1 Division of Virology, Department of Infection and Immunity, Jichi Medical School, Tochigi-Ken 329-0498, Japan
2 Koyadairamura Kokuho Clinic, Tokushima-Ken 777-0302, Japan
3 Kawakamimura Kokuho Clinic, Yamaguchi-Ken 758-0122, Japan
4 Yodakubo Hospital, Nagano-Ken 386-0603, Japan
5 Kitaibaraki Municipal General Hospital, Ibaraki-Ken 319-1704, Japan
6 Kamiichi General Hospital, Toyama-Ken 930-0391, Japan
7 Division of Pulmonary Medicine, Department of Medicine, Jichi Medical School, Tochigi-Ken 329-0498, Japan
Correspondence
Hiroaki Okamoto
hokamoto{at}jichi.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession numbers for the complete nucleotide sequences of isolates wbJSG1, wbJTS1 and wbJYG1 reported in this study are AB222182AB222184, respectively.
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
We obtained and analysed paired serum and liver specimens, serum only or liver tissues only from 87 wild boars (Sus scrofa leucomystax) that had been captured in the following prefectures (located from north to south in Japan): Ibaraki (n=15 boars), Toyama (n=2), Nagano (n=14), Gifu (n=1), Kanagawa (n=2), Tottori (n=19), Okayama (n=1), Wakayama (n=2) and Yamaguchi (n=10) on mainland Honshu, Kagawa (n=6) and Tokushima (n=14) on Shikoku Island, and Kumamoto Prefecture (n=1) on Kyushu Island, between November 2004 and May 2005. A total of 62 serum samples and 73 liver tissues, including 48 paired serum and liver specimens, were available from the 87 boars: all of the boars in the present study were different from the 41 boars in our previous study (Sonoda et al., 2004). The serum samples were tested for the presence of anti-HEV IgG by an in-house ELISA using purified recombinant ORF2 protein that had been expressed in the pupae of silkworm (Mizuo et al., 2002
) as the antigen probe, as described previously (Takahashi et al., 2003
). The serum samples and liver tissues were tested for the presence of HEV RNA by RT-PCR using the method described previously with primers targeting the ORF2 region (Mizuo et al., 2002
). The RT-PCR assay used has the capability to amplify all four known genotypes of HEV (Mizuo et al., 2002
; Takahashi et al., 2003
).
The A450 value of boar anti-HEV antibodies ranged from 0·002 to 0·645 and one (2 %) of the 62 serum samples had an A450 value of 0·300. The sample with an A450 value of 0·645 decreased to <30 % of the original value after absorption with the same recombinant ORF2 protein that was used as the antigen probe, confirming the specificity of the assay. However, the presence of anti-HEV antibody in three serum samples with an A450 value of 0·217, 0·240 or 0·276 was not confirmable by the absorption test. Therefore, only one serum sample from a female boar (wbJTS1) with a body weight of 50 kg that had been caught in Tokushima Prefecture on Shikoku Island on December 9, 2004, was regarded conservatively as being positive for boar anti-HEV IgG in the present study. Among all of the serum and liver specimens from the boars, HEV RNA was detected reproducibly in paired serum and liver specimens obtained from the above-mentioned boar with detectable anti-HEV IgG and in liver specimens obtained from another female boar (wbJYG1) with a body weight of 50 kg that had been caught in Yamaguchi Prefecture on mainland Honshu on February 6, 2005, although a serum sample was not available from the boar. The two HEV-viraemic boars identified in the present study weighed 50 kg, suggesting that the viraemic boars were approximately 2 years of age and that wild boars can acquire de novo HEV infection at older ages than domestic pigs, whose HEV RNA is generally detectable between 2 and 4 months of age. The two HEV-infected boars had no clinical manifestations. The HEV sequences amplified from the serum and liver tissue of the wbJTS1 boar were identical in a 412 nt sequence of the ORF2 region.
