1 Department of Gastroenterology, National Tokyo Hospital, Kiyose, Tokyo 204-0023, Japan
2 Japanese Red Cross Saitama Blood Center, Saitama-Ken 338-0001, Japan
3 Immunology Division and Division of Molecular Virology, Jichi Medical School, Tochigi-Ken 329-0498, Japan
Correspondence
Hiroaki Okamoto
hokamoto{at}jichi.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The entire nucleotide sequence of the HE-JA10 isolate reported herein has been assigned DDBJ/EMBL/GenBank accession no. AB089824.
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
To detect anti-HEV IgG and IgM antibodies, ELISAs were performed using purified recombinant ORF2 protein of HEV genotype IV that had been expressed in the pupae of silkworm, as described previously (Mizuo et al., 2002). For the anti-HEV IgA assay, peroxidase-labelled rabbit IgG (Fab')2 against human IgA (Dako) was used in place of the enzyme-labelled anti-human IgG or IgM antibodies. The cut-off absorbance value (read at a wavelength of 450 nm) used for the anti-HEV IgG assay was 0·152 and that for the anti-HEV IgM assay was 0·353. The tentative cut-off absorbance value for the anti-HEV IgA assay was determined to be 0·350, according to the method described previously (Mizuo et al., 2002
). The relative titres of anti-HEV IgG, IgM or IgA antibodies were determined by end-point ELISA; i.e. the serum dilution that would give the absorbance value (measured at a wavelength of 450 nm) of each cut-off point was estimated by testing multiple dilutions of the serum.
To quantify HEV RNA, total RNA was extracted from 100 µl of serum or its dilutions, reverse-transcribed and then subjected to nested PCR with ORF2-specific primers, as described previously (Mizuo et al., 2002). The highest dilution of serum (10N) found positive was estimated and converted to the titre ml-1 of serum.
To sequence the full length of the genome, a central 7 kb sequence of the HEV genome (HE-JA10) was divided into six overlapping sections and amplified by PCR. These six overlapping fragments were nt 431270 (1228 nt), 12382641 (1404 nt), 26233905 (1283 nt), 38995327 (1429 nt), 52736398 (1126 nt) and 63627145 (784 nt) (primer sequences excluded). The extreme 5' end sequence (nt 170) was determined by a modified RACE technique, RNA ligase-mediated RACE (RLM-RACE), using the First Choice RLM-RACE kit (Ambion). Amplification of the extreme 3' end sequence [nt 71197244, excluding the poly(A) tail] was attempted by RACE according to the method described previously (Okamoto et al., 2001). Amplification products were sequenced on both strands either directly or after cloning into the pT7BlueT vector (Novagen) and sequence analysis was performed as described previously (Takahashi et al., 2002b
). Phylogenetic trees were constructed using the neighbour-joining method (Saitou & Nei, 1987
) and final trees were obtained using the TREEVIEW program, version 1.6.6 (Page, 1996
).
In September 1993, a 40-year-old woman visited an internal medicine clinic and presented with general malaise and nausea. Because she had elevated liver function tests [1019 IU alanine aminotransferase (ALT) l-1 serum and 590 IU aspartate aminotransferase (AST) l-1 serum], she was admitted to our hospital on day 6 from disease onset and followed for 281 days. The results of the initial tests were as follows: ALT, 541 IU l-1; AST, 174 IU l-1; total bilirubin, 2·4 mg dl-1; and thymol turbidity test, 19·0 KU (normal, 04 KU). Liver pathology on biopsy specimens obtained on day 9 from disease onset showed typical findings of acute viral hepatitis, unaccompanied by morphological cholestasis. On admission, HEV RNA was detected at an endpoint dilution of 1:1000 and IgM, IgA and IgG classes of anti-HEV antibodies were detected, each with a relative titre of >1:2000 dilution. The titre of HEV RNA decreased rapidly and HEV viraemia continued up through day 23. The anti-HEV IgM antibody level was the highest (1:2400 dilution) on day 9 and then decreased rapidly, in parallel with anti-HEV IgA antibody levels. The relative titre of anti-HEV IgG antibodies was highest on day 145 (1:17 000 dilution) and then gradually decreased but remained at high titres (1:2200 dilution) even 8·7 years after the onset of hepatitis (Table 1).
