Short Communication. P. Billam, Z. F. Sun and X.-J. Meng

Journal of General Virology (2007), 88, 1538 1544 DOI 10.1099/vir.0.82754-0 Short Communication Analysis of the complete genomic sequence of an apparently avirulent strain of avian hepatitis E virus (avian HEV) identified major genetic differences compared with the prototype pathogenic strain of avian HEV P. Billam, Z. F. Sun and X.-J. Meng Correspondence X.-J. Meng xjmeng@vt.edu Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0342, USA Received 4 December 2006 Accepted 5 January 2007 Avian hepatitis E virus (HEV) was identified from chickens with hepatitis splenomegaly syndrome. In this study, the complete genomic sequence of an apparently avirulent strain of avian HEV was determined to be 6649 nt in length, excluding the poly(a) tail, which is 5 nt shorter than the prototype avian HEV. Sequence analyses revealed that the ORF1 has 89.6 % nucleotide sequence identity, with numerous non-silent mutations and deletions, compared to the prototype strain. The ORF2 capsid gene showed 90.7 % sequence identity with six non-silent mutations, and ORF3 had four non-silent mutations with 97 % sequence identity. Overall, the apparently avirulent strain shares only 90.1 % nucleotide sequence identity with the prototype strain. The identification of significant non-silent mutations in the capsid gene and other regions suggests that these mutations may play a role in HEV attenuation. This is the first report of the full-length sequence of an apparently avirulent strain of HEV. Hepatitis E virus (HEV) is responsible for the majority of enterically transmitted cases of non-a and non-b hepatitis in humans (Arankalle et al., 1994; Purcell & Emerson, 2001). Hepatitis E is an important human disease of public health importance in many developing countries. Sporadic cases of acute hepatitis E have also been reported in industrialized countries including the USA (Harrison, 1999; Meng, 2000, 2003, 2005; Purcell & Emerson, 2001; Schlauder et al., 1998; Wang et al., 2001). The disease mostly occurs in young adults and the mortality is generally low, but can reach up to 25 % in infected pregnant women (Khuroo & Kamili, 2003). HEV is primarily transmitted by the faecal oral route (Emerson & Purcell, 2003). Recently, zoonotic food-borne transmissions of HEV have been reported in Japan (Matsuda et al., 2003; Tamada et al., 2004; Tei et al., 2003). HEV is a single-stranded, positive-sense, non-enveloped RNA virus belonging to the genus Hepevirus (Emerson et al., 2004). The genome size is 7.2 kb and has three open reading frames (ORFs) (Emerson et al., 2001; Purcell & Emerson, 2001). The largest of the three ORFs is ORF1, The GenBank/EMBL/DDBJ accession number for the avirulent HEV sequence obtained in this study is EF206691. The GenBank accession numbers for all sequences and primers used in this study are available as supplementary material in JGV Online. which encodes the non-structural proteins, including the methyltransferase, protease, helicase and RNA-dependent RNA polymerase (RdRp), while ORF2 encodes the capsid protein. The small ORF3 encodes a cytoskeleton-associated phosphoprotein (Purcell & Emerson, 2001). The first animal strain of HEV, swine hepatitis E virus (swine HEV), was isolated from a pig in the USA in 1997 (Meng et al., 1997) and shown to be closely related to human HEV strains worldwide (Hsieh et al., 1999; Huang et al., 2002a; Nishizawa et al., 2003; Okamoto et al., 2001; Takahashi et al., 2003a, b; van der Poel et al., 2001; Wang et al., 2002). More recently, another animal strain of HEV, avian HEV, was identified from chickens with hepatitis splenomegaly (HS) syndrome in the USA (Haqshenas et al., 2001). Avian HEV is genetically and antigenically related to human HEV, and is associated with a hepatic disease. The clinical and pathological findings associated with avian HEV infection in chickens under the natural route of infection have been reported (Billam et al., 2005). The complete genomic sequence of avian HEV was determined (Huang et al., 2004). In spite of an approximately 50 % nucleotide sequence identity with mammalian HEVs, avian HEV shares many significant structural and functional features with human and swine HEVs. Avian HEV also shares approximately 80 % nucleotide sequence identity with the Australian chicken 1538 0008-2754 G 2007 SGM Printed in Great Britain

