bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 Emergence and Genetic Analysis of Avian Gyrovirus 2 Variants-Related Gyrovirus

2 in Farmed King-ratsnakes (Elaphe carinata): the First Report in China

3

4 Qianqian Wu1, Qinxi Chen1, Wen Hu1, Xueyu Wang1, Jun Ji1,*

5 1Henan Provincial Engineering Laboratory of Insects Bio-reactor, China-UK-NYNU-RRes

6 Joint Laboratory of Insect Biology, Nanyang Normal University, Nanyang, 473061, PR China

7

8

9 *Corresponding authors:

10 Jun Ji

11 Henan Provincial Engineering Laboratory of Insects Bio-reactor, Wolong Road 1638,

12 Nanyang Normal University, Nanyang, PR China

13 Tel: +8618537796628

14 Fax: +86(0) 37763525087

15 E-mail addresses: [email protected]

16 bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

17 ABSTRACT Avian gyrovirus 2 (AGV2), which is similar to chicken infectious anemia

18 virus, is a new member of the Circovirus genus. AGV2 has been detected not only in chicken

19 but also in human tissues and feces. In this study, a total of 91 samples (8 liver tissues and 83

20 faecal samples) collected from king-ratsnakes (Elaphe carinata) at 7 separate farms in Hubei

21 and Henan, China, were analyzed to detect AGV2 DNA via specific PCR. The results

22 indicated a low positive rate of AGV2 (6.59%, 6/91) in the studied animals, and all of the

23 positive samples were from the same farm. The AGV2 strain HB2018S1 was sequenced, and

24 the genome with a total length of 2376 nt contained three partially overlapping open reading

25 frames: VP1, VP2, and VP3. Phylogenetic tree analysis revealed that the HB2018S1,

26 NX1506-1 strain from chickens in China belong to the same clade, with nucleotide homology

27 as high as 99.5%. In total, 10 amino acid mutation sites, including 44R/K, 74T/A, 256 C/R,

28 279L/Q, and 373V/A in AGV2 VP1; 60I/T, 125T/I, 213D/N, and 215L/S in AGV2 VP2; and

29 83H/Y in AGV2 VP3, were found in the genome of HB2018S1 that were different from those

30 observed in most reference strains, suggesting that the differences are related to an

31 transboundary movement among hosts which needs to be further elucidated.

32 IMPORTANCE Recently, AGV2 has been detected in live poultry markets and human

33 blood in mainland China. Previous findings indicated future studies should investigate the

34 large geographic distribution of AGV2 and monitor the variants, the host range, and the

35 associated diseases. To the best of our knowledge, this study is the first report on AGV2

36 infected poikilotherm, suggested that cross-host transmission of viruses with circular

37 single-stranded DNA genomes would be a public health concern.

38

39 KEYWORDS Avian gyrovirus 2, variants-related gyrovirus, sequence analysis,

40 phylogenetic tree, amino acid sites

41

42 bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

43 INTRODUCTION

44 Avian gyrovirus 2 (AGV2) belongs to the viral genus Cyclovirus (family: Circoviridae),

45 and it was first detected in diseased chicken in Brazil in 2011[1]. AGV2 infections in chicken

46 can result in brain damage, mental retardation, and weight loss[2]. Although other specific

47 symptoms of AGV2 infections have not been confirmed, autopsy-based studies have reported

48 clinical manifestations such as hemorrhage, edema, glandular gastric erosion, and facial and

49 head swelling in infected chickens[3]. Varela et al. (2014) used a duplex quantitative real-time

50 PCR assay to detect commercially available poultry vaccines and suggested that the

51 widespread existence of AGV2 is associated with vaccine contamination [4]. AGV2 has also

52 been reported in different regions of Europe, Latin America, Africa, South America, and Asia,

53 which indicates its global distribution [5-8]. Further, AGV2 has been detected not only in

54 poultry but also in human blood samples [9]. In 2011, Sauvage et al. (2011) found human

55 gyrovirus, which is similar to AGV2, in a skin swab of a healthy person, suggesting that

56 AGV2 has public health significance [10]. In the present study, we detected AGV2 in farmed

57 snakes that was highly homologous to AGV2 and amplified its whole-genome sequence. We

58 subsequently performed an in-depth sequence analysis based on genetic evolution and amino

59 acid mutations between the strain and reference strains.

