Analysis of the full-length genome of hepatitis E virus isolates obtained from wild boars in Japan

Tsutomu Nishizawa1, Masaharu Takahashi1, Kazunori Endo1, Shinji Fujiwara2, Nobuo Sakuma3, Fumiaki Kawazuma4, Hirotsugu Sakamoto5, Yukihiro Sato6, Masashi Bando7 and Hiroaki Okamoto1

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


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Two (2·3 %) of 87 wild-caught boars in Japan had detectable hepatitis E virus (HEV) RNA. The two boar HEV isolates (wbJTS1 and wbJYG1) obtained in the present study and a previously reported isolate (wbJSG1) whose partial sequence had been determined were sequenced over the entire genome. The wbJSG1, wbJTS1 and wbJYG1 isolates comprised 7225 or 7226 nt, excluding the poly(A) tail, and segregated into genotype 3. They differed by 8·5–11·2 % from each other and by 8·6–18·4 % from 17 reported genotype 3 HEV isolates, including one boar isolate, in the full-length sequence. When compared with 191 reported genotype 3 HEV isolates whose partial sequences were known, these three boar isolates were closer to Japanese isolates than to isolates of non-Japanese origin (89·2±2·6 vs 85·9±2·2 %; P<0·0001). A proportion of wild boars in Japan are infected with markedly heterogeneous HEV strains that are indigenous to Japan and may serve as reservoirs of HEV.

The GenBank/EMBL/DDBJ accession numbers for the complete nucleotide sequences of isolates wbJSG1, wbJTS1 and wbJYG1 reported in this study are AB222182–AB222184, respectively.


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Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded, positive-sense RNA virus without an envelope (Purcell & Emerson, 2001). The genome of HEV is approximately 7·2 kb in size and contains three open reading frames (ORF1–3) (Tam et al., 1991). Extensive genomic diversity has been noted among HEV isolates and HEV sequences have tentatively been classified into four genotypes (genotypes 1–4) (Schlauder & Mushahwar, 2001). The main mode of transmission of HEV in developing countries in Asia, Africa and Latin America is the faecal–oral, water-borne route; large-scale outbreaks via drinking water that was contaminated with faeces containing HEV have been reported. However, in industrialized countries, including Japan, where the water supply and sewage water are treated and disinfected at water-treatment plants, the likelihood of water-borne infection is very low. Recent studies have documented that HEV-associated hepatitis occurs among individuals in industrialized countries with no history of travel to endemic countries (Harrison, 1999; Purcell & Emerson, 2001; Schlauder & Mushahwar, 2001; Smith, 2001) and that HEV is a zoonotic virus (Erker et al., 1999; Hsieh et al., 1999; Meng et al., 1997, 1998; Pina et al., 2000; Wu et al., 2002). It has recently been suggested that zoonotic food-borne transmission of HEV from domestic pigs, wild boars and wild deer to humans plays an important role in the occurrence of cryptic hepatitis E in Japan, where Japanese people have distinctive habits of eating raw fish (sushi or sashimi) and, less frequently, uncooked or undercooked meat (including the liver and colon/intestine of animals) (Matsuda et al., 2003; Tamada et al., 2004; Tei et al., 2003; Yazaki et al., 2003). The Meng isolate was the first strain of HEV isolated from an animal, namely from an infected pig in the USA in 1997 (Meng et al., 1997). Since then, many swine HEV isolates, which are genetically closely related to strains of human HEV, have been identified in many countries worldwide (Garkavenko et al., 2001; Hsieh et al., 1999; Huang et al., 2002; Meng, 2000, 2003; Nishizawa et al., 2003; Pina et al., 2000; Wang et al., 2002; Wu et al., 2002). In addition, we found a high prevalence of swine anti-HEV antibodies among Japanese pigs of 3–6 months of age (71 % or 2150/3009) and a high HEV viraemia rate among pigs of 2–4 months of age (11 % or 190/1798) (Takahashi et al., 2003, 2005). Thus, HEV is considered to be enzootic in pigs worldwide. Regarding HEV infection among wild deer, Tei et al. (2003) presented data that implicated deer products in the transmission of this disease. However, our previous study revealed that none of 132 wild deer in Japan, including two deer with anti-HEV antibody, had detectable HEV RNA (Sonoda et al., 2004), suggesting that HEV infection via consumption of raw meat or viscera from wild deer occurs very rarely. For HEV from wild boars, although the prevalence of HEV antibody has been reported to be 25 % (15/59) in Australia (Chandler et al., 1999) and 9 % (3/35) in Japan (Sonoda et al., 2004), the genomic characteristics of boar HEV isolates are not fully understood. Therefore, in the present study, we examined the prevalence of HEV infection among wild-caught boars in Japan, and determined and analysed the full-length genomic sequences of three boar HEV strains to clarify the genomic characteristics of the boar HEVs circulating in Japan.