Complete sequence of avirulent avian HEV big liver and spleen disease virus (Haqshenas et al., 2001; Payne et al., 1999). Recently we found that antibodies to avian HEV were also prevalent in healthy chicken flocks in the USA (Huang et al., 2002b), suggesting that chickens are subclinically infected. From a prospective study, we isolated an apparently avirulent strain of avian HEV from healthy chickens without clinical disease (Sun et al., 2004). Chickens became infected at about 3 4 months of age under natural conditions; however, the clinical signs of HS syndrome were absent. Since the avian HEV strain from healthy chickens does not cause any clinical disease in the field, it is important to determine its complete sequence and compare it to the pathogenic strain from chickens with HS syndrome. Thus far, the complete sequence for avian HEV is reported only for a single pathogenic strain (Huang et al., 2004). The original virus material used for the determination of the complete sequence of the apparently avirulent avian HEV was collected from healthy chickens in a prospective study (Sun et al., 2004). The original virus was further biologically amplified by infection of young specific-pathogen-free (SPF) chickens to produce a larger virus stock. Six out of seven inoculated chickens had seroconverted by 18 weeks post-inoculation. The faecal and bile suspension harvested from two of the SPF chickens at 3 weeks post-inoculation was positive for avian HEV RNA, and was pooled to generate a virus stock for use in this study. Primers used for the amplification and sequencing of genomic fragments of the avirulent avian HEV strain in this study were designed on the basis of the prototype pathogenic avian HEV (primer sequences are shown in Supplementary Table S1, available in JGV Online). Briefly, RNA was extracted with TRI reagent (MRC) from 200 ml virus stock. Total RNA was resuspended in 12.25 ml DNase-, RNase- and proteinase-free water. Reverse transcription was performed at 42 uc for 60 min with respective reverse primer and SuperScript II reverse transcriptase (Invitrogen). Five microlitres of the resulting cdna was amplified in a 50 ml reaction using Platinum PCR Supermix high fidelity (Invitrogen) by two-round nested PCR. To identify the extreme 39 genomic sequence, we employed the 39 rapid amplification of cdna ends (RACE) technique using a commercially available 39 adapter and outer and inner antisense primers (Ambion). Total RNA, extracted from 200 ml avirulent avian HEV material, was resuspended in 9 ml DNase-, RNase- and proteinase-free water (Invitrogen), and then reverse-transcribed at 55 uc using a thermostable reverse transcriptase (ThermoScript; Invitrogen) and a commercial 39 adapter. Outer 39 RACE antisense primer and avirulent avian HEV-specific forward primer F6227 were used in first-round PCR amplification with the following PCR conditions: initial incubation at 94 uc for 9 min, followed by 39 cycles of denaturation at 94 uc for 0.5 min, annealing at 46 uc for 0.5 min, extension at 72 uc for 1 min and a final extension at 72 uc for 7 min. Inner 39 RACE reverse primer and avirulent avian HEVspecific inner forward primer FF6307 were employed in a second-round nested PCR with essentially the same PCR conditions except for an annealing temperature of 48 uc. The sequence at the extreme 59 end was determined by using the 59 RACE technique. First-strand cdna synthesis was performed by reverse transcription at 55 uc using a thermostable reverse transcriptase and avirulent avian HEV-specific reverse primer R355. The cdna was then purified using a S.N.A.P. column (Invitrogen). The 