Taking the results of the present study with those of our previous study (Sonoda et al., 2004), three (2·3 %) of the 128 wild boars captured in 16 prefectures of Japan were viraemic for HEV. To determine the full-length sequence of the HEV isolates (wbJSG1, wbJTS1 and wbJYG1) from the three infected boars, total RNA was extracted from boar liver specimens (100 mg) by using TRIzol reagent (Invitrogen) and the RNA preparation thus obtained was reverse-transcribed and subjected to nested PCR. The central 7 kb sequence of each of the wbJSG1, wbJTS1 and wbJYG1 genomes was divided into seven overlapping sections and amplified: these were nt 431270 (1228 nt) (primer sequences excluded), nt 10812623 (1543 nt), nt 26053127 (523 nt), nt 31064700 (1595 nt), nt 46515975 (1325 nt), nt 59606380 (421 nt) and nt 63427199 (858 nt). The extreme 5'-end sequence (nt 150) was determined by a modified rapid amplification of cDNA ends (RACE) technique called RNA ligase-mediated RACE (RLM-RACE) with a First Choice RLM-RACE kit (Ambion), as described previously (Okamoto et al., 2001
). Amplification of the extreme 3'-end sequence [nt 71017225 or 71017226 excluding the poly(A) tail] was attempted by the RACE method, according to the method described previously (Okamoto et al., 2000
). To avoid contamination during PCR procedures, the guidelines of Kwok & Higuchi (1989)
were strictly observed. The amplification products were sequenced on both strands either directly or after cloning into pT7BlueT-Vector (Novagen) and sequence analysis was performed as described previously (Okamoto et al., 2001
). A phylogenetic tree was constructed by the neighbour-joining method (Saitou & Nei, 1987
) and bootstrap values were determined on 1000 resamplings of the datasets (Felsenstein, 1985
).
The wbJSG1 and wbJYG1 isolates had the same genomic length of 7226 nt, excluding the poly(A) tract at the 3' terminus, whereas wbJTS1 had a genomic length of 7225 nt, the difference in genomic length being attributed to a deletion of 1 nt in wbJTS1 in the 3' untranslated region (UTR). Each of the three isolates possessed three major ORFs, similar to reported human and swine HEV isolates (Meng et al., 1997; Tam et al., 1991
; Wang et al., 2000
). In each isolate, ORF1, ORF2 and ORF3 had a coding capacity of 1703 aa (nt 265134), 660 aa (nt 51727151) and 122 aa (nt 51345499), respectively. The 5' UTR of wbJSG1, wbJTS1 and wbJYG1 each comprised 25 nt, and the 3' UTR sequence of the wbJSG1, wbJTS1 and wbJYG1 genomes consisted of 74 or 75 nt [excluding the poly(A) tail]. The three isolates showed an overall nucleotide sequence identity of 88·891·5 %, with the highest identity of 100 % in the 5' UTR and the lowest identity of 85·3 % in the 3' UTR; they had nucleotide (amino acid) sequence identities of 88·191·2 % (96·797·8 %) in ORF1, 90·392·3 % (97·697·9 %) in ORF2 and 95·696·2 % (93·495·1 %) in ORF3. Comparison of the wbJSG1, wbJTS1 and wbJYG1 genomes against 52 reported HEV genomes of genotypes 14 whose entire or almost entire nucleotide sequences were known revealed that they were closest to JJT-Kan (a genotype 3 human HEV isolate of Japanese origin; see Fig. 1
for GenBank accession no.) and swJ570 (a genotype 3 HEV isolate obtained from a farm pig in Japan) with identities of 89·291·4 %, but were only 73·475·5 % similar to the prototype HEV isolates of genotypes 1, 2 and 4 (Sar-55, MEX-14 and T1, respectively) in the nucleotide sequence of the full genome. There were no nucleotides or deduced amino acids that were unique to boar HEV strains over the entire genome. The phylogenetic tree was constructed based on the overlapping, almost-complete genomic sequence of 55 HEV isolates obtained from humans, swine, wild boars and wild deer, using an avian HEV as an outgroup; the HEV-like virus infecting chickens shares only 50 % amino acid sequence identity with human and swine strains and is unlikely to infect humans, as it was not experimentally transmissible to rhesus macaques (Huang et al., 2004
). The tree confirmed that wbJSG1, wbJTS1 and wbJYG1 belonged to genotype 3 and segregated into a cluster consisting of human and swine HEV isolates that are presumed to be indigenous to Japan (Fig. 1
