Department of Medicine, Royal Free and University College Medical School, University College London, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK1
Department of Hepatitis, National Institute for the Control of Pharmaceuticals and Biological Products, Temple of Heaven, Beijing, PR China2
Author for correspondence: Tim Harrison. Fax +44 20 7433 2852. e-mail t.harrison{at}rfc.ucl.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The first complete genomic sequence was derived from virus implicated in an epidemic of hepatitis E in Burma (Reyes et al., 1990 ; Tam et al., 1991
). The genome is a positive-sense, polyadenylated RNA molecule of around 7500 nt. The largest open reading frame (ORF 1), located at the 5' end of the genome, is believed to encode a non-structural polyprotein which contains motifs recognizable as consensus elements of methyl transferase, protease, helicase and RNA-dependent RNA polymerase activities (Koonin et al., 1992
). ORF 2 is located at the 3' end of the genome and is believed to encode the major capsid protein. Unusually for a non-enveloped virus, the predicted polypeptide has a signal sequence at the amino terminus and sites for N-linked glycosylation. The short ORF 3 overlaps the other two and encodes a polypeptide of uncertain function.
Complete and partial nucleotide sequences have been determined for many HEV isolates. Complete viral sequences from Pakistan (Tsarev et al., 1992 ), China (Bi et al., 1993
; Yin et al., 1994
) and India (Donati et al., 1997
; Panda et al., 1995
) and partial sequences of isolates from Africa and the Asian republics of the former Soviet Union (Chatterjee et al., 1997
) have high identity (>90% nucleotide identity) to the Burmese prototype. In contrast, the sequence of a virus implicated as the cause of an epidemic of hepatitis E in Mexico (Huang et al., 1992
) shares less than 77% identity with Burmese-like viruses.
The concept of Old World and New World hepatitis E viruses was eclipsed by the discovery of a third genotype infecting pigs (Meng et al., 1997 ), and causing sporadic cases of acute hepatitis in humans (Kwo et al., 1997
), in the United States. Complete sequences of the US genotype have been reported (Schlauder et al., 1998
) and, although these are distinct from the Burmese-like group and the single isolate from Mexico, they share all of the characteristic features of the HEV genome. We reported recently that some isolates of HEV from China are distinct from the Burmese-like (genotype 1) viruses known to be endemic in that country and constitute a fourth genotype (Wang et al., 1999
). Recent reports of further divergent HEV sequences from Italy and Greece suggest that the virus is also present in Europe and that further genotypes may exist (Schlauder et al., 1999
).
The diagnostic assays for anti-HEV antibodies, which are commercially available, are based on recombinant proteins or synthetic peptides derived from ORFs 2 and 3 of the Burmese and Mexican genotypes (Yarbough et al., 1991 ; Dawson et al., 1992
). We failed to detect anti-HEV in sera from patients infected with genotype 4 HEV although, in some instances, the acute phase samples may have been taken prior to the development of detectable levels of antibody (Wang et al., 1999
). The availability of complete ORF 2 and 3 sequences from genotype 4 HEV should enable evaluation of sequence variation in the regions critical for antibody assays and help determine whether it is necessary to modify current assays to detect antibodies to this new genotype.
Here, we report the entire nucleotide sequence of a representative of the Chinese genotype of HEV. Typical of genetic variation in HEV, many of the variant nucleotides occur in the third base of the codons, so that the predicted amino acid sequences remain conserved. However, the Chinese genotype has a single base insertion which is likely to affect the translation of the ORF 2 and ORF 3 proteins. This insertion was confirmed in an independent isolate. The implications of this unique feature of the Chinese genotype of HEV are discussed.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
As described below, isolate HEV-T1 was found to have a single base insertion affecting the translational strategy of ORFs 2 and 3. In order to determine whether this change is present in other genotype 4 viruses, viral cDNA was amplified from a second stool sample and identified as genotype 4 using the above criteria. This sample, designated T11, was collected 2 days after the onset of jaundice in a 39-year-old male with acute hepatitis. Both cases of hepatitis were sporadic (community acquired) and were not associated with an epidemic.
Extraction of RNA.
One tenth volume of 10x PBS was added to the stool samples and the suspensions were mixed thoroughly and clarified by centrifugation at 3000 r.p.m. at room temperature for 10 min. The supernatants were stored at -20 °C for RNA extraction. HEV RNA was extracted from 560 µl stool suspension using a QIAamp Viral RNA kit (QIAGEN) and the manufacturers protocol for large sample volumes.
