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Short Communication |
1 Division of Virology, Department of Infection and Immunity, Jichi Medical School, Tochigi-Ken 329-0498, Japan
2 Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan
3 First Department of Internal Medicine, Mie University School of Medicine, Mie-Ken 514-8507, Japan
Correspondence
Hiroaki Okamoto
hokamoto{at}jichi.ac.jp
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The GenBank/EMBL/DDBJ accession numbers for the sequences of the HE-JA04-1911, swJ8-5 and swJ12-4 isolates determined in this work are AB248520–AB248522, respectively.
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), is the major cause of enterically transmitted non-A, non-B hepatitis. Transmission of HEV occurs primarily by the faecal–oral route via contaminated water supplies in developing countries where sanitation is suboptimal (Purcell & Emerson, 2001; Smith, 2001). Accumulating lines of evidence have indicated that hepatitis E is a zoonosis (Harrison, 1999; Meng, 2003; Meng et al., 1997, 1998, 2002; Nishizawa et al., 2003; Okamoto et al., 2001; Tei et al., 2003; Yazaki et al., 2003). In addition, recent studies have indicated that zoonotic food-borne transmission of HEV from domestic pigs, wild boar or wild deer to humans may occur as autochthonous infection in Japan, where some people ingest uncooked or undercooked meat or viscera (such as raw liver and colon/intestines) from pigs, wild boar and deer (Li et al., 2005; Matsuda et al., 2003; Tamada et al., 2004; Tei et al., 2003; Yazaki et al., 2003). The genome of HEV is a single-stranded, positive-sense RNA of approximately 7.2 kb and consists of a short 5' untranslated region (UTR), three open reading frames (ORF1–3) and a short 3' UTR terminated by a poly(A) tract (Tam et al., 1991). Based on the genomic variability noted among HEV isolates, HEV sequences have been classified into four genotypes: genotype 1 consists of epidemic strains in developing countries in Asia and Africa; genotype 2 has been described in Mexico and Africa; genotype 3 is widely distributed and has been isolated from sporadic cases of acute hepatitis E and/or domestic pigs in a total of 22 countries (Argentina, Australia, Austria, Cambodia, Canada, France, Germany, Greece, Italy, Japan, Korea, Kyrgyzstan, Mexico, The Netherlands, New Zealand, Russia, South Africa, Spain, Taiwan, Thailand, the UK and the USA); and genotype 4 contains strains from humans and/or domestic pigs in China, India, Indonesia, Japan, South Africa, Taiwan and Vietnam (Schlauder & Mushahwar, 2001; see Table 1).
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; Okamoto et al., 2001; Sonoda et al., 2004; Takahashi et al., 2001; Tei et al., 2003). Genotype 3 HEV isolates are provisionally classified into three phylogenetic clusters, with the highest nucleotide identity being 94.4–100 % between human and swine isolates in each cluster (Takahashi et al., 2003). Entire or almost entire genomic sequences have been determined for 16 HEV isolates: 13 HEV isolates obtained from humans, pigs, wild boar and a wild deer segregated into a cluster (provisionally designated cluster IIIjp in our previous study; Takahashi et al., 2003) consisting of predominantly Japan-indigenous strains represented by the JRA1 isolate (see Table 1
for GenBank accession no.) and three human HEV isolates were classified into a second cluster (cluster IIIus) with HEV isolates homologous to those in the USA (US1 and US2). The genomic characteristics and the possible origin of HEV isolates that have been classified into a third cluster (cluster IIIsp) and are 84.2–87.2 % similar to Spanish HEV isolates in the 304 nt ORF2 sequence remain unknown. In the present study, we determined the entire genomic sequences of three HEV isolates (HE-JA04-1911, swJ8-5 and swJ12-4) that belong to the third cluster within genotype 3 in Japan and differed by 89.8–94.2 % from each other in the 412 nt ORF2 sequence to investigate further the extent of the genomic heterogeneity of HEV and to compare them with all reported genotype 3 HEV isolates for which only partial sequences of 69–1866 nt in various genomic regions are available.
