Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Price's Fork Road, Blacksburg, VA 24061-0342, USA
Correspondence
X.-J. Meng
xjmeng{at}vt.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Swine HEV, the first animal strain of HEV, was discovered and characterized from a pig in the United States in 1997 (Meng et al., 1997). Since then, many swine HEV isolates have been identified worldwide and shown to be genetically closely related to strains of human HEV (Chandler et al., 1999
; Hsieh et al., 1999
; Pina et al., 2000
; Halbur et al., 2001
; Haqshenas & Meng, 2001
; Garkavenko et al., 2001
; Okamoto et al., 2001
; van der Poel et al., 2001
; Williams et al., 2001
; Huang et al., 2002b
; Kasorndorkbua et al., 2002
; Wu et al., 2002
; Takahashi et al., 2003a
, b
). Recently, another animal strain of HEV, avian HEV, was identified in the United States from chickens with hepatitissplenomegaly (HS) syndrome (Haqshenas et al., 2001
) and shown to be antigenically and genetically related to human and swine HEVs (Haqshenas et al., 2001
, 2002
; Huang et al., 2002a
). HS syndrome was first reported in 1991 in western Canada and has now been recognized in the United States (Ritchie & Riddell, 1991
; Shivaprasad & Woolcock, 1995
; Riddell, 1997
). It has been shown that avian HEV shares approximately 5060 % nucleotide sequence identity with the known human and swine HEVs and about 80 % sequence identity with the Australian chicken big liver and spleen disease virus (BLSV) (Payne et al., 1999
; Haqshenas et al., 2001
).
Our recent study showed that avian HEV antibody is highly prevalent in apparently healthy chicken flocks in the United States (Huang et al., 2002a). However, thus far avian HEV has only been genetically identified from chickens with HS syndrome (Haqshenas et al., 2001
; Huang et al., 2002a
; Sun et al., 2003
). Therefore, it is important to genetically identify and characterize avian HEV from chickens without clinical disease. In addition, the extent of genetic variability of the ORF2 capsid gene of avian HEV isolates from chickens with HS syndrome is not known, as only one strain of avian HEV has been sequenced thus far for its ORF2 gene. Thus, it is also important to characterize the capsid genes of additional avian HEV isolates from chickens with HS syndrome in different geographical regions.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Prospective study.
We have previously shown that avian HEV antibodies are highly prevalent not only in chicken flocks with HS syndrome but in healthy chicken flocks as well (Huang et al., 2002a). To identify genetically the virus responsible for the seropositivity in healthy chickens, we conducted a prospective study. Briefly, fourteen 12-week-old chickens were randomly selected from three healthy chicken flocks in a commercial farm in Virginia that had previously tested positive for avian HEV antibody (Huang et al., 2002a
). Each of the 14 chickens was tagged and mixed with other chickens in the same flock. The 14 study chickens were housed and raised under the same natural conditions as the other chickens in the flocks. Weekly or biweekly faecal swabs and serum samples were collected from the 14 chickens for 19 weeks and the chickens were 30 weeks of age at the end of the prospective study. Both the serum and faecal samples were tested by RT-PCR for avian HEV RNA and the serum samples were also tested by an ELISA for avian HEV antibody.
Primer design.
Primers used for genetic identification and characterization of avian HEV isolates were designed from the helicase gene region in ORF1 as well as from the ORF2 gene region based on multiple sequence alignments of the prototype avian HEV and other HEV isolates (Haqshenas et al., 2001; Huang et al., 2002a
; Sun et al., 2003
). For amplification of the helicase gene region, two nested sets of degenerate primers were used: external primer set AHEV F-1/SD, 5'-TGTTATT(C)ACACCCACCAAG(A)ACGT(C)TG-3', and Helic R, 5'-CCTCA(G)TGGACCGTA(T)ATCGACCC-3'; and internal primer set AHEV F-2/SD, 5'-GCCACGGCTG(A)TTACACCC(T)CAC(T)GT-3', and Helic R-2, 5'-GACCCA(G)GGA(G)TTCGACTGCTT-3'. The sizes of expected PCR products for the first and second rounds were 452 bp and 386 bp, respectively.
For amplification of the ORF2 capsid gene region, two nested sets of degenerate primers were used: external primer set AHEV ORF2/F-1/SD, 5'-TCGCCT(C)GGTAAT(C)ACA(T)AATGC-3', and AHEV ORF2/R-1/SD, 5'-GCGTTC(G)CCG(C)ACAGGT(C)CGGCC-3'; and internal primer set AHEV ORF2/F-2/SD, 5'-ACA(T)AATGCT(C)AGGGTCACCCG-3', and AHEV ORF2/R-2/SD, 5'-ATGTACTGA(G)CCA(G)CTG(C)GCCGC-3'. The sizes of the expected PCR products for the first and second rounds were 278 bp and 242 bp, respectively.
RT-PCR.
RNA was extracted with TriReagent (Molecular Research Center, Inc.) from 100 µl chicken faecal, bile or serum samples. Total RNA was resuspended in 12·25 µl DNase-free, RNase-free and proteinase-free water. Reverse transcription was performed at 42 °C for 60 min in the presence of a master mix consisting of 12·25 µl total RNA, 0·25 µl Superscript II reverse transcriptase (Invitrogen), 1 µl 10 µM antisense primer, 0·5 µl RNase inhibitor, 1 µl 0·1 M dithiothreitol, 4 µl 5x RT buffer and 1 µl 10 mM dNTPs. The resulting cDNA was amplified by PCR with appropriate primers and AmpliTaq Gold DNA polymerase (Applied Biosystems). The PCR reaction parameters consisted of an initial incubation at 95 °C for 9 min, followed by 39 cycles of 94 °C for 1 min, 42 °C for 1 min and 72 °C for 1·5 min, with a final incubation period at 72 °C for 7 min. The PCR products were examined on a 0·8 % agarose gel.
ELISA to detect avian HEV antibody in chickens.
A truncated recombinant ORF2 capsid protein of avian HEV was expressed in Escherichia coli and purified by affinity chromatography using the BugBuster His-Bind Purification Kit (Novagen) (Haqshenas et al., 2002). The purified protein was used as an antigen to standardize an ELISA to detect avian HEV antibodies in chickens, as reported previously (Huang et al., 2002a
). Sera from specific-pathogen-free (SPF) chickens were used as negative controls and convalescent sera from SPF chickens experimentally infected with avian HEV were included as positive controls. All sera were tested at least twice.
Sequence and phylogenetic analyses.
The PCR products were excised from a 0·8 % agarose gel, purified using the Geneclean III kit (Qbiogene, BIO 101 Systems) and directly sequenced at the Virginia Bioinformatics Institute Core Laboratory Facility with an automated DNA sequencer.
The PCR primer sequences were excluded from the resulting sequences. Only 269 bp of the resulting 386 bp partial helicase gene and 172 bp of the resulting 242 bp partial ORF2 gene sequences of these avian HEV isolates were used for comparison with the available corresponding regions of BLSV and swine and human HEVs. The sequences of avian HEV isolates from a healthy chicken and 14 avian HEV isolates from chickens with HS syndrome were analysed and compared with the corresponding regions of the prototype avian HEV isolate, BLSV and selected strains of swine and human HEVs by the MacVector computer program (Oxford Molecular Inc.).
Phylogenetic analyses were conducted with the aid of the PAUP program (David L. Swofford, Smithsonian Institute, Washington, DC). The branch-and-bound search and mid-point rooting options with 1000 replicates were used to generate the phylogenetic trees. Phylogenetic analyses were performed on two different genomic regions: a 269 bp fragment of the ORF1 helicase gene, for which the sequence of BLSV is also known, and a 172 bp fragment of the ORF2 capsid gene.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Sequence analysis revealed that the sequences of the helicase gene region of the avian HEV isolates recovered from faecal and serum samples of the same healthy chicken (2553) in three different weeks were identical (data not shown). However, they shared 86 % nucleotide sequence identity with the prototype avian HEV and 7997 % sequence identity with isolates recovered from chickens with HS syndrome. They also shared 75 % identity with the Australian chicken BLSV and 5154 % sequence identity with selected known representative strains of swine and human HEVs (data not shown).
Sequence analysis based on the 172 bp fragment of the ORF2 gene revealed similar results. The ORF2 gene sequences of the isolates recovered from the faecal and serum samples of the same healthy chicken in three different weeks (2553-26F, 2553-26S, 2553-27F, 2553-27S, 2553-28F and 2553-28S) were 99100 % identical (Table 2). They shared 90 % nucleotide sequence identity with the prototype avian HEV and 7595 % sequence identity with avian HEV isolates from chickens with HS syndrome. They also shared 4751 % sequence identity with selected known representative strains of swine and human HEVs (Table 2
).