PCR amplification.
Primers for the amplification of the Chinese genotype of HEV, and for 5' and 3' RACE, are listed in Table 1. Some primers were based on sequences conserved between the Burmese and Mexican genotypes, others were designed on the basis of sequence information from the T1 isolate. cDNA was synthesized from 9 µl purified RNA using AMV or superscript RT and the outer, antisense PCR primer. First-round PCRs were carried out in a 50 µl reaction using 10 µl cDNA, 25 pmol of each outer primer, 5 µl 10x PCR buffer, 2 mM MgCl2 and 5 U Taq polymerase. Second-round reactions were carried out in 50 µl volumes with 5 µl first-round product, 25 pmol of each inner primer and 5 U Taq polymerase.
|
RACE.
To amplify the 5' end, first-strand cDNA was synthesized using primer E1EO (Table 1). First-strand cDNA (20 µl) was treated with 1 µl RNase H (Promega) at 37 °C for 30 min and purified using QIAEX II (QIAGEN) according to the manufacturers instructions, yielding 20 µl purified cDNA. Purified cDNA (10 µl) was used in each of two homopolymer tailing reactions: 20 µM dATP or dGTP and 20 U terminal deoxynucleotidyl transferase (Promega), using the buffer supplied by the manufacturer, and incubated at 37 °C for 30 min. The tailed cDNA was purified using a QIAEX II kit (QIAGEN) and amplified in the first-round PCR with primers E1EO and R2 (dG-tailed) or E1EO and R1 (dA-tailed). Five microlitres of each first-round PCR product was amplified further in second-round, semi-nested PCR with primers E1EI and R2 or R1.
To amplify the 3' end, first-strand cDNA was synthesized with primer R2 in a 20 µl reaction. First-strand cDNA (10 µl) was used in a first-round PCR with primers E2BO and R1 and 5 µl first-round product was used in a second-round PCR (semi-nested) with primers E2BI and R1.
Cloning and sequencing of amplicons.
The products of PCR amplification and RACE were cloned into pGEM-T (Promega) or pCRII (Invitrogen). Recombinant plasmids were purified and the inserts were sequenced, either manually, using Sequenase (version 2.0; Amersham Pharma Biotec), or using an ABI Prism dRhodamine terminator cycle sequencing ready reaction kit (PE Applied Biosystems) and an ABI Prism 310 genetic analyser. The complete sequence of the T1 isolate has been deposited in the EMBL and GenBank nucleotide databases (accession no. AJ272108).
Phylogenetic analysis.
The nucleotide sequences were aligned using PILEUP and compared using GAP (Wisconsin Sequence Analysis Package; Genetics Computer Group, Madison, Wisconsin, version 9.0) or Clustal X from the European Bioinformatics Institute (EBI). These alignments were analysed using the DNADIST program of PHYLIP (version 3.5c; Felsenstein, 1993 ) or Clustal X to calculate the evolutionary distances between sequences.
The following full-length HEV sequences were used for analysis. Genotype 1: Burmese prototype [B1, accession no. M73218 (Tam et al., 1991 )]; Burmese isolate [B2, D10330 (Tam et al., 1991
; Aye et al., 1993
)]; Pakistan isolate [P1, M80581 (Tsarev et al., 1992
)]; Chinese isolates [C1, L25547 (Bi et al., 1993
), C2, M94177 (S. R. Yin and others, unpublished results) and C3, D11093 (T. Uchida and others, unpublished results)]; Indian isolates [I1, X98292 (Donati et al., 1997
) and I2, X99441 (A. Von Brunn and others, unpublished results)]. Genotype 2: the Mexican prototype [M1, M74506 (Huang et al., 1992
)]. Genotype 3: US isolates [US1 and US2, AF060668 and AF060669, respectively (Schlauder et al., 1998
)].
![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCR products were cloned into pGEM-T or pCR2.1 and two or three clones were sequenced in each direction, either manually using the -40 or reverse sequencing primers and Sequenase version 2.0, or automatically using an ABI model 310 DNA sequencer and the ABI ready reaction sequencing kit. The full-length sequence was assembled with removal of overlaps between adjacent PCR products. If the identity within each overlapping region failed to reach 99%, the amplification, cloning and sequencing were repeated.