). Among 208 HEV isolates of genotype 3 that were retrievable from GenBank/EBML/DDBJ as of July 2005, the entire or almost-entire sequence of only 17 isolates (8·2 %) obtained in four countries, i.e. USA, Canada, Kyrgyzstan and Japan, had been determined. When compared with the remaining 191 HEV isolates obtained in the 13 countries listed in Table 1
, the wbJSG1, wbJTS1 and wbJYG1 genomes obtained in the present study were closer to human and swine HEV isolates of Japanese origin than to those of non-Japanese origin, with identities of 89·2±2·6 vs 85·9±2·2 % (P<0·0001; MannWhitney U-test). Of note, wbJSG1 was most closely related to the Japanese human HEV isolate HE-JHD1980 (GenBank accession no. AB175484), although it differed by 6·3 % (412 nt in ORF2); wbJTS1 was closest to the Japanese swine HEV isolates swJHR1-1 (AB194528) and swJOK1-1 (AB194530), with a difference of 4·9 % (412 nt in ORF2); and wbJYG1 was nearest to the Japanese human HEV isolate HE-JHD1979 (AB175483), with a difference of 5·3 % (412 nt in ORF2). The three boar HEV isolates obtained in the present study were most similar to human or swine HEV isolates of Japanese origin, including those that had circulated in the years 1979 or 1980.
|
|
|
![]() |
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]
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 characterization 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]
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.
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, 16091618.
Kwok, S. & Higuchi, R. (1989). Avoiding false positives with PCR. Nature 339, 237238.[CrossRef][Medline]
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.[CrossRef][Medline]
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, 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 swine hepatitis E virus. J Virol 72, 97149721.
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.
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., Nishizawa, T., Tawara, A., Takahashi, M., Kishimoto, J., Sai, T. & Sugai, Y. (2000). TT virus mRNAs detected in the bone marrow cells from an infected individual. Biochem Biophys Res Commun 279, 700707.[CrossRef][Medline]
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]
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. (2001). Hepatitis E virus. In Fields Virology, 4th edn, pp. 30513061. Edited by D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman & S. E. Straus. Philadelphia, PA: Lippincott Williams & Wilkins.
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]
Smith, J. L. (2001). A review of hepatitis E virus. J Food Prot 64, 572586.[Medline]
Sonoda, H., Abe, M., Sugimoto, T. & 7 other authors (2004). Prevalence of hepatitis E virus (HEV) infection in wild boars and deer and genetic identification of a genotype 3 HEV from a boar in Japan. J Clin Microbiol 42, 53715374.
Takahashi, M., Nishizawa, T., Miyajima, H., Gotanda, Y., Iita, T., Tsuda, F. & Okamoto, H. (2003). 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, K., Kitajima, N., Abe, N. & Mishiro, S. (2004). Complete or near-complete nucleotide sequences of hepatitis E virus genome recovered from a wild boar, a deer, and four patients who ate the deer. Virology 330, 501505.[CrossRef][Medline]
Takahashi, M., Nishizawa, T., Tanaka, T., Tsatsralt-Od, B., Inoue, J. & Okamoto, H. (2005). Correlation between positivity for immunoglobulin A antibodies and viraemia of swine hepatitis E virus observed among farm pigs in Japan. J Gen Virol 86, 18071813.
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.[CrossRef][Medline]
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, 869873.[CrossRef][Medline]
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]
Tei, S., Kitajima, N., Ohara, S., Inoue, Y., Miki, M., Yamatani, T., Yamabe, H., Mishiro, S. & Kinoshita, Y. (2004). Consumption of uncooked deer meat as a risk factor for hepatitis E virus infection: an age- and sex-matched case-control study. J Med Virol 74, 6770.[CrossRef][Medline]
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., 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]
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]
Yazaki, Y., Mizuo, H., Takahashi, M., Nishizawa, T., Sasaki, N., Gotanda, Y. & Okamoto, H. (2003). Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food. J Gen Virol 84, 23512357.
Received 1 August 2005;
accepted 30 August 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |