computing likelihood scores and ranking the 88 model tests according to the Akaike 168
Independent (corrected) Criterion (AIcC) calculated with the jModelTest software version 169
0.1.1 (43). The best matrix fitted model was then used as tree prior in following analyzes. 170
Other specified priors included a relaxed uncorrelated lognormal clock and the Yule 171
speciation process. Matrices were submitted to a maximum of 30,000,000 iterations in order 172
to allow the Markov chain to converge whenever possible. These analyzes were conducted 173
using the BEAST software version 1.6.1 (10). Posterior ESS values and other statistics were 174
extracted to the output files using TreeAnnotator 1.6.1 and investigated using Tracer 1.5 from 175
the Beast package. Resulting trees were edited and visualized in FigTree version 1.3.1 176
(BEAST package). 177
178
Detection of SPaV1 by one-step RT-PCR of 3D gene 179
Nucleic acids were extracted as previously described, except that no DNAse treatment was 180
applied to the fecal filtrates. Primers for prevalence study were selected within the 3D RNA 181
dependant-RNA polymerase gene of SPaV1 genome by using Primer Pro 3.4 software 182
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(www.changbioscience.com) (SPaV1.3D.151F: 5’- AAACCATGGCCTGGTGTGCGT-3’ and 183
SPaV1.3D.151R: 5’- TGCCAATCGCAGAGTCAACCT-3). Reverse transcription and PCR 184
were performed using the Superscript One-step RT-PCR Platinum Taq Kit (Invitrogen) 185
according to the manufacturer’s instructions. PCR products of 151 nt were sequenced with 186
both primers to confirm the detection and assess sequence variation. 187
188
Cell culture 189
The micro filtrated (0.22 µm, Sartorius, Goettingen, Germany) fecal filtrates resuspended in 190
PBS were incubated on Vero E6 grown to sub-confluence in MEM media supplemented with 191
120 µg/mL of streptomycin, 120 units/mL of penicillin and 10% of Fœtal Calf Serum (FCS). 192
The occurrence of any cytopathic effect (CPE) was checked on a daily basis during 12 days. 193
The supernatants were extracted and tested by PCR following the protocol described in the 194
previous section. 195
196
RESULTS 197
198
Identification of SPaV1 genome by HTS 199
The Illumina sequencing generated a total of 27,146,966 reads with a mean length of 96 pb. 200
After the host genome filtration and BLAST analyzes against bacterial, viral and generalist 201
NCBI databases, 725 reads matching with various Picornaviridae genomes were assembled 202
into seven contigs (ranging from 206 pb to 3034 pb). These contigs showed a maximum of 42 203
% of amino acids (aa) identities with the best hits reported within the nr NCBI database: the 204
rodent Parechovirus (Ljungan virus - LV), the human parechoviruses type 1 (HPeV1) and 205
type 5 (HPeV5) and the Duck hepatitis A virus (DHV). Based on the sequence of the contigs 206
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distributed along the genome, twelve primers pairs (Table 1) were designed and used to 207
generate overlapping PCR products validated on both WTA and cDNA products. The 208
sequencing by the Sanger method gave a resulting sequence of 6896 nt (after excluding the 209
polyadenylated tract), with a 5’ partial untranslated region (UTR) of 378 nt, an open reading 210
frame of 6402 nt encoding a potential polyprotein precursors of 2133 aa and a 3’ UTR of 116 211
nt (Figure 1). The available SPaV1 genomic sequence showed a G + C content of 43.3%, 212
which was similar to the values obtained for the corresponding region of the parechoviruses 213
(HPeV1: 40% and LV : 42%) and related clades (DHV: 43%, Seal Aquamavirus A1: 44%, 214
Porcine teschovirus 1: 45% and turdivirus 1: 47%). A BLASTx analysis on the complete 215
genome of SPaV1 provided 31% aa identity and 50% aa similarity to the LV strain 145 SL. A 216
sliding window analysis on the polyprotein of SPaV1 showed that the identity with the 217
members of closer genera never exceeded 50% (Figure S1, supplemental data). 218
219
Genome organization and coding region of SPaV1 220
The partial 5’ UTR had no sequence homology to any virus recorded in GenBank. This 221
region precedes two putative initiator methionine codons found at nucleotide positions 199 222
and 379. Only the initiator codon at position 379 was surrounded by an optimal Kozak 223