Genome organization of genotype 4 HEV
The HEV-T1 genome comprises 7232 nt, excluding the 3' poly(A) tail (Table 2). The 5' nontranslated region (NTR) comprises 25 bases and the 3' NTR, 68 bases. The presence of three major ORFs was confirmed. ORF 1 begins at nt 26 and ends at nt 5146 (5121 nt in length) and potentially encodes a product of 1707 aa. ORF 2 (nt 51467161) comprises 2016 nt and encodes 672 aa. The predicted size of ORF 2 for HEV-T1 is 14 codons longer than for other isolates in the sequence databases. ORF 1 and ORF 2 in HEV-T1 overlap by one base, whereas the ORF 2 of other isolates begins 41 bases downstream of ORF 1. This variation is caused by the insertion of a single nucleotide (U) at position nt 5159. This insertion also affects ORF 3, which starts at nt 5174 and ends at nt 5509 (336 nt), and potentially encodes a polypeptide of 112 aa. ORF 3 of HEV-T1 starts 28 bases downstream of ORF 1, whereas ORF 1 and ORF 3 in other isolates reportedly overlap by one base.
|
|
To date, full-length sequences for three genotypes of HEV have been deposited in the nucleotide sequence databases. The Burmese-like isolates are considered as genotype 1, the single Mexican isolate, genotype 2, and the US isolates, genotype 3 (Wang et al., 1999 ). The nucleotide identities, based on full-length sequences, are approximately 75·0% between genotypes 1 and 2, 74·0% between genotypes 1 and 3, and 74·0% between genotypes 2 and 3. HEV-T1, as a representative of genotype 4, shows 74·575·8% identity to the Burmese-like isolates, 74·5% to the Mexican isolate and 75·376·3% to the US isolates (Table 3
). These comparisons support our hypothesis that T1 and related viruses constitute a novel genotype of HEV.
|
The length of the 3' NTR of HEV-T1, at 68 nt, is similar to that of other isolates (6574 nt), with the exception of the Pakistan isolate (Table 2). The sequence of the 3' NTR is highly variable with some deletions, although these are not located in the same positions in the various isolates. There is more than 92% identity between Burmese-like isolates in the overlapping region. HEV-T1 has 7579%, 77% and 61% identity to the Burmese-like isolates, the Mexican and US strains, respectively, whereas Burmese-like isolates have 7072% and 7276% identity to the Mexican and US strains, respectively.
Analysis of ORF 1
The predicted ORF 1 polypeptide of HEV-T1 is 1708 aa. The HEV-T1 polypeptide has 85·486·7%, 85% and 88·588·7% identity, at the amino acid level, to the Burmese-like, Mexican and US strains, respectively, whereas the ORF 1 polypeptides of Burmese-like strains have more than 95% identity to each other, 81·583·0% identity to the US strains and 81·884·2% identity to the Mexican strain (Table 4). The HEV-T1 ORF 1 polyprotein is 1416 aa longer than the Burmese-like and Mexican strains, 9 aa longer than HEV-US1 and 1 aa shorter than HEV-US2. The variability in ORF 1 length was mostly because of insertions within the hypervariable region (nt 21452376). This region was amplified independently with two different primer sets. The overlapping sequences of the two PCR products covered the entire length of the hypervariable region and were identical.
|
Analysis of ORF 2
The HEV-T1 ORF 2 product is 672 aa in length and is most highly conserved. The HEV-T1 ORF 2 is 91·692·4%, 91·993% and 90·1% identical at the amino acid level to Burmese-like, US and Mexican strains, respectively, whereas the identity between the HEV-US and Mexican strains is approximately 90% (Table 5). Within genotypes, the identity between two isolates within the Burmese genotype is more than 98% and, between US1 and US2, is 98%.
|
Analysis of ORF 3
The HEV-T1 ORF 3 is 336 nt long and encodes 112 aa. Because of the single nucleotide (U) insertion at position nt 5160, the likely initiation codon of ORF 3 in HEV-T1 is 28 nt downstream of that predicted for HEV isolates described previously and the ORF 3 in HEV-T1 is shorter than in other HEV strains. The overlapping regions in ORF 3 of HEV-T1 and other isolates were aligned and the identity was calculated using the Clustal X program. The ORF 3 of HEV-T1 was 83·383·9%, 83·1% and 85·687·1% identical at the nucleotide level to Burmese-like, Mexican and HEV-US strains, respectively, whereas the Burmese-like isolates were 86·287% and 89·590·9% identical to the HEV-US and Mexican isolates within this region (Table 6).