60 RESULTS

61 Positive rates of AGV2 in snakes in China. To increase our understanding of the infection

62 of AGV2 in farmed snakes in China, a total of 91 king-ratsnakes (Elaphe carinata) samples

63 were collected and tested. The results indicated that 5 faecal samples (6.02%, 5/83) and only

64 one liver tissues (12.5%, 1/8) were positive for AGV2. All the 6 positive samples were

65 collected from the same snake farm in Hubei, China. Detailed information on the snake

66 samples testing positive for AGV2 DNA is listed in Table 1.

67 Sequence analysis of whole-genome. Based on the results of sequencing and

68 contig-assembly, the AGV2 strain was named HB2018S1 and was found to contain a whole

69 genome with a full length of 2376 nt. The nucleotide homology of HB2018S1 with the same bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

70 cluster of the NX1506-1, NX1506-2, NX1510, and JL1508 reference strains was up to 99.5%.

71 The lowest nucleotide homology of 95.3% was detected between HB2018S1 and S53/It from

72 chickens. In addition, the nucleotide homologies between HB2018S1 and each of the three

73 human strains 915F06007FD, CL33, and JQ69076 3 were 95.6%, 97.1%, and 96.8%,

74 respectively.

75 Phylogenetic analysis. Phylogenetic tree analysis revealed that AGV2 sequences of the

76 present study and the 25 full-length genome sequences downloaded from GenBank could

77 have two branches (Fig. 1). HB2018S1 and most of the downloaded sequences belonged to

78 branch I, which was subsequently divided into four clusters. Excluding one cluster containing

79 two strains of the human virus 915F06007FD and CL33, and G13 from ferrets, the remaining

80 three clusters were found to represent AGV2 mainly from chickens.

81 Major mutation analysis of mutated amino acids. There are three overlapping ORFs in

82 the AGV2 genome, which encode VP1, VP2, and VP3 proteins. ORF3, which is located at

83 sites 953–2335 and encodes VP1, comprises 460 amino acid residues. VP1 of HB2018S1

84 shared 97.4%–98.9% homology of deduced amino acid with the reference strains. VP1 of all

85 strains contained 20 substituted amino acids (displayed in gray in Table 2), including at sites

86 36, 44, 74, 95, 154, 212, 242, 256, 270, 279, 288, 293, 310, 311, 314, 373, 383, 401, 416, and

87 459, and VP1 of HB2018S1 had five unique amino acid mutations at sites 44R/K, 74T/A,

88 256C/R, 279L/Q, and 373V/A.

89 The ORF1 encoding AGV2 VP2 was located at sites 450–1145 and encoded 231 amino

90 acid sequences, and VP2 sequences of HB2018S1 shared 94.0%–98.3% amino acid homology

91 with those of the reference strains. There were 15 hyper-mutated amino acid sites in all

92 sequences (14N/T, 60I/T, 125T/I, 141R/Q, 156–158GKR/RRG, 161Y/H, 165A/T, 167T/S,

93 174–175EE/DD, 179A/V, 213D/N, and 215L/S), whereas G17 from ferrets had an S insertion

94 at site 162. In addition, the substitutions 60I/T, 125T/I, 213D/N, and 215L/S only occurred in

95 HB2018S1 (displayed in gray in Table 3).

96 ORF2 encodes VP3, which is a non-structural protein comprising 124 amino acid residues.

97 HB2018S1 VP3 had 92.0%–99.2% homology with the other strains. Compared with the bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

98 amino acid substitution sites of the reference strains, HB2018S1 had only amino acid

99 substitutions at site 83H/Y, excluding G17, which had an R insertion at site 122. The other

100 major amino acid mutation sites were as follows: 7R/H, 9R/Q, 12T/I, 14Q/R, 28S/C, 54Y/S,

101 65A/V, 69D/A, 71G/E, 79V/A, 81L/S, 85R/K,99A/S,101K/R, 103Q/R, 104Q/R, 115N/E,

102 120K/R, and 124L/V (displayed in gray in Table 4).

103 The 5′-untranslated regions (UTRs) of HB2018S1 were between sites 224 and 351, which

104 comprised six closely matching direct repetition (DR) regions with lengths of 22 nt. Among

105 these, five DRs were identical (5′-GTACAGGGGGGTACGTCACCAT-3′). The different DR

106 was the last DR. The three bases at its 3′ end were AGC of HB2018S1, which was similar

107 with the reference strains.