39 end of cdna was tailed with homopolymeric dctp using terminal deoxynucleotidyl transferase (TdT) by incubating at 37 uc for 10 min. The cdna was denatured at 94 uc for 3 min before TdT tailing to disrupt potential secondary structure. Nested PCR was performed on dc-tailed cdna. First-round PCR was done with avirulent avian HEVspecific reverse primer R339 and abridged anchor primer (Invitrogen) with the following PCR conditions: initial incubation at 94 uc for 9 min, followed by 10 cycles of denaturation at 94 uc for 0.5 min, annealing at 50 uc for 0.5 min, extension at 72 uc for 1 min, followed by 25 cycles of denaturation at 94 uc for 0.5 min, annealing at 55 uc for 0.5 min, extension at 72 uc for 1 min and a final extension at 72 uc for 7 min. Second-round nested PCR was done using reverse primer R252 and abridged universal amplification primer (Invitrogen) with the following PCR conditions: initial incubation at 94 uc for 9 min, followed by 39 cycles of denaturation at 94 uc for 0.5 min, annealing at 58 uc for 0.5 min, extension at 72 uc for 1 min and a final extension at 72 uc for 7 min. The PCR products were excised from 0.8 % agarose gel, purified using a Geneclean III kit (Qbiogene) and sequenced at the Virginia Bioinformatics Institute (Blacksburg, VA, USA). The primer-walking strategy was employed to determine the complete genomic sequence of both DNA strands. The sequence was assembled and analysed using Lasergene version 6 (DNASTAR) and MacVector version 9.0 (MacVector, Inc.) computer programs. The consensus sequence was obtained by assembling approximately 19 overlapping sequences. Each sequence is based, in a majority of cases, on at least four sequence reads, two each from forward and reverse primers. Multiple nucleotide and amino acid alignments were analysed using CLUSTAL W of the MacVector program. Phylogenetic analyses were performed using the PAUP program version 4.0 (David Swofford, Smithsonian Institute, Washington, DC, USA). The complete genomic sequence of the apparently avirulent strain of avian HEV has been deposited in the GenBank database with accession no. EF206691. GenBank accession nos for other HEV strains used in the study are available in Supplementary Table S2 in JGV Online. The complete genome of the avirulent strain of avian HEV, excluding the 39 poly(a) tail, is 6649 nt in length, 5 nt shorter than the prototype avian HEV genome and 605 nt shorter than mammalian HEV genomes. The 59 non-coding http://vir.sgmjournals.org 1539

P. Billam, Z. F. Sun and X.-J. Meng region (NCR) is 24 nt and is the same length as that of the prototype avian HEV genome. The ORF1 encodes a polyprotein of 1530 aa in length, 1 aa shorter than that of ORF1 of the prototype avian HEV. ORF2 is 1821 nt in length and encodes a putative capsid protein of 606 aa, identical in length to that of prototype avian HEV. ORF3 comprises 264 nt and encodes a small, 87 aa long protein. The 39 NCR, excluding the poly(a) tail, is 128 nt in length, 2 nt shorter than that of the prototype strain. The nucleotide sequence identities of the avirulent strain of avian HEV with mammalian HEVs and prototype pathogenic avian HEV are presented in Table 1. Sequence comparisons identified major genetic differences between the prototype pathogenic strain of avian HEV and the apparently avirulent strain. In ORF1, a total of 41 mutations were observed in the avirulent strain compared to the prototype strain (Fig. 1), with nucleotide sequence identity ranging from 89 to 92 % in different functional domains. Two non-silent mutations, V187I and S201T, were identified in the methyl transferase domain. Compared to the prototype strain, the papain-like cysteine protease domain of the avirulent strain had a single mutation, K498R, while the helicase gene region underwent three mutations, V854I, I936M and R950K. The RdRp domain encoded 483 aa and had six non-silent mutations (L1071S, V1106A, I1171V, G1179E, D1382E and R1402H). Multiple deletions and insertions were also noted