|
Comparison with partial ORF 1 sequences of other HEV variants
Partial sequences have been reported of two HEV isolates (G9 and G20) from Guangzhou province, China (Huang et al., 1995 ) and of three isolates (TW4, TW7 and TW8) from Taiwan (Wu et al., 1998
). Analysis of 196 nt within the RNA-dependent RNA polymerase region of ORF 1 suggests that these isolates are distinct from the Burmese-like and Mexican strains (Wu et al., 1998
). These sequences also are distinct from the HEV-US1 and HEV-US2 viruses (Schlauder et al., 1998
). Seventeen sequences of 196 nt, within the RNA-dependent RNA polymerase region of ORF 1, were aligned using the Clustal X program. HEV-T1 has 7583% identity to these other sequences at the nucleotide level. The identity between TW4 and TW8 is 98%. TW7 has 8790% identity to G9 and G20, whereas it has 78%, 84% and 84% identity to HEV-T1, TW4 and TW8, respectively. Genetic distances were also calculated with DNADIST. The genetic distance between TW4 and TW8 was 0·0153 and the distances between TW4 and TW7, G20, G9 and HEV-T1 were more than 0·150. The distances of TW7 to G9 and G20 were 0·122 and 0·0918, respectively. The distance of HEV-T1 to the five variants was also more than 0·150. In addition, four short sequences, also reported from Taiwan (Hsieh et al., 1998
), show approximately 88% nucleotide identity to T1 whereas, over the same short stretch of sequence, T1 has 89% identity to S15 (genotype 4; Wang et al., 1999
) and 83% or less to other HEV genotypes, including those reported recently from Europe (Schlauder et al., 1999
). HEV variants isolated from China, and which do not fall into genotype 1 (Burmese-like) may, therefore, be divided into at least three groups. T1 and related viruses, including those reported by Hsieh et al. (1998)
, form the first group (genotype 4), TW4 and TW8 form the second and TW7, G9 and G20 the third, although Wu et al. (1998)
consider that G9 and G20 belong to distinct groups. Thus, there seem to be at least two further genotypes of HEV in China. It is not clear whether these may be related to new genotypes identified recently in Europe (Schlauder et al., 1999
).
Comparison with partial ORF 2 sequences of other HEV variants
Recently, three other variants of HEV were reported from Chinese patients with acute hepatitis, T21 (accession no. AF151963; Yang et al., 2000 ) and LZ-105 and HF-044 (accession nos AF103940 and AF134812; Li et al., 2000
). In order to investigate whether these three isolates also belonged to genotype 4, 16 partial ORF 2 sequences were aligned using Clustal X. HEV-T1 was 85%, 85%, 87% and 88% identical to T21, T11, LZ-105 and HF-044, respectively, and 7678%, 74% and 7779% identical to Burmese-like, Mexican and US isolates, respectively. The genetic distances were also calculated with DNADIST: the distances of HEV-T1 to T21, HF-044, T11 and LZ-105 were 0·150, 0·147, 0·123 and 0·120, respectively (all equal to or less than 0·150), whereas the distances of HEV-T1 to Burmese-like, Mexican and US isolates were 0·2270·250, 0·215 and 0·2130·227, respectively (all greater than 0·150). An unrooted tree was drawn using Treeview and the variants T21, T11, LZ-105 and HF-044 clustered with HEV-T1. All the results indicate that these four variants belong to genotype 4 with HEV-T1.
Diagnosis of HEV genotype 4 infections
The question as to whether current diagnostic assays require modification to detect genotype 4 HEV infections must be answered experimentally using relevant convalescent sera. However, some insight may be gained by comparing the genotype 4 amino acid sequences with the prototype strains. Table 7(a) shows the immunoreactive domains 4-2 (ORF 3) and 3-2 (ORF 2) identified by Yarbough et al. (1991)
and which constitute the basis of commercially available antibody assays. With the exception of the amino-terminal end of 4-2, T1 shows considerable conservation, particularly with genotype 2 (recombinant proteins from genotypes 1 and 2 are included in the antibody assays). Linear, immunoreactive epitopes which have been identified in the ORF 2 protein are located predominantly in the carboxyl-terminal region. Peptide sequences identified by Kaur et al. (1992)
and Khudyakov et al. (1994a
, b
) are shown in Table 7
(b
and c
), respectively. With the exception of the single epitope in the amino-terminal region of ORF 2, T1 again seems well conserved with the other genotypes. Current antibody assays may be suitable for the detection of IgG responses to genotype 4 infections but this will require confirmation using validated sera. Serological tests for HEV show poor concordance and those based on synthetic peptides, in particular, may be unreliable (Mast et al., 1998
).