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 43–1270 (1228 nt) (primer sequences excluded), nt 1081–2623 (1543 nt), nt 2605–3127 (523 nt), nt 3106–4700 (1595 nt), nt 4651–5975 (1325 nt), nt 5960–6380 (421 nt) and nt 6342–7199 (858 nt). The extreme 5'-end sequence (nt 1–50) 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 7101–7225 or 7101–7226 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 26–5134), 660 aa (nt 5172–7151) and 122 aa (nt 5134–5499), 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 88·8–91·5 %, 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 88·1–91·2 % (96·7–97·8 %) in ORF1, 90·3–92·3 % (97·6–97·9 %) in ORF2 and 95·6–96·2 % (93·4–95·1 %) in ORF3. Comparison of the wbJSG1, wbJTS1 and wbJYG1 genomes against 52 reported HEV genomes of genotypes 1–4 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 89·2–91·4 %, but were only 73·4–75·5 % 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; Mann–Whitney 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.



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Fig. 1. Phylogenetic tree constructed by the neighbour-joining method based on the nucleotide sequence of 55 HEV isolates obtained from humans, swine, wild boars and wild deer, using an avian HEV isolate (GenBank accession no. AY535004) as an outgroup. As the length of the sequence that has been identified for different isolates varies, the overlapping 7146 nt sequences of 52 reported human, swine, boar and deer HEV isolates and those of the wbJSG1, wbJTS1 and wbJYG1 isolates obtained in the present study (indicated in bold) were compared. GenBank accession numbers are shown in parentheses. After the slash, the name of the country other than Japan where the HEV strain was isolated is shown. Swine HEV isolates, boar HEV isolates and a deer HEV isolate are indicated by asterisks (*, ** and ***, respectively). Bootstrap values are indicated for the major nodes as a percentage of the data obtained from 1000 resamplings. Bar, 0·1 substitutions per site.

 

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Table 1. Comparison of three boar HEV isolates of genotype 3 with 191 reported human and swine HEV isolates of the same genotype whose partial sequence is known

 
Upon comparison with reported HEV sequences obtained from wild boars (Table 2), the wbJSG1, wbJTS1 and wbJYG1 isolates shared nucleotide identities of 88·8–90·2 % with the JBOAR1-Hyo04 isolate of genotype 3 (Takahashi et al., 2004) over the entire genome and nucleotide identities of only 79·6–80·8 % with two boar isolates of genotype 4 in the 412 nt ORF2 sequence. Among the HEV strains recovered from patients who developed hepatitis E after ingestion of uncooked or undercooked meat or liver from wild boars, the wbJSG1, wbJTS1 and wbJYG1 isolates were closest to two HEV isolates of genotype 3 (ENK-NGS03 and EMN-NGS03) that had been recovered from two patients who had ingested grilled meat from wild boars in Nagasaki Prefecture on Kyushu Island (Tamada et al., 2004), with nucleotide identities of 90·2–93·4 % in the 317 nt ORF1 sequence. Furthermore, the wbJSG1, wbJTS1 and wbJYG1 isolates were merely 75·2–75·8 % similar in the full-length sequence to the JSF-Tot03 isolate of genotype 4 that had been recovered from a patient who had eaten uncooked liver from wild boar in Tottori Prefecture on mainland Honshu (Matsuda et al., 2003). In addition, the three boar HEV isolates obtained in the present study shared only 88·9–90·3 % identity with a single HEV isolate from a leftover portion of the deer meat that had been kept frozen to be eaten in the future and those from four patients who had acquired hepatitis E after consumption of raw meat in Hyogo Prefecture on mainland Honshu (Tei et al., 2003). These results suggest that heterogeneous strains of HEV of genotype 3 or 4 are circulating among wild boars in Japan.


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Table 2. Comparison of three boar HEV isolates obtained in the present study with seven reported HEV isolates obtained from wild boars or deer or from patients who contracted hepatitis E after ingestion of meat or liver from wild boars

 
In conclusion, although the route(s) of transmission of HEV among wild boars in Japan remains unknown, a proportion of wild boars are infected with markedly heterogeneous HEV strains that are closer to Japanese human and swine HEV strains than to those of non-Japanese origin. To gain a better understanding of hepatitis E as a zoonosis and to prevent zoonotic transmission of HEV to humans, further clinical, ecological and virological studies on HEV infection in humans and candidate animals are warranted to clarify how domestic HEV strains circulating in animal species are transmitted to animals and humans.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Ministry of Health, Labour and Welfare of Japan. We thank Masayuki Nakayama (Nanakai-mura Kokuho Clinic, Ibaraki), Hideki Shinnou (Kamitaira Clinic, Toyama), Toshinori Tanaka (Jichi Medical School, Tochigi), Yoshinori Koga (Kasuga-mura Clinic, Gifu), Naoki Doi (Sagamiko-machi Kokuho Clinic, Kanagawa), Kazuyoshi Murao (Hino Hospital, Tottori), Hiroto Tanaka (Wakayama Medical University, Wakayama), Sumi Yoshino (Takinomiya General Hospital, Kagawa), Tatsuhiko Shiraishi (Tokushima Prefectural Miyoshi Hospital, Tokushima), Takuya Hashimoto (Kokuho Nishiiyayamason Clinic, Tokushima), Hitoshi Nishimura (Nishiki Central Hospital, Yamaguchi), Akira Yoshimura (Hongouson Clinic, Yamaguchi) and Makoto Nishimura (Soyo Hospital, Kumamoto) for supplying serum and/or liver specimens obtained from wild-caught boars.


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Received 1 August 2005; accepted 30 August 2005.



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