context (RNNAUGG) (29) and therefore interpreted as the start codon of the polyprotein. In 224
picornaviruses, the polyprotein precursor is cleaved by viral protease(s) to yield the mature 225
viral structural and non-structural proteins. Putative cleavage sites of SPaV1 were determined 226
by aligning the aa sequence with the closest known virus (LV) and submitting it to the 227
NetPicoRNA prediction server (4). The predicted cleavage sites of the SPaV1 polyprotein 228
were consistent with that of LV’s polyprotein (strain 145SL) described by Johansson (24). 229
These cleavage sites showed a typical molecular organization of picornaviruses with three 230
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structural proteins (VP0, VP3 and VP1) followed by seven non-structural proteins (2A, 2B, 231
2C, 3A, 3B, 3Cpro and 3Dpol) (Figure 1). As observed for Avihepatovirus, Enterovirus, 232
Hepatovirus, Parechovirus, Tremovirus, “Aquamavirus”, “Cosavirus” and “Megrivirus”, 233
SPaV1 does not contain any identifiable leader protein (L). Two predicted cleavage sites, 234
E787/E and Q1372/A define the P1, P2 and P3 coding regions of SPaV1 (Figure 1), which share 235
respectively 17 to 34%, 17 to 29%, and 21 to 29% of aa identities with representatives of 236
other picornaviruses genera (Table S1, supplemental data). The highest identities of the 237
polyprotein were observed with parechoviruses and particularly with LV (33.6, 28.9, 29.2% - 238
for P1, P2, P3 respectively), and with “seal Aquamavirus A1” (22.4, 24.4, 24.6%) and DHV 239
(25.6, 21.3, 27.6%) (Table3). 240
The P1 coding region of SPaV1 contains the “picornavirus capsid protein domain like” (pfam 241
entry: cd00205) and is predicted to be cleaved after Q253 (VP0/VP3), H497 (VP3/VP1) and 242
E787 (VP1/2A) (Figure 1). As observed for the related groups Avihepatovirus, Parechovirus, 243
“Aquamavirus” and more distant groups such as Porcine kobuvirus and others (46, 58), the 244
“VP0” of SPaV1 is probably not cleaved into VP4 and VP2 based on sequence alignment. 245
Similarly to parechoviruses, VP0 does not display the conserved Gxxx[ST] motif for 246
myristylation (6) (Figure 2A), and VP1 does not contain the characteristic [PS]ALxAxETG 247
motif. In addition, VP1 lacked the integrin binding RGD motif [involved in receptor binding] 248
similarly to HPeV3 and LV but in contrast to HPeV1 (57). Consistently to the LV and by 249
contrast to HPeVs, the VP1 protein of SPaV1 contains 2 N-terminal insertions (11- and 4-aa-250
long) and a unique C-terminal extension of 41 aa (43 aa for LV) (Figure 2C). Interestingly, the 251
N-terminal extremity of the VP3 protein (40% aa identity to parechoviruses – Table 3), 252
contained the highly conserved KxKxxRxK motif at position 263 (Figure 2B), recognized to 253
date as a distinctive feature of parechoviruses (57). 254
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The P2 polyprotein of SPaV1 was hypothesized to be cleaved after Q916 (2A/2B), Q1041 255
(2B/2C) and Q1372 (2C/3A). The 2A protein shared 43.6% aa identities with the LV, while this 256
score falls to 13% with DHV and “seal Aquamavirus A1” and to 10% with human 257
parechoviruses (Table 3). Furthermore, this 2A protein possessed the canonical cleavage site 258
DXEXNPG804P (47), which is present in Avihepathovirus and “Aquamavirus” as well as in 259
LV but not in HePV. This enzymatic cleavage releases a small 2A1 protein (17 aa) and a 2A2 260
protein (112 aa) of similar sizes than those of LV (20 and 135 aa, respectively). The conserved 261
H-box and NC-box motifs, which are involved in the control of cell proliferation (23), and a 262
putative transmembrane domain are all present in DHV and parechoviruses and absent from 263
the 2A protein of SPaV1. The conserved GXCG motif [characteristic of a trypsin-like 264
proteolytic activity, (31)] was also absent in the 2A of SPaV1. As for other picornaviruses, the 265
2C protein displayed the NTPase motif G1181XXGXGKS (14) and the D1232DLXQ motif 266
required for helicase activity (13). Similarly to DHV, the leucine (L) of the conserved 267
DDLXQ motif was replaced by phenylalanin (F). 268
The P3 of SPaV1 was predicted to be cleaved after Q1462 (3A/3B), Q1487 (3B/3C) and Q1678 269
(3C/3D). Consequently, the P3 polyprotein encodes the characteristic proteins 3A, 3B (VPg, a 270
small genome-linked protein), 3Cpro (protease) and 3Dpol (RNA- dependant RNA polymerase). 271
A pairwise aa sequence analysis showed that the 3A and 3B proteins of SPaV1 shares the 272
highest identities with HPeV1 (24.2%) and HPeV3 (40%), respectively (Table 3). As observed 273
in all picornaviruses described to date, 3B (25 aa) displayed the conserved tyrosine (Y) at 274
position 3 from the putative N-terminus. This amino acid is necessary to covalently link the 5’ 275
UTR extremity of the viral RNA to VPg that acts as a RNA replication primer (1). Similar to 276
the DHV, the “seal Aquamavirus A1” and parechoviruses, the 3C protein of SPaV1 contained 277
the catalytic triad formed by the amino acids H-D-C (12) found at positions 1525, 1563 and 278
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1638, respectively. As for other picornaviruses, the GXCG (G1636MCG) and GXH (G1654LH) 279
motifs required for proteolytic activity were identified in 3C of SPaV1 (12). In addition, 3C 280
did not contain the RNA binding motif K[FY]RDI (17). As for all members of the family 281
Picornaviridae, the 3D protein of SPaV1 displayed the four characteristic conserved motifs 282
K1839DELR, GG[LMN]PSG (G1986GMASG, where P is replaced by A), Y2003GDD and 283
F2047LKR (28). 284
285
Phylogenetic analyzes 286
No putative recombination breakpoint was identified in the genome of SPaV1 using RDP3 287
software. According to several regions of the genome (VP0, VP3, VP1, 2C, 3C and 3D), 288
Picornaviridae clusters in three major clades: i) The group infecting fish, used as outgroup, ii) 289
the cluster composed by the genera Parechovirus, Avihepatovirus and “Aquamavirus”, 290
infecting birds and mammals, iii) and the clade that clustered all other genera (Figures 3A-3C 291
and S2-S4). Phylogenetic analyzes constantly grouped SPaV1 with Parechovirus and at a 292
lesser extent with Avihepatovirus, “Aquamavirus” and Hepatovirus, enlightening the 293
particular relationship between these clades and the basal origin of these groups within the 294
Picornaviridae family. The analysis of the non-structural proteins (2C, 3C and 3D) identified 295
three to four major clades among Picornaviridae. Among these major clades, SPaV1 296
belonged to the most basal one according to the 2C (Figure S2, supplemental data) and the 3C 297
(Figure S3, supplemental data) but diverged itself from this clade and rooted all others 298
Picornaviridae members according to the 3D (Figure 3A). Despite these differences between 299
proteins, SPaV1 always found its origin closely to the ones of Avihepatovirus and 300
“Aquamavirus” (detected in seal), and, according to the 3C, clustered with Aquamavirus in 301
the sister clade of all parechoviruses (Figure S3, supplemental data). By contrast, analyzes of 302
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the capsid proteins (VP0, VP3 and VP1) rooted parechoviruses with SPaV1 without clustering 303
it with another taxon, making the group SPaV1/parechoviruses monophyletic (Figures 3B, 3C 304
and Figure S4 in supplemental data). These capsid proteins phylogenies were globally 305
congruent but differences noted between these fairly distinguishable topologies remained 306
statistically well supported. Other proteins such as the 2A and the 3A did not provide 307
significant results despite several adjustments of the priors and 30,000,000 iterations. 308
Likewise, the 2B region remained of no interest for phylogenetic reconstruction considering 309
the poor significance of the alignment and low posterior probabilities obtained from analyzes 310
of these data (data not shown). 311
312
Prevalence study and genetic variation of SPaV1 313
Fecal samples from healthy sows and piglets from the same herd (25 and 75, respectively) of 314
ages ranging from 4 to 28 weeks old were analyzed for the presence of SPaV1 by one-step 315
RT-PCR targeting a 3D region of 151 pb. SPaV1 was detected in fecal samples from 51/75 316
healthy piglets (68% of animals) and in none of the 25 fecal samples from healthy sows. The 317
prevalence in piglets aged four to eight weeks was 45% (9/20), in piglets aged nine to 318
fourteen weeks 89.47% (17/19), in piglets aged fifteen to twenty weeks 88.23% (15/17) and 319
in piglets aged twenty-one to twenty-eight weeks 52.63% (10/19) (Figure 4). This distribution 320
is reminiscent of the enteric viruses transmitted after the disappearance of maternal 321