|
Since the submission of this paper, Buisson et al. (2000) have reported HEV isolates from Nigeria that fall into the Mexican genotype but were unable to compare these sequences with genotype 4. Pairwise comparisons using GAP (GCG10) revealed 76·9% nucleotide identity between T1 and Nigerian isolates 1, 4, 5, 6 and 7 (accession nos AF172999, AF17300, AF17301, AF173230 and AF173231, respectively) and 76·0% between T1 and Nigerian isolate 9 (AF173232), in the ORF 2 region. These data are consistent with the assignment of T1 and these Nigerian isolates to separate genotypes.
In contrast to our usage of Arabic numerals, Buisson et al. (2000) use Roman numerals for the four genotypes. Furthermore, they label the Mexican and US genotypes III and II (our 2 and 3). Such inconstancies require resolution and agreement with the International Committee on Taxonomy of Viruses (ICTV).
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bi, S. L., Purdy, M. A., McCaustland, K. A., Margolis, H. S. & Bradley, D. W. (1993). The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China. Virus Research 28, 233-247.[Medline]
Bradley, D. W. (1992). Hepatitis E: epidemiology, aetiology and molecular biology. Reviews in Medical Virology 2, 19-28.
Buisson, Y., Grandadam, M., Nicand, E., Cheval, P., van Cuyck-Gandre, H., Innis, B., Rehel, P., Coursaget, P., Teyssou, R. & Tsarev, S. (2000). Identification of a novel hepatitis E virus in Nigeria. Journal of General Virology 81, 903-909.
Chatterjee, R., Tsarev, S., Pillot, J., Coursaget, P., Emerson, S. U. & Purcell, R. H. (1997). African strains of hepatitis E virus that are distinct from Asian strains. Journal of Medical Virology 53, 139-144.[Medline]
Clayson, E. T., Innis, B. L., Myint, K. S., Narupiti, S., Vaughn, D. W., Giri, S., Ranabhat, P. & Shrestha, M. P. (1995). Detection of hepatitis E virus infections among domestic swine in the Kathmandu Valley of Nepal. American Journal of Tropical Medicine and Hygiene 53, 228-232.[Medline]
Dawson, G. J., Chau, K. H., Cabal, C. M., Yarbough, P. O., Reyes, G. R. & Mushahwar, I. K. (1992). Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides. Journal of Virological Methods 38, 175-186.[Medline]
Donati, M. C., Fagan, E. A. & Harrison, T. J. (1997). Sequence analysis of full length HEV clones derived directly from human liver in fulminant hepatitis E. In Viral Hepatitis and Liver Disease, pp. 313-316. Edited by M. Rizzetto, R. H. Purcell, J. L. Gerin & G. Verme. Torino: Edizioni Minerva Medica.
Felsenstein, J. (1993). PHYLIP inference package version 3.5c. Department of Genetics, University of Washington, Seattle, WA, USA.
Harrison, T. J. (1999). Hepatitis E virus an update. Liver 19, 171-176.[Medline]
Hsieh, S. Y., Yang, P. Y., Ho, Y. P., Chu, C. M. & Liaw, Y. F. (1998). Identification of a novel strain of hepatitis E virus responsible for sporadic acute hepatitis in Taiwan. Journal of Medical Virology 55, 300-304.[Medline]
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, 550-558.[Medline]
Huang, R. T., Nakazono, N., Ishii, K., Kawamata, O., Kawaguchi, R. & Tsukada, Y. (1995). II. Existing variations on the gene structure of hepatitis E virus strains from some regions of China.Journal of Medical Virology 47, 303-308.[Medline]
Kaur, M., Hyams, K. C., Purdy, M. A., Krawczynski, K., Ching, W. M., Fry, K. E., Reyes, G. R., Bradley, D. W. & Carl, M. (1992). Human linear B-cell epitopes encoded by the hepatitis E virus include determinants in the RNA-dependent RNA polymerase. Proceedings of the National Academy of Sciences, USA 89, 3855-3858.[Abstract]
Khudyakov, Y. E., Favorov, M. O., Jue, D. L., Hine, T. K. & Fields, H. A. (1994a). Immunodominant antigenic regions in a structural protein of the hepatitis E virus. Virology 198, 390-393.[Medline]
Khudyakov, Y. E., Favorov, M. O., Khudyakova, N. S., Cong, M. E., Holloway, B. P., Padhye, N., Lambert, S. B., Jue, D. L. & Fields, H. A. (1994b). Artificial mosaic protein containing antigenic epitopes of hepatitis E virus. Journal of Virology 68, 7067-7074.[Abstract]
Koonin, E. V., Gorbalenya, A. E., Purdy, M. A., Rozanov, M. N., Reyes, G. R. & Bradley, D. W. (1992). Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis-e virus delineation of an additional group of positive-strand RNA plant and animal viruses. Proceedings of the National Academy of Sciences, USA 89, 8259-8263.[Abstract]
Kwo, P. Y., Schlauder, G. G., Carpenter, H. A., Murphy, P. J., Rosenblatt, J. E., Dawson, G. J., Mast, E. E., Krawczynski, K. & Balan, V. (1997). Acute hepatitis E by a new isolate acquired in the United States. Mayo Clinic Proceedings 72, 1133-1136.[Medline]
Li, K., Zhuang, H. & Zhu, W. (2000). Partial nucleotide sequencing of hepatitis E viruses isolated from 14 cities of China: identification of 2 major variants of hepatitis E virus. Journal of Medical Virology (in press).