The HE-JA10 isolate had a genomic length of 7244 nt, excluding the poly(A) tract at the 3' terminus, and possessed three major ORFs, similar to those reported for human and swine HEV isolates. ORFs 13 have a coding capacity of 1709 (nt 265152), 660 (nt 51907169) and 122 aa (nt 51525517), respectively. Comparison of the HE-JA10 genome against reported HEV genomes of genotypes IIV, whose entire or nearly entire nucleotide sequences are known (see legend to Fig. 1), revealed that HE-JA10 was most closely related to JMY-Haw, with identities of 95·4, 95·2, 96·0 and 98·4 % in the nucleotide sequence of the full genome, ORF1, ORF2 and ORF3, respectively. The HE-JA10 isolate was closely related to a genotype III isolate in the United States (US1) with 92.2% identity over the full-length genome and only 73·575·6 % similar to the human and swine HEV isolates of genotypes I, II and IV. The phylogenetic tree constructed based on the full genomic sequence confirmed that HE-JA10 belongs to genotype III and is most closely related to JMY-Haw (data not shown). The 5' UTR of HE-JA10 comprised 25 nt, with a sequence beginning GCAGACCAC, similar to the 5' UTRs of the P1 strain of genotype I, the MEX-14 strain of genotype II, the swine strain of genotype III (swJ570) and the HE-JI4 strain of genotype IV, which had been determined by RLM-RACE (Fig. 1a
), the presence of a cap structure being taken into consideration (Kabrane-Lazizi et al., 1999
). Therefore, it is very likely that functional HEV genomes of all four genotypes have the extreme 5' end sequence starting with GCAGACCAC and that the extra nucleotides of G, AG or TCGACAGGGG at the very extreme 5' end are not essential. The 3' UTR of the HE-JA10 genome consisted of 72 nt and this region showed appreciable diversity, differing by 4·219·4 % compared with the seven HEV isolates of genotype III and by 26·436·8 % compared with the 18 HEV isolates of the other three genotypes whose extreme 3' end sequences are known (Fig. 1b
). In the amino acid sequences of ORFs 13, the HE-JA10 isolate also had highest identities with JMY-Haw at 99·1, 99·2 and 98·4 %, respectively. As illustrated in Fig. 1(c)
, HE-JA10 was most closely related to JMY-Haw and JKN-Sap in the amino acid sequence of the hypervariable region of the ORF1 protein, differing by only 7 aa in each case, but differing from the remaining five isolates of the same genotype in this particular region by 1833 aa. The hypervariable region of HE-JA10 ORF1 displayed only up to 1 aa difference among the five clones obtained, in contrast with the N terminus of the E2 protein of hepatitis C virus (Hijikata et al., 1991
; Weiner et al., 1991
) and the V3 loop of the gp120 protein of human immunodeficiency virus type 1 (Meyerhans et al., 1989
).
|
|
Anti-HEV IgM and IgA antibodies were detectable in our patient for more than 6 months after the onset of disease. Furthermore, anti-HEV IgG antibodies continued to be detected and remained at high levels for more than 8 years after the onset of disease. Khuroo et al. (1993) reported that anti-HEV IgG antibodies were detected in 47 % of patients with HEV infection after 14 years. However, as Krawczynski & Aggarwal (1999)
pointed out, it might be difficult to determine the exact duration of persistence of anti-HEV IgG antibodies in endemic areas because of repeated exposure to HEV. On the other hand, in Japan, a country with low endemicity and where clinical HEV infection rarely occurs, there seems to be little or no repeated exposure. The chance of repeated exposure may be negligible in our patient, as supported further by the fact that the patient's husband who lives with the patient was negative for anti-HEV IgG antibodies both in 1993 and 2002. Therefore, we speculate that anti-HEV IgG antibodies persist for more than 10 years after the onset of sporadic acute hepatitis E in industrialized countries with low endemicity as well. Regarding the duration of seropositivity for anti-HEV IgM, it has been reported that, of sera collected from patients during various hepatitis E outbreaks 34 and 612 months after the onset of jaundice, 50 and 40 %, respectively, were positive for anti-HEV IgM antibodies (Favorov et al., 1996
). Our patient continued to be positive for anti-HEV IgM antibodies for more than 9 months during the convalescent phase. Taken together, seropositivity for anti-HEV IgM antibodies can be regarded as the marker of choice as a diagnostic indicator of recent HEV infection in both developing and industrialized countries. As for anti-HEV IgA antibodies, it is unclear whether our assay is detecting both dimeric secretory IgA and monomeric IgA. However, it seems that anti-HEV IgA antibodies can be utilized as an additional confirmatory antibody for recent HEV infection, as suggested by Chau et al. (1993)
who detected anti-HEV IgA antibodies in serum samples obtained from patients who had acute waterborne hepatitis in southern Somalia and Pakistan.
In conclusion, the finding that genotype III HEV was present in Japan in the early 1990s raises the questions of when variant HEV strains emerged and how widespread they were in industrialized countries. The increasing globalization of the world marketplace and increased overseas travel may facilitate the spread of HEV variants into industrialized countries that were believed to be non-endemic. The reported high prevalence of anti-HEV antibodies in a number of animal species, such as pigs, rats and mice, may suggest that multiple sources of exposure to HEV may exist in the general population in industrialized countries that are not at apparent risk for exposure to HEV (Purcell & Emerson, 2001b). Whether the domestic spread of HEV infection in industrialized countries is via zoonosis and/or food deserves further analysis.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
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., Khudyakov, Y. E., Mast, E. E. & 7 other authors (1996). IgM and IgG antibodies to hepatitis E virus (HEV) detected by an enzyme immunoassay based on an HEV-specific artificial recombinant mosaic protein. J Med Virol 50, 5058.[CrossRef][Medline]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783791.
Garkavenko, O., Obriadina, A., Meng, J., Anderson, D. A., Benard, H. J., Schroeder, B. A., Khudyakov, Y. E., Fields, H. A. & Croxson, M. C. (2001). Detection and characterisation of swine hepatitis E virus in New Zealand. J Med Virol 65, 525529.[CrossRef][Medline]
Harrison, T. J. (1999). Hepatitis E virus: an update. Liver 19, 171176.[Medline]
Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M., Ohkoshi, S. & Shimotohno, K. (1991). Hypervariable regions in the putative glycoprotein of hepatitis C virus. Biochem Biophys Res Commun 175, 220228.[Medline]
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, F. F., Haqshenas, G., Guenette, D. K., Halbur, P. G., Schommer, S. K., Pierson, F. W., Toth, T. E. & Meng, X. J. (2002). 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, 13261332.
Kabrane-Lazizi, Y., Meng, X. J., Purcell, R. H. & Emerson, S. U. (1999). Evidence that the genomic RNA of hepatitis E virus is capped. J Virol 73, 88488850.
Khuroo, M. S., Kamili, S., Dar, M. Y., Moecklii, R. & Jameel, S. (1993). Hepatitis E and long-term antibody status. Lancet 341, 1355.[Medline]
Krawczynski, K. & Aggarwal, R. (1999). Hepatitis E. In Schiff's Diseases of the Liver, 8th edn, vol. 1, pp. 849860. Edited by E. R. Schiff, M. F. Sorrell & W. C. Maddrey. Philadelphia: Lippincott Raven.
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 Clin Proc 72, 11331136.[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 swine hepatitis E virus. J Virol 72, 97149721.
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 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.
Meyerhans, A., Cheynier, R., Albert, J., Seth, M., Kwok, S., Sninsky, J., Morfeldt-Manson, L., Asjo, B. & Wain-Hobson, S. (1989). Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations. Cell 58, 901910.[Medline]
Mizuo, H., Suzuki, K., Takikawa, Y. & 8 other authors (2002). Polyphyletic strains of hepatitis E virus are responsible for sporadic cases of acute hepatitis in Japan. J Clin Microbiol 40, 32093218.
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]
Page, R. D. M. (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357358.[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. & Emerson, S. U. (2001a). Hepatitis E virus. In Fields Virology, 4th edn, pp. 30513061. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Purcell, R. H. & Emerson, S. U. (2001b). Animal models of hepatitis A and E. ILAR J 42, 161177.[Medline]
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, 13351339.[Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406425.[Abstract]
Schlauder, G. G. & Mushahwar, I. K. (2001). Genetic heterogeneity of hepatitis E virus. J Med Virol 65, 282292.[CrossRef][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. 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]
Schlauder, G. G., Frider, B., Sookoian, S., Castano, G. C. & Mushahwar, I. K. (2000). Identification of 2 novel isolates of hepatitis E virus in Argentina. J Infect Dis 182, 294297.[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., Hino, K. & Mishiro, S. (2002a). 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., Yoshikawa, A., Sato, S., Isoda, N., Ido, K., Sugano, K. & Okamoto, H. (2002b). Identification of two distinct genotypes of hepatitis E virus in a Japanese patient with acute hepatitis who had not travelled abroad. J Gen Virol 83, 19311940.
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, 120131.[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., 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, 706709.[CrossRef][Medline]
Wang, Y., Zhang, H., Xia, N. & 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]
Weiner, A. J., Brauer, M. J., Rosenblatt, J. & 8 other authors (1991). Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NS1 proteins and the pestivirus envelope glycoproteins. Virology 180, 842848.[Medline]
Worm, H. C., Schlauder, G. G., Wurzer, H. & Mushahwar, I. K. (2000). Identification of a novel variant of hepatitis E virus in Austria: sequence, phylogenetic and serological analysis. J Gen Virol 81, 28852890.
Wu, J. C., Chen, C. M., Chiang, T. Y., Tsai, W. H., Jeng, W. J., Sheen, I. J., Kin, 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 30 August 2002;
accepted 20 September 2002.