antibodies, as observed for the Hepatitis E virus (8). Among the 51 positive fecal samples for 322
SPaV1, 22 were sequenced to assess genetic diversity. Nucleotide differences between 323
samples ranged from 0.7% to 9.3%. These results suggested the existence of a wide variety of 324
strains in the tested industrial farm. 325
326
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Cell culture 327
Vero E6 cells were inoculated with the fecal supernatants of the two index piglets from which 328
the virus was identified. Cytopathic effect (CPE) was not observed either during the first or 329
the second passages and the PCRs on the supernatants were negative. 330
331
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DISCUSSION 332
We report the nucleotide sequence and the predicted polyprotein of a novel swine 333
picornavirus identified in stool samples of healthy piglets by an HTS method. A recent study 334
has shown that RNA viruses and more precisely Picornaviridae represent the majority of the 335
fecal virome in piglets (50). The genome of this novel virus, called SPaV1 presents the typical 336
genome organization of a member of the Picornaviridae family, mixing characteristics of the 337
two Parechovirus sub-clades: the Human Parechovirus (HPeV) and the Rodent Parechovirus 338
Ljungan virus (LV) species. Interpreted in the light of phylogeny of each protein, these 339
characteristics may reflect the common origin of parechoviruses and SPaV1. Considering the 340
P1, P2 and the P3 proteins, SPaV1 shares less than 40% identity with the LV, the closest taxon 341
described to date. The ICTV recommend less than 40% aa identity in the P1 and P2, and less 342
than 50% in the P3 for genus demarcation in Picornaviridae (52). This new taxon fulfills 343
these criteria and can therefore be considered as a new genus in Picornaviridae. 344
The recent discovery of a high ranking taxonomy level represented by the SPaV1 illustrates 345
that our picture of the diversity of this family is still partial. Major viral genera of 346
Picornaviridae are represented in several avian or mammalian species and this host diversity 347
may contribute to viral diversity in addition to other factors such as typical error prone RNA 348
replication system (9). Overall, the diversity of both virus family (12 genera) and hosts (fish, 349
reptiles, mammals and birds) depicts the dynamism of the evolution of Picornaviridae. For 350
the most studied groups such as Enterovirus, recombinations were shown to play a master role 351
in shaping their genome, and this was not restricted to the intra-species level (49). Moreover, 352
the structural and non-structural parts of the genome of enteroviruses were shown to evolve 353
independently, P1 being so far less subjected to recombination (37, 49). Albeit recombination 354
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between taxa and even genera might have occurred throughout the genome during the 355
evolution of SPaV1, it seems unlikely that traces of such ancient event would still remain 356
detectable. The SPaV1 and therefore the higher ranked “Pasivirus” genus, originated from one 357
of the earliest differentiated and major clade of the Picornaviridae (Figure 3A, 3B, 3C and S2, 358
S3, S4 in supplemental data). Given the host diversity pattern observed for several most 359
studied clades clustering sometimes several viral genera, it is probable that other pasiviruses 360
may infect birds, rodents primates or other animals (Figure 3A, 3B, 3C and S2, S3, S4 in 361
supplemental data). 362
The clear phylogenetic relationship between the SPaV1 and parechoviruses is consistent with 363
numerous similarities of these taxa. Despite being a potential new genus of Picornaviridae, 364
SPaV1 exhibits features that were considered to date to be characteristic of Parechovirus and 365
more specifically of the LV. The low G+C percentage is consistent with those of 366
parechoviruses and related clades and contrast with those of other Picornaviridae. SPaV1 367
contains only three capsid proteins (VP0, VP3 and VP1) exhibiting remarkable features 368
resembling with those of parechoviruses and seems to be lacking a leader protein. VP3 369
contains the conserved KxKxxRxK motif, considered to date as a characteristic signature of 370
parechoviruses (Figure 2B). This motif belongs to a basic amino acid rich region described as 371
immunogenic in HPeVs (25). Moreover, the VP3 of SPaV1 shares more than 40% identity 372
with the one of LV (Table 3) and the characteristics of other capsid proteins reinforce the 373
proximity of SPaV1 and LV. Among those propinquities, the N-terminal extremity of VP0 is 374
shorter than those of HPeVs and lacks the myristoylation site (Figure 2A). Therefore, this site 375
described as mandatory for efficient viral infectivity of poliovirus (30), is not required for LV 376
and SPaV1. Another capsid protein, the VP1, exhibits two insertions of unknown function at 377
Page 18
the N-terminal extremity: i) one counting 11 aa previously described in LV (24), and a second 378
motif of 4 aa identified by multi-alignment of SPaV1, LVs and HPeVs (Figure 2C). The C-379
terminal extremity of VP1 contains a unique 41-aa extension (43 aa for the VP1 of the LV) 380
and no RGD motif but a long C-terminal extremity (Figure 2C). To date, RGD is the unique 381
motif associated with the viral entry mediated by integrin within parechoviruses. Among 382
parechoviruses lacking RGD motif, the well-studied HPeV3 has been associated with 383
neuropathology (18). Nevertheless, no strict association between the RGD presence/absence 384
and the neurovirulence of parechoviruses has been demonstrated. The absence of the RGD 385
motif implies the existence of an alternative cell receptor. By contrast with HPeVs, LV shares 386
with SPaV1 a cleaved 2A resulting in the 2A1 and the 2A2 proteins. The 2A1 of SPaV1 387
exhibits a strong homology with the one of the LV. Due to the absence of the GXCG region, 388
the 2A lacks a proteolytic activity and SPaV1 therefore possess a single 3C protease as 389
described for LV. One of the main differences between the 2A2 protein of SPaV1 and 390
parechoviruses consists in the absence of both the H-box/NC motifs and the putative 391
transmembrane domain. 392
No pathogenicity was noted in infected piglets, that is reminiscent of the high frequency of 393
asymptomatic infections for related parechoviruses infecting human or animals. Nevertheless, 394
HPeVs are pathogens frequently associated to various enteric, nervous or respiratory 395
syndromes in young children (48, 53). Another Parechovirus, the LV, was identified in bank 396
voles (Myodes glareolus) in Europe and the United states (19). Interestingly, LV has been 397
proposed as a potential environmental trigger for human type 1 diabetes on the basis of the 398
presence of LV antibodies while LV RNA detection remained negative, suggesting that the 399
etiologic agent of the disease could be a cross reactive virus (40, 41, 54). The spill-over 400
Page 19
likelihood of such a virus could be greater from domestic animals than from wild animals, as 401
seen for the Hepatitis E virus genotype 3, which is very prevalent but clinically silent in pigs 402
and which frequently infect humans (42). Therefore, SPaV1 or another “Pasivirus” could be a 403
more relevant trigger than LV. 404
Major neutralizing antigenic sites have been located within exposed BC and EF-loops of the 405
capsid proteins and are therefore suspected to shape the immunogenic specificity of 406
picornaviruses (45). These BC- and EF-loops of SPaV1 (Figures 2A-C), exhibit notable 407
differences with LV and other parechoviruses suggesting that cross reactions are unlikely. 408
Therefore, without experimental data, it is difficult to state that cross reaction between LV and 409
SPaV1 or other yet unknown member(s) of this new genus is impossible. 410
SPaV1 was identified in apparently healthy piglets suggesting that this virus present a silent 411
circulation in the investigated farm. Furthermore, the detection in the same farm of several 412
strains (0.7% - 9.3% divergent from SPaV1) suggested that swine is the natural host of this 413
novel and foreseen diversified genus of Picornaviridae. At the individual level, sequencing 414
revealed several polymorphisms within the 3D, denoting a consistent variability. A better 415
picture of the SPaV1 biology, would be achieved through the study of prevalence, tropism, 416
geographic distribution and genetic variation of this new virus. If the zoonotic potential of 417
SPaV1 is attested and despite the absence of any pathogenicity in piglets, the threat to human 418
health should be evaluated considering its circulation in the vicinity of human populations. 419