in the avirulent strain. The hypervariable region (HVR) of the avirulent strain was positioned 8 aa ahead that of the prototype strain. The HVR contains 23 mutations. The ORF2 capsid gene of the avirulent strain shares 91 % nucleotide sequence identity with that of the prototype strain. A total of six non-silent mutations were observed in the ORF2 (C4R, A288T, M359L, S436A, S511N and R600K) and one of them (C4R) was in the predicted signal peptide region. A R600K mutation occurred in the antigenic domain IV (Haqshenas et al., 2001). The tetrapeptide APLT is conserved, as is the case in other strains of HEV. A 54 aa deletion present in the prototype avian HEV compared to mammalian HEVs was also observed in the avirulent strain. The ORF3 shared 97 % nucleotide sequence identity with the prototype avian HEV with four non-silent mutations (A45V, A48V, I54T and A59V). The 59 NCR of the avirulent strain is identical in length to that of the prototype avian HEV, but the 39 NCR has two deletions at positions corresponding to nt 6558 and 6612, respectively, of the prototype strain. Phylogenetic analyses revealed that the avirulent avian HEV clustered together with the prototype avian HEV but was distantly related to mammalian HEVs (Fig. 2a). The avian HEV isolates from different geographical locations, the prototype avian HEV and the apparently avirulent avian HEV isolate are heterogeneic in the capsid gene (Fig. 2b). The discovery of avian HEV associated with a hepatic disease provided a homologous small animal model system to study HEV pathogenesis and replication (Billam et al., 2005). Unfortunately, only a single strain of avian HEV has thus far been fully sequenced (Huang et al., 2004). The prototype avian HEV shares significant structural and functional motifs with mammalian HEVs (Haqshenas et al., 2001; Huang et al., 2004), indicating that it belongs to the same genus (Hepevirus) as human and swine HEVs (Emerson et al., 2004). Like the prototype avian HEV, the avirulent strain shares significant features with mammalian HEV strains. Apart from the deletions and mutations observed in the ORF1 of prototype avian HEV, an additional 3 nt deletion (coding for a glycine) with no change in the reading frame was also noted in the avirulent strain. The HVR was the most divergent region not only with the mammalian HEVs, but also with the prototype avian HEV strain. ORF1 of the avirulent strain contains the majority of mutations compared to the prototype avian HEV. A total of 41 mutations occurred in ORF1, with nine of them in helicase and RdRp genes, the regions responsible for virus replication. It has been demonstrated that a single or a few amino acid changes can alter the virulence and attenuation phenotypes, tissue tropism and cell culture adaptation of numerous viruses. For examples, the Y73H mutation in the 3D pol region of a temperature-resistant mutant S138C 5 of Sabin type 1 poliovirus strain was found to contribute to the neurovirulence phenotype in monkeys. This mutation was also found in the neurovirulent wild-type Mahoney strain, but not in the attenuated phenotype Sabin type 1 (Christodoulou et al., 1990). Point mutations leading to G108V or G108D amino acid changes in the putative N- terminal cleavage site of the porcine transmissible gastroenteritis virus replicase resulted in a 3 log reduction in virus titre (Galán et al., 2005). The identified critical mutations in the functional domains of ORF1, encoding proteins and enzymes involved in virus replication of the avirulent avian HEV, may influence the replicative competence and may be responsible for naturally occurring attenuation phenotype of avian HEV. Clearly, additional studies are warranted to systematically determine which mutation(s) in the ORF1 are critical for virus attenuation. Six unique non-silent mutations were identified in the capsid gene of the avirulent strain. One of the non-silent mutations (R600K) is located in the putative antigenic domain IV of the prototype avian HEV and