|
|
Phylogenetic analysis of the ORF2 gene region revealed that avian HEV isolates from chickens with HS syndrome in different geographical regions are heterogeneic. Minor branches, indicating heterogeneity, exist among avian HEV isolates regardless of their geographical origins. Avian HEV isolates from both a healthy chicken and from chickens with HS syndrome are more distantly related to BLSV (Fig. 2).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The data from the prospective study confirmed our previous seroepidemiological study showing that avian HEV is enzootic in chicken flocks (Huang et al., 2002a) and provided convincing evidence that subclinical avian HEV infection occurs in the majority of chickens in the United States. We demonstrated that, under natural conditions, chickens become infected at approximately 34 months of age. We speculate that the nature of subclinical infection in the majority of chickens is due to the relatively low doses of virus that can be transmitted among chickens via the faecaloral route. It has been documented that human HEV infection in primates is dose-dependent: primates that received higher doses of human HEV developed biochemical and virological evidence of hepatitis, whereas primates that received lower doses had only subclinical infection, as evidenced by seroconversion to anti-HEV antibodies (Tsarev et al., 1994
). This dose-dependent hypothesis may explain why there are only sporadic cases of HS syndrome in chickens, even though most chickens in the United States are infected with avian HEV (Huang et al., 2002a).
Sequence and phylogenetic analyses revealed that the capsid gene sequences of avian HEV isolates from chickens with HS syndrome in four different states were heterogeneic, regardless of their geographical origins. This observation is consistent with reports on swine and human HEVs, which are also heterogeneic. Sequence analyses based on the ORF1 helicase gene and ORF2 capsid gene regions showed that the sequences of the avian HEV isolates recovered from a healthy chicken in three different weeks were nearly identical. Phylogenetic analysis also revealed that the avian HEV isolates recovered from a healthy chicken clustered together and were genetically related to, but different from, the prototype avian HEV and avian HEV isolates recovered from chickens with HS syndrome.
In summary, the genetic identification of avian HEV isolates from a healthy chicken farm and the demonstration of subclinical avian HEV infection in chicken flocks further complicates the causal relationship between avian HEV infection and HS syndrome in chickens. Although we believe that avian HEV infection is dose-dependent and that only chickens infected with higher doses of the virus develop HS syndrome, we cannot rule out the possibility that the avian HEV isolate identified from healthy chickens may represent an avirulent strain and that the subclinical infections may be caused by an avirulent strain of avian HEV. Further studies are warranted to fully characterize this avian HEV isolate recovered from a healthy chicken, both experimentally and genetically.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 34283432.[Abstract]
Chandler, J. D., Riddell, M. A., Li, F., Love, R. J. & Anderson, D. A. (1999). Serological evidence for swine hepatitis E virus infection in Australian pig herds. Vet Microbiol 68, 95105.[CrossRef][Medline]
Clemente-Casares, P., Pina, S., Buti, M., Jardi, R., Martin, M., Bofill-Mas, S. & Girones, R. (2003). Hepatitis E virus epidemiology in industrialized countries. Emerg Infect Dis 9, 448454.[Medline]
Emerson, S. U., Zhang, M., Meng, X. J., Nguyen, H., Huang, Y. & 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 U S A 98, 1527015275.
Erker, J. C., Desai, S. M., Schlauder, G. G., Dawson, G. J. & Mushahwar, I. K. (1999). A hepatitis E virus variant from the United States: molecular characterization and transmission in cynomolgus macaques. J Gen Virol 80, 681690.[Abstract]
Favorov, M. O., Kosoy, M. Y., Tsarev, S. A., Childs, J. E. & Margolis, H. S. (2000). Prevalence of antibody to hepatitis E virus among rodents in the United States. J Infect Dis 184, 449455.
Garkavenko, O., Obriadina, A., Meng, J., Anderson, D. A., Benard, H. J., Schroeder, B. A., Khudaakov, Y. E., Fields, H. A. & Croxson, M. C. (2001). Detection and characterization of swine hepatitis E virus in New Zealand. J Med Virol 65, 525529.[CrossRef][Medline]
Halbur, P. G., Kasorndorkbua, C., Gilbert, C., Guenette, D., Potters, M. B., Purcell, R. H., Emerson, S. U., Toth, T. E. & Meng, X. J. (2001). Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human. J Clin Microbiol 39, 918923.
Haqshenas, G. & Meng, X. J. (2001). Determination of the nucleotide sequences at the extreme 5' and 3' ends of swine hepatitis E virus genome. Arch Virol 146, 24612467.[CrossRef][Medline]
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 hepatitissplenomegaly syndrome in the United States. J Gen Virol 82, 24492462.