420
Page 20
ACKNOWLEDGMENTS 421
422 This study was mainly supported by Programme Transversal de Recherche (PATHODISC 423
301) from the Institut Pasteur (Paris, France) and by grants from region Ile de France. We 424
would like to acknowledge Francis Delpeyroux (Unité postulante Biologie des virus 425
entériques, Institut Pasteur, Paris, France) for fruitful 426
discussions. We also deeply thanks Mickael Hoffman and Marisa Barbknecht (department of 427
microbiology, University of Wisconsin – La Crosse) for kindly providing us with their 428
sequence of the Bluegill picornavirus.429
Page 21
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Page 29
FIGURE LEGENDS 652
653
Figure 1 654
Schematic representation of predicted SPaV1 genome organization, including 5' UTR, 3' 655
UTR, P1, P2 and P3 regions. Primer pairs designed from contigs and used to generate 656
overlapping PCR products are distributed along the nucleotidic sequence. Conserved motifs 657
and predicted cleavage sites are indicated along the polyprotein. 658
659
Figure 2 660
Two dimensions structure predictions of the capsid proteins, VP0 (2A), VP3 (2B), VP1 (2C), 661
of the SPaV1 aligned with representatives of parechoviruses [HPeV1 and HPeV3, American 662
(LV64-7855) and Swedish strains (LV87-012, 174F and 145SL) of LV] performed by Jalview 663
11.0. Predicted α-helices and β-strands in SPaVL1 capsid proteins are represented by red and 664
green arrows, respectively. The positions of BC and EF loops are noted above the alignments. 665
Double-headed arrow indicates the shorter N-terminal extremity of VP0 in figure 2A. In 666
figure 2B, the conserved KxKxxRxK motif of VP3 is framed. In the VP1 depicted in figure 667
2C, the two N-terminal aa insertions and the C-terminal aa extension are located by 668
respectively a double pin-headed lines and a dotted double-headed arrow. 669
670
Figures 3 (A, B and C) 671
Phylogenetic analysis of complete VP0, VP1 and 3D (B, C and A, respectively) nucleotide 672
sequences under GTR+G model and relaxed uncorrelated clock implemented in BEAST 673
package. The scale bar unit is expressed in number of substitutions per site, posterior 674
probabilities are reported at the nodes and red color highlights most supported nodes. 675
676
Figure 4 677
Prevalence of SPaV1 on fecal samples from 25 healthy sows and from 75 healthy piglets 678
ranging from 4 to 28 weeks old. The hatched pattern represents weakly positive animals. 679
680
681
Page 30
Nucleotide position
P1 P2 P3
379 1138 1870 2740 3127 3502 44954765 4840
5413
2A1
6780
A(n)5’UTR 3’UTR
3B
VP0 VP3 2A2 2B 2C 3A 3CPro 3DPolVP1
Contigsposition
Amino acid position
253 497 787 916 1041 13721462
16781487
1
2A1
2133position
VP0 VP3 2A2 2B 2C 3A 3CPro 3DPolVP1Q/G H/G E/E Q/G Q/S Q/A
B
Q/R Q/GQ/G
DxExNPGP GxxGxGKS
DDFGQ GxCG KDELRKxKxxRxK
YGDDFLKR
GGMASG
2133 aa
Rhv-like domain Rhv-like domain
Figure 1
Page 31
VP0
BC-loop
βB βCβB βC
EF-loop
βE α
Figure 2A
Page 32
BC lBC-loop
αZ βB βC
EF-loop
βE αB βF
Figure 2B
Page 33
BC loopBC-loop
βB βC αAβB βC αA
Figure 2CFigure 2C
Page 37
89 88100
53
80
s (%
)
4553
40
60
tive
anim
als
020
40
tage
of p
osit
0
Sows0
4-8 9-14 15-20 21-28
Perc
ent
Age range in weeks of piglets
Figure 4
Page 38
Primer name Sequence of forward primer (5’-3’)
Primer name Sequence of reverse primer (5’-3’)
PCR product (bp)
SPaV1.1F
5’-gcttttgaccagtggctctgg-3’
SPaV1.1R
5’-agccgtaggagcagcactatg-3’
531
SPaV1.2F 5’-tgatactgctgaatctggcgg-3’ SPaV1.2R 5’-acccgcagtcagaagaatcag-3’ 566
SPaV1.3F 5’-tcaggtcaatgctgctgcagg-3’ SPaV1.3R 5’-agctgtgaacggtagcaaagg-3’ 598
SPaV1.4F 5’-ctagtgttgcaggcacgagag-3’ SPaV1.4R 5’-cttgacagtgtcaccgcatgg-3’ 572
SPaV1.5F 5’-gttgaaacccgattggctcac-3’ SPaV1.5R 5’-ggagcctcaggcactaacttc-3’ 746
SPaV1.6F 5’-tattcctggtcgccattgcgg-3’ SPaV1.6R 5’-catacatcaagacagggccag-3’ 403
SPaV1.7F 5’-cccccattatggggatattcct-3’ SPaV1.7R 5’-atttcaggagggtacgatccc-3’ 807
SPaV1.8F 5’-cttctgctatggagttgctgg-3’ SPaV1.8R 5’-ccccatacgtggtaaaaccct-3’ 630
SPaV1.9F 5’-ttgaaggattgtgccaccacc-3’ SPaV1.9R 5’-gctagcgcaatagtcgaacac-3’ 900
SPaV1.10F 5’-ccttcttggccctgctgttc-3’ SPaV1.10R 5’-gacaccatctccaaggtctcc-3’ 619
SPaV1.11F 5’-gggtgcttgactataatgggtc-3’ SPaV1.11R 5’-tgccaatcacagagtcaacctc-3’ 597
SPaV1.12F 5’-tcaaggactagtcaccgacac-3’
SPaV1.12R 5’- ccggaacagcttgcaaaagac -3’ 598
Table 1: List of primers designed from contigs used to acquire the genome of SPaV1.