108 DISCUSSION

109 To the best of our knowledge, AGV2 has been detected in three different hosts: chickens,

110 humans, and ferrets. Most strains of AGV2 are mainly detected in chickens, which is its

111 original host. In the present study, AGV2 was detected, for the first time, in a cold-blooded

112 farmed snake; however, how the snake was infected remains unknown. Farmed snakes are

113 mainly fed on raw meats and specific feed. In this study, only one snake farm was positive for

114 AGV2 DNA. According to the feed survey, the snakes in the positive farm were usually fed

115 on chickens. Whether snakes are infected with AGV2 because of alimentary transmission

116 following chicken consumption requires further investigation. In 2012, AGV2 was detected in

117 the feather sacs of two adjacent chicken flocks in southwestern Brazil, suggesting that there is

118 a horizontal transmission mode of AGV2 although more studies are required to demonstrate

119 this phenomenon. From 2011 to 2013, some researchers conducted epidemiological analysis

120 of AGV2 in serum and feces of some individuals to determine whether AGV2 could be

121 replicated in humans. Whether the human AGV2 is attributable to metabolic residues in

122 poultry food remains to be clarified. Phylogenetic tree analysis showed that the sequence of

123 HB2018S1 exhibited high homology with the sequences of NX1506-1, NX1506-2, and bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

124 NX1510 from China. However, the regions are far from the site of collection of HB2018S1,

125 and the AGV2 transmission mode requires further investigation.

126 The AGV2 genome is 2376 nt in length, which is similar to the AGV2 genome from

127 chickens in China, with three overlapping ORFs similar to the ones in chicken infectious

128 anemia virus (CIAV) encoding VP1, VP2, and VP3. At the protein/amino acid level, the

129 homology of VP1, VP2, and VP3 proteins in AGV2 corresponding with VP1, VP2, and VP3

130 proteins in CIAV was 38.8%, 40.3%, and 32.2%, respectively [1]. 5′-UTR, which is located

131 between the polyadenylation site and the transcription beginning, is similar to the structure of

132 CIAV [11, 12].

133 The VP1 protein of CIAV is the only capsid protein of the virus, which constitutes the

134 neutral antigen site of the virus. There are three conserved replication motifs on the VP1

135 sequence in CIAV, including FATLT (at 313–317 residues), QRWHTLV (at 351–357 residues),

136 and YALK (at 402–405 residues)[13]. Similar sequences were observed in the HB2018S1

137 genome: FAALS (325–329 residues), RRWLTLV (363–369 residues), and KAMA (412–415

138 residues). These sites are completely conserved in the currently reported AGV2 sequences.

139 Based on a part of the AGV2 sequences, Yao et al. (2016) hypothesized that there was a

140 high-variant region in the middle and back of the VP1 protein, ranging from 288 to 314

141 residues. In the present study, only eight strains of viruses, including GS1512, HLJ1508, G13,

142 JL1511 HM590588, JQ690763, RS/BR/15/25, and G17, had mutations, and the mutation sites

143 were not identical. Because of the limited number of sequences, it was not possible to predict

144 the high variation regions accurately. However, further investigations are required to verify

145 whether the five mutations in the VP1 protein in HB2018S1, which are different from the

146 other sequences, affect the host selectivity of the virus.

147 The VP2 protein in CIAV is a dual-specific protein phosphatase [14]. The

148 WX7HX3CXCX5H sequence of the phosphatase motif is also highly conserved in CIAV,

149 TTV, and TTV-like TLMV [15]. A similar motif in the VP2 protein of AGV2 is

150 WLRQCARSHDEICTCGRWRSH (95–115 residue) [14] performed a site-directed

151 mutagenesis of VP2 and observed that it affected the replication of viral particles, resulted in bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

152 cytopathological changes, and downregulated the levels of MHC I in infected cells. VP1 and

153 VP2 were necessary for the replication of viral particles. Noteborn [16] also observed

154 interaction between VP1 and VP2 proteins based on an immuncoprecipitation test, which

155 further confirmed that the non-structural protein VP2 plays a scaffold role in virus particle

156 assembly. In the VP2 protein, HB2018S1 has four different amino acid mutations and a S

157 insertion site of G17 from ferrets. Whether such mutations alter VP2 function and, in turn,

158 affect virus replication and whether they affect the scaffold function of the VP2 protein

159 require further study.

160 The VP3 protein in CIAV is also known as apoptin. In normal cells, the VP3 protein

161 exists in the cytoplasm. However, in cancerous cells, it exists in the nucleus and induces

162 apoptosis [17, 18]. It has also been demonstrated that the VP3 protein in AGV2 induces

163 specific apoptotic functions in tumor cells, while the change in molecular function caused by

164 a mutation at site 83H/Y of VP3 protein in HB2018S1 still requires further experimental

165 evidence.

166 In the present study, in addition to the AGV2 virus from chickens, humans, and ferrets,

167 an AGV2 strain from a cold-blooded farmed snake was found, which could provide insight

168 into the modes of transmission of AGV2 and its host diversity.

169

170 MATERIALS AND METHODS

171 Samples and virus detection. In 2018, a total of 91 samples (8 liver tissues from snakes

172 died from bacterial infection and 83 faecal samples) from 7 separate king-ratsnakes (Elaphe

173 carinata) farms in Hubei and Henan, China, were collected and tested for AGV2 by PCR

174 using the AGV2-specific primers AGV2-F (5′-CGTGTCCGCCAGCAGAAAC-3′) and

175 AGV2-R (5′-GGTAGAAGCCAAAGCGTCCAC-3′) as previously described [9]. The samples

176 were washed with phosphate-buffered saline, repeatedly frozen and thawed thrice, immersed

177 in liquid nitrogen, and ground into a homogenate. The homogenate was centrifuged at 5000

178 rpm for 10 min, and 0.2 mL of the supernatant was obtained for nucleic acid extraction. DNA bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

179 and RNA were extracted using a DNA/RNA extraction kit (TransGen Biotech, Beijing, China)

180 according to the manufacturer’s instructions, and the extracted DNA was stored at −20°C until

181 use.

182 Whole-genome sequencing of AGV2. Whole-genome sequencing of AGV2 was

183 conducted using three pairs of overlapping primers designed by Yao et al (2016). including

184 primers for the first segment (1F: 5′-ATT TCCTAGCACTCAAAAACCCATT-3′ and 1R:

185 5′-TCTGGGCGTGCTCAATTCTGATT-3′; from nucleotides 1960–379), second segment (2F:

186 5′-TCACAGCCAATCAGAATT GAGCACG-3′ and 2R:

187 5′-TTCTACGCGCATATCGAAATTTACC-3′; from nucleotides 349–1082), and third

188 segment (3F: 5′-TATTCCCGGAGGGGTAAATTTCGAT-3′ and 3R:

189 5′-CCCCTGTCCCCGTGATGGAATGTTT-3′; from nucleotides 1046–2027); the amplified

190 fragments were 802, 733, and 981 bp in length, respectively. The PCR amplification product

191 mixed to obtain a total reaction volume of 20 μL contained a template DNA, reaction buffer,

192 GC enhancer, 6 pmol of upstream/downstream primers, 0.4 mM of each dNTP (3 µL), and

193 PrimeSTAR HS DNA polymerase (TaKaRa Biotechnology Co., Ltd., Dalian, China).

194 Sequence amplification was performed under the following cycling conditions: initial

195 denaturation at 98°C for 5 min followed by 30 cycles of denaturation at 98°C for 10 s,

196 annealing at 60°C for 15 s, and extension at 72°C for 10 s, with final extension at 72°C for 10

197 min. The PCR products of the three fragments were cloned into a pMD18-T easy vector

198 (TaKaRa Biotechnology Co., Ltd., Dalian, China) for later sequencing (Syn-Biotechnology,

199 Suzhou, China). PCR and sequencing were performed at least thrice.

200 Sequence and phylogenetic analysis. SeqMan (DNASTAR, Lasergene®, Madison,

201 Wisconsin) was used for contig-assembly of the partial sequences. The whole-genome

202 sequences of the snake-originated strain were submitted to GenBank with the accession

203 number MK840982 After sequence alignment of the 25 related reference sequences available

204 at GenBank using Clustalx v1.83, a phylogenetic tree based on nucleic acids and the deduced

205 amino acid sequences was constructed using Mega v.6.0 using the neighbor-joining method, bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

206 Kimura 2-parameter model, pairwise deletion, and bootstrap analysis of 1000 replicates [19].

207 In addition, mutation sites based on the amino acid sequences of three open reading frames

208 (ORFs) were compared.

209 Acknowledgments

210 This study was supported by the National Natural Science Foundation of China (Grant no.

211 31870917), the Scientific and Technological Project of Henan Province (Grant nos.

212 182107000040and 182102110084), the Key Scientific and Technological Project of the

213 Education Department of Henan province (Grant no. 18A230012), Key Scientific and

214 Technological Project of Nanyang City (Grant nos. KJGG2018144 and KJGG2018069), and

215 the Technological Project of Nanyang normal university (Grant nos. 16134, 18046, and

216 2018CX014).

217 Declaration of interest statement

218 The authors have no competing interests to declare. bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

219 REFERENCES 220 221 1. Rijsewijk FA, Dos SHF, Teixeira TF, et al. 2011. Discovery of a genome of a distant 222 relative of chicken anemia virus reveals a new member of the genus Gyrovirus. Arch 223 Virol 156:1097-1100. https://doi.org/10.1007/s00705-011-0971-6. 224 2. dos SHF, Knak MB, de Castro FL, et al. 2012. Variants of the recently discovered 225 avian gyrovirus 2 are detected in Southern Brazil and The Netherlands. Vet Microbiol 226 155:230-236. https://doi.org/10.1016/j.vetmic.2011.09.021. 227 3. Shuai Yao TT, Xiang Gao CH, You Li YG, Yanping Zhang. 2016. Avian gyrovirus 2 in 228 poultry, China, 2015–2016. Emerg Microbes Infect 5. 229 4. Varela AP, Dos SHF, Cibulski SP, et al. 2014. Chicken anemia virus and avian 230 gyrovirus 2 as contaminants in poultry vaccines. Biologicals 42:346-350. 231 https://doi.org/10.1016/j.biologicals.2014.08.002. 232 5. Abolnik C, Wandrag DB. 2014. Avian gyrovirus 2 and avirulent Newcastle disease 233 virus coinfection in a chicken flock with neurologic symptoms and high mortalities. 234 Avian Dis 58:90-94. https://doi.org/10.1637/10657-090313-Reg.1. 235 6. Feher E, Pazar P, Kovacs E, et al. 2014. Molecular detection and characterization of 236 human gyroviruses identified in the ferret fecal virome. Arch Virol 159:3401-3406. 237 https://doi.org/10.1007/s00705-014-2203-3. 238 7. Maggi F, Macera L, Focosi D, et al. 2012. Human gyrovirus DNA in human blood, 239 Italy. Emerg Infect Dis 18:956-959. https://doi.org/10.3201/eid1806.120179. 240 8. Phan TG, Li L, O'Ryan MG, et al. 2012. A third gyrovirus species in human faeces. J 241 Gen Virol 93:1356-1361. https://doi.org/10.1099/vir.0.041731-0. 242 9. Ye J, Tian X, Xie Q, et al. 2015. Avian Gyrovirus 2 DNA in Fowl from Live Poultry 243 Markets and in Healthy Humans, China. Emerg Infect Dis 21:1486-1488. 244 https://doi.org/10.3201/eid2108.150203. 245 10. Sauvage V, Cheval J, Foulongne V, et al. 2011. Identification of the first human 246 gyrovirus, a virus related to chicken anemia virus. J Virol 85:7948-7950. 247 https://doi.org/10.1128/JVI.00639-11. 248 11. Noteborn MH, de Boer GF, van Roozelaar DJ, et al. 1991. Characterization of cloned 249 chicken anemia virus DNA that contains all elements for the infectious replication 250 cycle. J Virol 65:3131-3139. 251 12. Phenix KV, Meehan BM, Todd D, McNulty MS. 1994. Transcriptional analysis and 252 genome expression of chicken anaemia virus. J Gen Virol 75 ( Pt 4):905-909. 253 https://doi.org/10.1099/0022-1317-75-4-905. 254 13. Peng YH, Nishizawa T, Takahashi M, Ishikawa T, Yoshikawa A, Okamoto H. 2002. 255 Analysis of the entire genomes of thirteen TT virus variants classifiable into the fourth 256 and fifth genetic groups, isolated from viremic infants. Arch Virol 147:21-41. 257 14. Peters MA, Jackson DC, Crabb BS, Browning GF. 2002. Chicken anemia virus VP2 is 258 a novel dual specificity protein phosphatase. J Biol Chem 277:39566-39573. 259 https://doi.org/10.1074/jbc.M201752200. 260 15. Takahashi K, Iwasa Y, Hijikata M, Mishiro S. 2000. Identification of a new human 261 DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and 262 chicken anemia virus. Arch Virol 145:979-993. 263 16. Noteborn MH, Verschueren CA, Koch G, Van der Eb AJ. 1998. Simultaneous 264 expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins 265 VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope. J Gen 266 Virol 79 ( Pt 12):3073-3077. https://doi.org/10.1099/0022-1317-79-12-3073. 267 17. Cheng JH, Sheu SC, Lien YY, et al. 2012. Identification of the NLS and NES motifs 268 of VP2 from chicken anemia virus and the interaction of VP2 with mini-chromosome 269 maintenance protein 3. BMC Vet Res 8:15. https://doi.org/10.1186/1746-6148-8-15. 270 18. Taebunpakul P, Sayan BS, Flinterman M, et al. 2012. Apoptin induces apoptosis by 271 changing the equilibrium between the stability of TAp73 and DeltaNp73 isoforms 272 through ubiquitin ligase PIR2. Apoptosis 17:762-776. bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