Serum samples were obtained from two domestic pigs (swJ8-5 and swJ12-4; Takahashi et al., 2003) raised in Hokkaido, Japan, and from a 52-year-old Japanese male who contracted sporadic acute hepatitis E in July 2004. His laboratory data on admission showed an elevated total bilirubin level of 4.1 mg dl–1, an aspartate aminotransferase level of 4680 IU l–1 and an alanine aminotransferase level of 3026 IU l–1. He had IgM and IgA antibodies to HEV detectable by an in-house ELISA (Takahashi et al., 2005) and HEV RNA detectable by RT-PCR (Mizuo et al., 2002). He had no history of travel abroad. To determine the full-length sequence of the three HEV isolates, total RNA was extracted from 350 µl (HE-JA04-1911) or 750 µl (swJ8-5 and swJ12-4) of serum using TRIzol LS (Invitrogen) and the RNA preparation was reverse-transcribed and subjected to nested PCR. Seven overlapping regions excluding the extreme 5' and 3' termini were amplified (primer sequences excluded): nt 37–1270 (1234 nt), nt 1239–2074 (836 nt), nt 2020–3241 (1222 nt), nt 3110–4513 (1404 nt), nt 4324–6020 (1697 nt), nt 6011–6422 (412 nt) and nt 6383–7167 (785 nt): the nucleotide numbers were in accordance with HE-JA04-1911. The extreme 5'-end sequence (nt 1–53) was determined by a modified rapid amplification of cDNA ends (RACE) technique called RNA ligase-mediated RACE (RLM-RACE) using the First Choice RLM-RACE kit (Ambion), as described previously (Okamoto et al., 2001). Amplification of the 3'-end sequence [nt 7143–7264 excluding the poly(A) tail] was attempted by the RACE method described previously (Okamoto et al., 2001). The amplification products were sequenced on both strands, either directly or after cloning into pT7Blue T-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 HE-JA04-1911 isolate had a genomic length of 7264 nt, excluding the poly(A) tract at the 3' terminus. This is the longest among all known HEV isolates whose entire sequence has been determined. The swJ8-5 and swJ12-4 isolates both had a genomic length of 7225 nt, the difference in genomic length of HE-JA04-1911 being attributed to an insertion of 39 nt in the hypervariable region of ORF1 in this isolate. These three isolates each possessed three major ORFs similar to all other reported HEV isolates including an avian HEV isolate (Tam et al., 1991; Meng et al., 1997; Huang et al., 2004). ORF1, -2 and -3 had coding capacities of 1717 or 1704 aa (nt 26–5176 or 26–5137), 660 aa (nt 5214–7193 or 5175–7154) and 122 aa (nt 5176–5541 or 5137–5502), respectively. The 5' UTRs of the three isolates comprised 25 nt, and swJ8-5 had a unique substitution of C at nt 11, whereas the other two isolates had T at this position. The 3' UTRs of the three isolates each consisted of 71 nt [excluding the poly(A) tail], which was shorter than those in the 21 reported genotype 3 isolates due to a deletion of 5 nt after nt 7232.