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Primer name Sequence of forward primer (5’-3’) Primer name Sequence of reverse primer (5’-3’) SpaV1-EP1 PHO-5’-gagaaagagggatctcgtgcc-3’ SPaV1-P2.CircL1 5’-gagaagattgaacaaggccttac-3’ SPaV1-P3.CircL1 5’-taagctcatctttaaggtgacag-3’ SPaV1-P1.CircL1 5’-aagagtcttttgcaagctgttcc-3’ SPaV1-EP2 PHO-5’-ccacgtcagctcatgatagatg-3’ SPaV1-P2.CircL2 5’-tggaatggcttcaggatcacc-3’ SPaV1-P3.CircL2 5’-ggttacctaccacatgctgtg-3’ SPaV1-P1.CircL2 5’-ggaggagggtgttgagtatac-3’ SPaV1-EP3 PHO-5’-gtcaagcttgatggtgattatcc-3’ SPaV1-P2.CircL3 5’-gtcggtcagtgagtgttttcc-3’ SPaV1-P3.CircL3 5’-gccaacttccatcgctccaac-3’ SPaV1-P1.CircL3 5’-caattaaattcagcaagtatag-3’ Table 2 : List of primers used to complete the end of the SPaV1 genome. Primers EP (SPaV1-EP1, SPaV1-EP2 and SPaV1-EP3) are 5’end phosphorylated (PHO).
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Protein
SPaV1
(JQ316470)
Pairwise amino acid identities (%) between SPaV1 and the most closely related picornaviruses
Position Length (aa)
LV145 SL (AF327922)
HPeV1 (FM178558)
HPeV3 (AB084913)
SPeV-1 (NC_009891)
Duck hepatitis A virus 1
(NC_008250)
VP0 1Met-Gln253 253 29.3 30.9 32.3 24.5 30.1
VP3 254Gly-497His 244 40.5 41.1 40.8 26.7 27.5
VP1 498Gly-Glu787 290 26.5 25.6 25.3 16.5 18.8
P1 1Met-Glu787 787 33.6 32.4 32.5 22.4 25.6
2A 788Glu- Gln916 119 43.6 9.9 9.6 11.9 13.0
2B 917Gly-Gln1041 125 22.7 25.0 26.4 12.5 22.1
2C 1042Ser-Gln1372 331 35.6 36.2 36.6 30.4 32.4
P2 788Glu-Gln1372 585 28.9 27.6 28.4 24.4 21.3
3A 1373Ala-Gln1462 90 18.0 24.2 22.7 1.0 18.0
3B 1463Arg-Gln1487 25 27.3 26.7 40.0 8.3 21.1
3C 1488Gly-Gln1678 191 26.1 28.6 27.7 23.3 24.4
3D 1679Gly-2133Ser 455 34.3 30.3 32.4 28.7 32.0
P3 1373Ala- 2133Ser 761 29.2 29.0 29.6 24.6 27.6
Table 3: Pairwise amino acids identities between the predicted proteins of SpaV1 and the related picornaviruses. Pairwise amino acid identities were calculated with the complete 2A sequence (2A1 and 2A2). Precursor protein region 1, 2 and 3: P1, P2 and P3.