273 https://doi.org/10.1007/s10495-012-0720-7. 274 19. Chenna R, Sugawara H, Koike T, et al. 2003. Multiple sequence alignment with the 275 Clustal series of programs. Nucleic Acids Res 31:3497-3500. 276 277

278

279 Table 1. Information regarding the samples of snake in our study 280 Number of Positive rates Farm Province samples Faecal samples Liver samples A Henan 10 0/9 0/1 B Hubei 13 5/11 1/2 C Hubei 5 0/5 0/0 D Hubei 21 0/20 0/1 E Henan 19 0/17 0/2 F Hubei 11 0/10 1/1 G Hubei 12 0/11 1/1 Total 91 5/83 1/8 281 bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

282 Table 2. Mutations at different amino acid sites in AGV2 VP1

36 44 74 95 154 212 242 256 270 279 288 293 310 311 314 373 383 401 416 459 Strains G/S R/K T/A K/T A/S K/R R/G C/R S/A L/Q V/Q G/Q E/Q D/E R/K V/A P/Q V/M L/I N/T HB2018S1 G K A K A K R R S Q V G E D R A P V L N NX1510 G R T K A K R C S L V G E D R V P V L N NX1506-2 G R T K A K R C S L V G E D R V P V L N NX1506-1 G R T K A K R C S L V G E D R V P V L T JL1508 G R T K A K R C S L V G E D R V P V L N LN1511 G R T K A K R C S L V G E D R V P V L T JX1602 G R T K A K R C S L V G E D R V P V L N GZ1601 S R T K A K R C S L V G E D R V P V L N HLJ1603-2 G R T K A K R C S L V G E D R V P V L N HLJ1603-1 G R T K A K R C S L V G E D R V P V L T HE1511 G R T K A K R C S L V G E D R V P V L T HLJ1506-2 G R T K A K R C S L V G E D R V P V L N GS1512 G R T K A K R C S L V G E D K V P V L N HLJ1508 G R T T S K R C S L Q Q E D R V P V L N G13 G R T K A K R C S L V G E E R V P V L N JL1511 G R T T S K R C S L Q Q E D R V P V L N HM590588 G R T T S R G C A L V G Q D R V Q M I N JQ690763 G R T K A K R C S L Q Q E D R V P V L N RS/BR/15/2S G R T K S K R C A L V G Q D R V Q M I N HLJ1506-1 S R T K A K R C S L V G E D R V P V L N 915F06007FD G R T K A K R C S L V G E D R V P V L N G17 S R T K S K R C S L Q Q E D K V P V L N CL33 G R T K A K R C S L V G E D R V P V L N S53/It G R T K A K R C S L V G E D R V P V L N HLJ1510 G R T K A K R C S L V G E D R V P V L N 283 bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