Haqshenas, G., Huang, F. F., Fenaux, M., Guenette, D. K., Pierson, F. W., Larsen, C. T., Shivaprasad, H. L., Toth, T. E. & Meng, X. J. (2002). The putative capsid protein of the newly identified avian hepatitis E virus shares antigenic epitopes with that of swine and human hepatitis E viruses and chicken big liver and spleen disease virus. J Gen Virol 83, 22012209.
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, 38283834.
Huang, C. C., Nguyen, D., Fernandez, J., Yun, K. Y., Fry, K. E., Bradley, D. W., Tam, A. W. & Reyes, G. R. (1992). Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV). Virology 191, 550558.[Medline]
Huang, F. F., Haqshenas, G., Shivaprasad, H. L. & 7 other authors (2002a). Heterogeneity and seroprevalence of the newly identified avian hepatitis E virus from chickens in the United States. J Clin Microbiol 40, 41974202.
Huang, F. F., Haqshenas, G., Guenette, D. K., Halbur, P. G., Schommer, S., Pierson, F. W., Toth, T. E. & Meng, X. J. (2002b). Detection by RT-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the US. J Clin Microbiol 40, 13261332.
Hussaini, S. H., Skidmore, S. J., Richardson, P., Sherratt, L. M., Cooper, B. T. & O'Grady, J. G. (1997). Severe hepatitis E infection during pregnancy. J Viral Hepat 4, 5154.[CrossRef][Medline]
Kabrane-Lazizi, Y., Fine, J. B., Elm, J. & 7 other authors (1999). Evidence for wide-spread infection of wild rats with hepatitis E virus in the United States. Am J Trop Med Hyg 61, 331335.
Kasorndorkbua, C., Halbur, P. G., Guenette, D. K., Toth, T. E. & Meng, X. J. (2002). Use of a swine bioassay and a RT-PCR assay to assess the risk of transmission of swine hepatitis E virus in pigs. J Virol Methods 101, 7178.[CrossRef][Medline]
McCrudden, R., O'Connell, S., Farrant, T., Beaton, S., Iredale, J. P. & Fine, D. (2000). Sporadic acute hepatitis E in the United Kingdom: an underdiagnosed phenomenon? Gut 46, 732733.
Mast, E. E., Kuramoto, I. K., Favorov, M. O., Schoening, V. R., Burkholder, B. T., Shapiro, C. N. & Holland, P. V. (1997). Prevalence of and risk factors for antibody to hepatitis E virus seroreactivity among blood donors in Northern California. J Infect Dis 176, 3440.[Medline]
Meng, X. J. (2000a). Zoonotic and xenozoonotic risks of hepatitis E virus. Infect Dis Rev 2, 3541.
Meng, X. J. (2000b). Novel strains of hepatitis E virus identified from humans and other animal species: is hepatitis E a zoonosis? J Hepatol 33, 842845.[CrossRef][Medline]
Meng, X. J. (2003). Swine hepatitis E virus: cross-species infection and risk in xenotransplantation. Curr Top Microbiol Immunol 278, 185216.[Medline]
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, 98609865.
Meng, X. J., Halbur, P. G., Shapiro, M. S., Govindarajan, S., Bruna, J. D., Mushahwar, I. K., Purcell, R. H. & Emerson, S. U. (1998). Genetic and experimental evidence for cross-species infection by the swine hepatitis E virus. J Virol 72, 97149721.
Meng, X. J., Dea, S., Engle, R. E. & 10 other authors (1999). Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population. J Med Virol 58, 297302.[CrossRef]
Meng, X. J., Wiseman, B., Elvinger, F., Guenette, D. K., Toth, T. E., Engle, R. E., Emerson, S. U. & Purcell, R. H. (2002). Prevalence of antibodies to the hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries. J Clin Microbiol 40, 117122.
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, 12451251.
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, 929936.[CrossRef][Medline]
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, 119125.[CrossRef][Medline]
Pina, S., Buti, M., Cotrina, M., Piella, J. & Girones, R. (2000). HEV identified in serum from humans with acute hepatitis and in sewage of animal origin in Spain. J Hepatol 33, 826833.[CrossRef][Medline]
Purcell, R. H. (1996). Hepatitis E virus. In Field Virology, 3rd edn, vol. 2, pp. 28312843. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Reyes, G. R. (1997). Overview of the epidemiology and biology of the hepatitis E virus. In Viral Hepatitis, pp. 239258. Edited by R. A. Willson. New York: Marcel Dekker.
Riddell, C. (1997). Hepatitissplenomegaly syndrome. In Diseases of Poultry, p. 1041. Edited by B. W. Calnek, H. J. Barnes, C. W. Beard, L. R. McDougald & Y. M. Saif. Ames, IA: Iowa State University Press.
Ritchie, S. J. & Riddell, C. (1991). "Hepatitissplenomegaly" syndrome in commercial egg laying hens. Can Vet J 32, 500501.
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, 447456.[Abstract]
Schlauder, G. G., Desai, S. M., Zanetti, A. R., Tassopoulos, N. C. & Mushahwar, I. K. (1999). Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV. J Med Virol 57, 243251.[CrossRef][Medline]
Shivaprasad, H. L. & Woolcock, P. R. (1995). Necrohemorrhagic hepatitis in broiler breeders. In Proceedings of the 44th Western Poultry Diseases Conference, 57 March, 1995, p. 6. Sacramento, CA, USA.
Sun, Z. F., Huang, F. F., Halbur, P. G., Schommer, S. K., Pierson, F. W., Toth, T. E. & Meng, X. J. (2003). Use of heteroduplex mobility assays (HMA) for pre-sequencing screening and identification of variant strains of avian and swine hepatitis E viruses. Vet Microbiol 96, 165176.[CrossRef][Medline]
Takahashi, K., Iwata, K., Watanabe, N., Hatahara, T., Ohta, Y., Baba, K. & Mishiro, S. (2001). Full-genome nucleotide sequence of a hepatitis E virus strain that may be indigenous to Japan. Virology 287, 912.[CrossRef][Medline]
Takahashi, K., Kang, J. H., Ohnishi, S. & Mishiro, S. (2002). Genetic heterogeneity of hepatitis E virus recovered from Japanese patients with acute sporadic hepatitis. J Infect Dis 185, 13421345.[CrossRef][Medline]
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, 851862.
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, 13421343.
Tei, S., Kitajima, N., Takahashi, K. & Mishiro, S. (2003). Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 362, 371373.[CrossRef][Medline]
Thomas, D. L., Yarbough, P. O., Vlahov, D., Tsarev, S. A., Nelson, K. E., Saah, A. J. & Purcell, R. H. (1997). Seroreactivity to hepatitis E virus in areas where the disease is not endemic. J Clin Microbiol 35, 12441247.[Abstract]
Tsarev, S. A., Emerson, S. U., Reyes, G. R., Tsareva, T. S., Legters, L. J., Malik, I. A., Iqbal, M. & Purcell, R. H. (1992). Characterization of a prototype strain of hepatitis E virus. Proc Natl Acad Sci U S A 89, 559563.[Abstract]
Tsarev, S. A., Tsareva, T. S., Emerson, S. U., Yarbough, P. O., Legters, L. J., Moskal, T. & Purcell, R. H. (1994). Infectivity titration of a prototype strain of hepatitis E virus in cynomolgus monkeys. J Med Virol 43, 135142.[Medline]
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, 970976.[Medline]
Wang, Y., Ling, R., Erker, J. C., Zhang, H., Li, H., Desai, S., Mushahwar, I. K. & Harrison, T. J. (1999). A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis. J Gen Virol 80, 169177.[Abstract]
Wang, Y., Zhang, H., Ling, R., Li, H. & Harrison, T. J. (2000). The complete sequence of hepatitis E virus genotype 4 reveals an alternative strategy for translation of open reading frames 2 and 3. J Gen Virol 81, 16751686.
Wang, Y. C., Zhang, H. Y., Xia, N. S. & 11 other authors (2002). Prevalence, isolation and partial sequence analysis of hepatitis E virus from domestic animals in China. J Med Virol 67, 516521.[CrossRef][Medline]
Williams, T. P. E., Kasorndorkbua, C., Halbur, P. G., Haqshenas, G., Guenette, D. K., Toth, T. E. & Meng, X. J. (2001). Evidence of extrahepatic sites of replication of the hepatitis E virus in a swine model. J Clin Microbiol 39, 30403046.
Wu, J. C., Chen, C. M., Chiang, T. Y., Tsai, W. H., Jeng, W. J., Sheen, I. J., Lin, C. C. & Meng, X. J. (2002). Spread of hepatitis E virus among different-aged pigs: two-year survey in Taiwan. J Med Virol 66, 488492.[CrossRef][Medline]
Zanetti, A. R., Schlauder, G. G., Romano, L., Tanzi, E., Fabris, P., Dawson, G. J. & Mushahwar, I. K. (1999). Identification of a novel variant of hepatitis E virus in Italy. J Med Virol 57, 356360.[CrossRef][Medline]
Received 18 August 2003;
accepted 7 November 2003.