another in the putative signal peptide (C4R) that is necessary for translocation of the peptide into the endoplasmic reticulum. The mutation from a hydrophobic cysteine to a highly charged hydrophilic arginine (C4R) could potentially alter the signal peptide s secondary structure and function. The R600K mutation in the antigenic domain IV might have an effect on the virulence characteristics of this isolate. Virus capsid is the major determinant of virus virulence and it is well known that mutations in the capsid gene can attenuate viruses. It has been shown that the genetic basis of the attenuation phenotype of poliovirus, serotype 3, lies in an S91F amino acid substitution in the coat protein VP3, along with a point mutation in the non-coding region (Westrop et al., 1989). Another Sabin 3-specific amino acid 1540 Journal of General Virology 88

http://vir.sgmjournals.org 1541 Table 1. Pairwise sequence comparison of the full-length genomic sequence of the apparently avirulent avian HEV with other selected representative HEV strains Values indicate nucleotide sequence identities (%). Genotype 1 Genotype 2 Genotype 3 Genotype 4 Avian HEV Burma Hyderabad Morocco Mexico Prototype swine HEV US1 US2 Arkell T1 swj13-1 JAK-Sai JKK-Sap Prototype avian HEV Genotype 1 Sar-55 93 92 89 76 73 73 73 73 74 75 75 75 48 48 Burma 96 89 76 73 73 73 73 74 75 75 74 47 47 Hyderabad 88 75 73 72 72 73 74 74 74 74 47 47 Morocco 76 73 73 74 74 74 75 74 75 48 48 Genotype 2 Mexico 73 73 73 73 73 73 73 73 47 47 Genotype 3 Prototype swine HEV 91 91 87 75 74 75 75 47 47 US1 91 87 74 74 75 75 46 47 US2 88 75 75 75 75 47 47 Arkell 74 75 75 75 47 46 Genotype 4 T1 84 84 84 47 47 swj13-1 89 88 47 47 JAK-Sai 88 47 47 JKK-Sap 47 47 Avian HEV Prototype avian HEV 90 Avirulent avian HEV Complete sequence of avirulent avian HEV

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Complete sequence of avirulent avian HEV Fig. 1. Amino acid sequence alignment of the ORF1 of the prototype avian HEV and the apparently avirulent avian HEV. Identical amino acids are indicated by grey shading and the mutations are shown by asterisks. Deletions are denoted by dashes. The first and the last nucleotide positions of putative functional domains and HVR region are indicated by arrows. HVR is underlined. The putative functional domains are: Methyl, methyl transferase; PLP, papain-like cysteine protease; HVR, hypervariable region; Helicase, helicase; RdRp, RNAdependent RNA polymerase. mutation of capsid protein VP1 (I6T) was also found to be contributing to the attenuating phenotype of this vaccine strain (Tatem et al., 1992). It is interesting to note that just a few amino acid mutations drastically diminished the neurovirulence of this strain and made it safe enough to use as a vaccine. In foot-and-mouth disease virus, the amino acid arginine at codon 56 in the capsid protein VP3 was reported to be critical for cell tropism and plaque morphology, making the variant bearing the arginine residue attenuated in vivo in cattle (Sa-Carvalho et al., 1997). The identification of six unique non-silent mutations in the capsid gene of the avirulent strain suggests that they may play a potential role in determining the attenuation phenotype of avian HEV. Further experiments, beyond the scope of this study, to characterize these six non-silent mutations will be helpful in understanding the genetic basis of HEV attenuation. It will now be important to definitively assess the pathogenic nature of this apparently avirulent strain and compare its pathogenicity with the prototype avian HEV strain under laboratory conditions in SPF chickens. The availability of the complete genomic sequence of the apparently avirulent strain will now afford us the opportunity to study the genetic determinants of HEV virulence and aid HEV vaccine development efforts in the future. Acknowledgements This study was supported by grants to X.-J. M. from the National Institutes of Health (AI050611 and AI065546). References Fig. 2. (a) A phylogenetic tree based on the complete genomic sequences of the avirulent avian HEV, the prototype avian HEV and selected mammalian HEV strains. (b) A phylogenetic tree based on the partial capsid gene sequences of the avirulent avian HEV, prototype avian HEV and other avian HEV isolates from the USA and Canada. The trees were constructed with the aid of the PAUP program (version 4.0) using branch-and-bound search with midpoint rooting option. A scale bar representing the number of character state changes proportional to the genetic distance is shown. The HEV genotypes are indicated in parentheses. Arankalle, V. A., Chadha, M. S., Tsarev, S. A., Emerson, S. U., Risbud, A. R., Banerjee, K. & Purcell, R. H. (1994). Seroepidemiology of water-borne hepatitis in India and evidence for a third enterically-transmitted hepatitis agent. Proc Natl Acad Sci U S A 91, 3428 3432. Billam, P., Huang, F. F., Sun, Z. F., Pierson, F. W., Duncan, R. B., Elvinger, F., Guenette, D. K., Toth, T. E. & Meng, X. J. (2005). Systematic pathogenesis and replication of avian hepatitis E virus in specific-pathogen-free adult chickens. J Virol 79, 3429 3437. Christodoulou, C., Colbere-Garapin, F., Macadam, A., Taffs, L. F., Marsden, S., Minor, P. & Horaud, F. (1990). Mapping of mutations associated with neurovirulence in monkeys infected with Sabin 1 poliovirus revertants selected at high temperature. JVirol64, 4922 4929. Emerson, S. U. & Purcell, R. H. (2003). Hepatitis E virus. Rev Med Virol 13, 145 154. Emerson, S. U., Zhang, M., Meng, X. J., Nguyen, H., St Claire, M., Govindarajan, S., Huang, Y. K. & Purcell, R. H. (2001). Recombinant hepatitis E virus genomes infectious for primates: importance of capping and discovery of a cis-reactive element. Proc Natl Acad Sci USA98, 15270 15275. Emerson, S. U., Anderson, D., Arankalle, A., Meng, X. J., Purdy, M., Schlauder, G. G. & Tsarev, S. A. (2004). Hepevirus.InVirus Taxonomy. Eigth Report of the International Committee on Taxonomy of Viruses, pp. 853 857. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier/Academic Press. Galán, C., Enjuanes, L. & Almazán, F. (2005). A point mutation within the replicase gene differentially affects coronavirus genome versus minigenome replication. J Virol 79, 15016 15026. http://vir.sgmjournals.org 1543

P. Billam, Z. F. Sun and X.-J. Meng Haqshenas, G., Shivaprasad, H. L., Woolcock, P. R., Read, D. H. & Meng, X. J. (2001). Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis-splenomegaly syndrome in the United States. J Gen Virol 82, 2449 2462. Harrison, T. J. (1999). Hepatitis E virus an update. Liver 19, 171 176. Hsieh, S. Y., Meng, X. J., Wu, Y. H., Liu, S. T., Tam, A. W., Lin, D. Y. & Liaw, Y. F. (1999). Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus. J Clin Microbiol 37, 3828 3834. Huang, F. F., Haqshenas, G., Guenette, D. K., Halbur, P. G., Schommer, S. K., Pierson, F. W., Toth, T. E. & Meng, X. J. (2002a). Detection by reverse transcription-pcr and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. J Clin Microbiol 40, 1326 1332. Huang, F. F., Haqshenas, G., Shivaprasad, H. L., Guenette, D. K., Woolcock, P. R., Larsen, C. T., Pierson, F. W., Elvinger, F., Toth, T. E. & Meng, X. J. (2002b). Heterogeneity and seroprevalence of a newly identified avian hepatitis e virus from chickens in the United States. J Clin Microbiol 40, 4197 4202. Huang, F. F., Sun, Z. F., Emerson, S. U., Purcell, R. H., Shivaprasad, H. L., Pierson, F. W., Toth, T. E. & Meng, X. J. (2004). Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV. J Gen Virol 85, 1609 1618. Khuroo, M. S. & Kamili, S. (2003). Aetiology, clinical course and outcome of sporadic acute viral hepatitis in pregnancy. J Viral Hepat 10, 61 69. Matsuda, H., Okada, K., Takahashi, K. & Mishiro, S. (2003). Severe hepatitis E virus infection after ingestion of uncooked liver from a wild boar. J Infect Dis 188, 944. Meng, X. J. (2000). Novel strains of hepatitis E virus identified from humans and other animal species: is hepatitis E a zoonosis? J Hepatol 33, 842 845. Meng, X. J. (2003). Swine hepatitis E virus: cross-species infection and risk in xenotransplantation. Curr Top Microbiol Immunol 278, 185 216. Meng, X. J. (2005). Hepatitis E as a zoonosis. In Viral Hepatitis, 3rd edn, pp. 611 623. Edited by H. Thomas, A. Zuckermann & S. Lemon. Oxford, UK: Blackwell Publishing Ltd. Meng, X. J., Purcell, R. H., Halbur, P. G., Lehman, J. R., Webb, D. M., Tsareva, T. S., Haynes, J. S., Thacker, B. J. & Emerson, S. U. (1997). A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci U S A 94, 9860 9865. Nishizawa, T., Takahashi, M., Mizuo, H., Miyajima, H., Gotanda, Y. & Okamoto, H. (2003). Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99 % identity over the entire genome. J Gen Virol 84, 1245 1251. Okamoto, H., Takahashi, M., Nishizawa, T., Fukai, K., Muramatsu, U. & Yoshikawa, A. (2001). Analysis of the complete genome of indigenous swine hepatitis E virus isolated in Japan. Biochem Biophys Res Commun 289, 929 936. Payne, C. J., Ellis, T. M., Plant, S. L., Gregory, A. R. & Wilcox, G. E. (1999). Sequence data suggests big liver and spleen disease virus (BLSV) is genetically related to hepatitis E virus. Vet Microbiol 68, 119 125. Purcell, R. H. & Emerson, S. U. (2001). Hepatitis E virus. In Fields Virology, 4th edn, pp. 3051 3061. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins. Sa-Carvalho, D., Rieder, E., Baxt, B., Rodarte, R., Tanuri, A. & Mason, P. W. (1997). Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle. J Virol 71, 5115 5123. Schlauder, G. G., Dawson, G. J., Erker, J. C., Kwo, P. Y., Knigge, M. F., Smalley, D. L., Rosenblatt, J. E., Desai, S. M. & Mushahwar, I. K. (1998). The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States. J Gen Virol 79, 447 456. Sun, Z. F., Larsen, C. T., Dunlop, A., Huang, F. F., Pierson, F. W., Toth, T. E. & Meng, X. J. (2004). Genetic identification of avian hepatitis E virus (HEV) from healthy chicken flocks and characterization of the capsid gene of 14 avian HEV isolates from chickens with hepatitissplenomegaly syndrome in different geographical regions of the United States. J Gen Virol 85, 693 700. Takahashi, M., Nishizawa, T., Miyajima, H., Gotanda, Y., Iita, T., Tsuda, F. & Okamoto, H. (2003a). Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus. J Gen Virol 84, 851 862. Takahashi, M., Nishizawa, T. & Okamoto, H. (2003b). Identification of a genotype III swine hepatitis E virus that was isolated from a Japanese pig born in 1990 and that is most closely related to Japanese isolates of human hepatitis E virus. J Clin Microbiol 41, 1342 1343. Tamada, Y., Yano, K., Yatsuhashi, H., Inoue, O., Mawatari, F. & Ishibashi, H. (2004). Consumption of wild boar linked to cases of hepatitis E. J Hepatol 40, 869 870. Tatem, J. M., Weeks-Levy, C., Georgiu, A., DiMichele, S. J., Gorgacz, E. J., Racaniello, V. R., Cano, F. R. & Mento, S. J. (1992). A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating. J Virol 66, 3194 3197. Tei, S., Kitajima, N., Takahashi, K. & Mishiro, S. (2003). Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 362, 371 373. van der Poel, W. H., Verschoor, F., van der Heide, R., Herrera, M. I., Vivo, A., Kooreman, M. & de Roda Husman, A. M. (2001). Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerg Infect Dis 7, 970 976. Wang, Y., Levine, D. F., Bendall, R. P., Teo, C. G. & Harrison, T. J. (2001). Partial sequence analysis of indigenous hepatitis E virus isolated in the United Kingdom. J Med Virol 65, 706 709. Wang, Y. C., Zhang, H. Y., Xia, N. S., Peng, G., Lan, H. Y., Zhuang, H., Zhu, Y. H., Li, S. W., Tian, K. G. & other authors (2002). Prevalence, isolation, and partial sequence analysis of hepatitis E virus from domestic animals in China. J Med Virol 67, 516 521. Westrop, G. D., Wareham, K. A., Evans, D. M., Dunn, G., Minor, P. D., Magrath, D. I., Taffs, F., Marsden, S., Skinner, M. A. & Schild, G. C. (1989). Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine. J Virol 63, 1338 1344. 1544 Journal of General Virology 88