Mast, E. E., Alter, M. J. & Holland, P. V. (1998). Evaluation of assays for antibody to hepatitis E virus by a serum panel. Hepatology 27, 856-861.
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. Proceedings of the National Academy of Sciences, USA 94, 9860-9865.
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 swine hepatitis E virus. Journal of Virology 72, 9714-9721.
Nielsen, H., Engelbrecht, J., von Heijne, G. & Brunak, S. (1996). Defining a similarity threshold for a functional protein sequence pattern: the signal peptide cleavage site. Proteins 24, 165-177.[Medline]
Panda, S. K., Nanda, S. K., Zafrullah, M., Ansari, I. U. H., Ozdener, M. H. & Jameel, S. (1995). An Indian strain of hepatitis E virus (HEV): cloning, sequence, and expression of structural region and antibody responses in sera from individuals from an area of high-level HEV endemicity. Journal of Clinical Microbiology 33, 2653-2659.[Abstract]
Reyes, G. R., Purdy, M. A., Kim, J. P., Luk, K. C., Young, L. M., Fry, K. E. & Bradley, D. W. (1990). Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis. Science 247, 1335-1339.[Medline]
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. Journal of General Virology 79, 447-456.[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. Journal of Medical Virology 57, 243-251.[Medline]
Tam, A. W., Smith, M. M., Guerra, M. E., Huang, C. C., Bradley, D. W., Fry, K. E. & Reyes, G. R. (1991). Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 185, 120-131.[Medline]
Torresi, J., Li, F., Locarnini, S. A. & Anderson, D. A. (1999). Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells. Journal of General Virology 80, 1185-1188.[Abstract]
Tsarev, S. A., Emerson, S. U., Reyes, G. R., Tsareva, T. S., Legters, L. J., Malik, I. A. & Purcell, R. H. (1992). Characterization of a prototype strain of hepatitis E virus. Proceedings of the National Academy of Sciences, USA 89, 559-563.[Abstract]
Wang, Y. C., Ling, R., Erker, J. C., Zhang, H. Y., Li, H. M., Desai, S., Mushahwar, I. K. & Harrison, T. J. (1999). A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis. Journal of General Virology 80, 169-177.[Abstract]
Wu, J. C., Sheen, I. J., Chiang, T. Y., Sheng, W. Y., Wang, Y. J., Chan, C. Y. & Lee, S. D. (1998). The impact of travelling to endemic areas on the spread of hepatitis E virus infection: epidemiological and molecular analyses. Hepatology 27, 1415-1420.[Medline]
Yang, J., Zhang, H., Wang, Y., Mao, Q. & Li, H. (2000). Partial sequence comparison of three sporadic hepatitis E variants. Chinese Journal of Virology (in press).
Yarbough, P. O., Tam, A. W., Fry, K. E., Krawczynski, K., McCaustland, K. A., Bradley, D. W. & Reyes, G. R. (1991). Hepatitis E virus: identification of type-common epitopes. Journal of Virology 65, 5790-5797.[Medline]
Yin, S. R., Purcell, R. H. & Emerson, S. U. (1994). A new Chinese isolate of hepatitis E virus: comparison with strains recovered from different geographical regions. Virus Genes 9, 23-32.[Medline]
Received 15 December 1999;
accepted 21 March 2000.