284 Table 3. Mutations at different amino acid sites in AGV2 VP2

285 14 60 125 141 156-158 161 162 165 167 174-175 179 213 215 Strains N/T I/T T/I R/Q GKR/RRG Y/H - A/T T/S EE/DD A/V D/N L/S HB2018S1 N T I R GKR Y - A T EE A N S NX1510 N I T R GKR Y - A T EE A D L NX1506-2 N I T R GKR Y - A T EE A D L NX1506-1 N I T R GKR Y - A T EE A D L JL1508 N I T R GKR Y - A T EE A D L LN1511 N I T R GKR Y - A T EE A D L JX1602 N I T R GKR Y - A T EE A D L GZ1601 T I T Q RRG H - T T DD V D L HLJ1603-2 T I T Q RRG H - T T DD V D L HLJ1603-1 T I T Q RRG H - T T DD V D L HE1511 T I T Q RRG H - T T DD V D L HLJ1506-2 N I T R GKR Y - A T EE A D L GS1512 N I T R GKR Y - A T EE A D L HLJ1508 N I T Q RRG H - A T DD V D L G13 N I T R GKR Y - A T EE A D L JL1511 N I T Q RRG H - A T DD V D L HM590588 T I T Q RRG H - T T DD V D L JQ690763 N I T Q RRG H - A T DD V D L RS/BR/15/2S T I T Q RRG H - T T DD V D L HLJ1506-1 T I T Q RRG H - T T DD V D L 915F06007FD N I T R GKR Y - A T EE A D L G17 T I T R RRG Y S A S EE A D L CL33 N I T R GKR Y - A T EE A D L S53/It N I T R GKR Y - A T EE A D L HLJ1510 N I T R GKR Y - A T EE A D L bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

286 Table 4. Mutations at different amino acid sites in AGV2 VP3

7 9 12 14 28 54 65 69 71 79 81 83 85 99 101 103 104 115 120 122 124 Strains R/H R/Q T/I Q/R S/C Y/S A/V D/A G/E V/A L/S H/Y R/K A/S K/R Q/R Q/R N/E K/R -- L/V HB2018S1 R R T Q S Y A D G V L Y R A K Q Q N K - L NX1510 R R T Q S Y A D G V L H R A K Q Q N K - L NX1506-2 R R I Q S Y A D G V L H R A K Q Q N K - L NX1506-1 R R T Q S Y A D G V L H R A K Q Q N K - L JL1508 R R T Q S Y A D G V L H R A K Q Q N K - L LN1511 R R T Q S Y A D G V L H R A K Q Q N K - L JX1602 R R T Q S Y A D G V L H R A K Q Q N K - L GZ1601 R Q T R C Y A D G A S H R S K R R E K - L HLJ1603-2 R Q T R C Y A D G A S H R S K R R E K - L HLJ1603-1 R Q T R C Y A D G A S H R S K R R E K - L HE1511 R Q T R C Y A D G A S H R S K R R E K - L HLJ1506-2 R R T Q S Y A D G V L H R A K Q Q N K - L GS1512 R R T Q S Y A D G V L H R A K Q Q N K - L HLJ1508 R R T Q C S V A E V L H R S R Q R E K - L G13 R R T Q S Y A D G V L H R A K Q Q N K - L JL1511 R R T Q C S V A E V L H R S R Q R E K - L HM590588 R Q T R C Y A D G A S H R S K Q R E K - L JQ690763 R Q T R C Y A D G V L H R S K Q R E K - L RS/BR/15/2S R Q T R C Y A D G A S H R S K Q R E K - L HLJ1506-1 R Q T R C Y A D G A S H R S K Q R E K - L 915F06007FD R R T Q S Y A D G V L H R A K Q Q N K - L G17 H Q T R S Y A D G V L H K A K Q R E R R V CL33 R R T Q S Y A D G V L H R A K Q Q N K - L S53/It R R T Q S Y A D G V L H R A K Q Q N K - L HLJ1510 R R T Q S Y A D G V L H R A K Q Q N K - L 287 bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

288

289 FIGURE LEGEND

290 Figure 1. Phylogenetic analysis of the nucleotide sequences of HB2018S1 and 25 reference genomes available at GenBank.

291 “△” indicates that the host of the strain is chicken. “◆” indicates that the host of the strain is human. “■” indicates that the host of the strain is ferret. “▲”

292 indicates the strain from snake in the present study.

293

294

295

296 bioRxiv preprint doi: https://doi.org/10.1101/629980; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.