On comparison of the entire genome, the HE-JA04-1911, swJ8-5 and swJ12-4 isolates had nucleotide identities of 89.7–92.9 % with each other and amino acid identities of 96.7–98.5 % in ORF1, 98.0–98.8 % in ORF2 and 97.5–99.2 % in ORF3 with each other. Following comparison with the 67 reported HEV isolates from humans, swine, wild boar and a wild deer whose entire or almost entire sequences have been determined, the HE-JA04-1911, swJ8-5 and swJ12-4 isolates were found to be 73.9–74.8 % similar to 17 reported genotype 1 isolates, 73.4–73.7 % similar to one genotype 2 isolate and 74.7–75.9 % similar to 28 reported genotype 4 isolates. Of note, the three HEV isolates obtained in the present study were closest to the Osh205 isolate recovered from a pig in Kyrgyzstan (Lu et al., 2004) among the 21 reported genotype 3 HEV isolates, with identities of only 82.8–83.0 % (Table 1), and were 80.7–81.8 % similar to the remaining 20 HEV isolates of genotype 3. A phylogenetic tree was constructed based on the full-length genomic sequences of genotype 1–4 HEV isolates and confirmed that HE-JA04-1911, swJ8-5 and swJ12-4 belonged to genotype 3 and were genetically distinct from the genotype 3 strains prevalent in Japan and the USA (Fig. 1). Using CLUSTAL W (version 1.8) (Thompson et al., 1994), sequence alignments were generated using all 70 HEV isolates of genotypes 1–4 including the three HEV isolates whose full-length sequences were determined in this study. Following alignment of the 70 HEV isolates of genotypes 1–4, conserved nucleotides were recognized in 2352 (45.7 %) of 5151 nt in ORF1, 1167 (58.9 %) of 1980 nt in ORF2 and 230 (62.8 %) of 366 nt in ORF3, which included the well-conserved area in ORF3 that was reported to be suitable for designing primers and a probe for sensitive detection of HEV RNA by a broadly reactive real-time RT-PCR (Jothikumar et al., 2006). On alignment of the deduced amino acid sequences of the 70 HEV genomes, conserved amino acids were recognized in 1050 (61.2 %) of 1717 aa in ORF1, 497 (75.3 %) of 660 aa in ORF2 and 65 (53.3 %) of 122 aa in ORF3. The amino acid sequence of the ORF2 region encoding a capsid protein of HEV virions was well conserved, which probably contributes to the presence of a single serotype for HEV.
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). Among the 245 genotype 3 HEV isolates of non-Japanese origin, the HE-JA04-1911 isolate had the highest similarity of 92.4 % with a UK swine isolate (P354/1/02) in the 473 nt ORF2 sequence and the lowest similarity of 76.6 % with a Taiwanese swine isolate (TW3SW) in the 304 nt ORF2 sequence. Compared with non-Japanese isolates of genotype 3, the swJ8-5 and swJ12-4 isolates were closest to UK human (UK7518) and swine (P143/11/02) isolates with identities of 90.9–92.0 % and had the lowest similarity of 77.9–79.2 % with the Ar2 isolate in Argentina. The swJ8-5 isolate was also close to a Greek human isolate (Gr2) with an identity of 92.1 %. The phylogenetic tree constructed based on the common 233 nt ORF1 sequence of genotype 3 HEV isolates using a genotype 4 isolate (T1) as an outgroup revealed that the HE-JA04-1911, swJ8-5 and swJ12-4 isolates segregated into a cluster consisting of UK, Greek and Japanese isolates (Fig. 2a). The phylogenetic tree constructed based on the common 280 nt ORF2 sequence of genotype 3 HEV isolates confirmed that the three genotype 3 isolates obtained in the present study were located on the same branch as 11 human and swine HEV isolates obtained in the UK (Fig. 2b).
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) and have been recovered from not only humans but also from various animals such as swine (Meng et al., 1997; Okamoto et al., 2001), wild deer (Tei et al., 2003; Takahashi et al., 2004), wild boar (Nishizawa et al., 2005; Sonoda et al., 2004; Takahashi et al., 2004) and a wild mongoose (Nakamura et al., 2006). In contrast, genotype 4 HEV strains circulate mainly in Asian countries and have been recovered so far from humans, swine and wild boar (Hsieh et al., 1999; Wang et al., 1999).
The genotype 3 HEV isolates in Japan segregate into three clusters: the most predominant cluster (n=203, as of 27 January 2006) represented by the JRA1 isolate is apparently indigenous to Japan; the second most predominant cluster (n=97) consists of US-isolate-like HEV isolates; and the third minor cluster (n=24) is represented by the HE-JA04-1911, swJ8-5 and swJ12-4 isolates whose entire nucleotide sequences were determined in the present study. Inter-cluster and intra-cluster divergences were 11.9–19.3 and 0–11.4 %, respectively, over the entire genome. Pairwise comparison revealed that the HE-JA04-1911, swJ8-5 and swJ12-4 isolates belonging to the third cluster within genotype 3 are most closely related to human and swine HEV isolates circulating in the UK among the HEV isolates reported outside Japan (Table 1). On phylogenetic analysis, these three isolates were interspersed among 11 UK human and swine isolates and four other Japanese human and swine isolates in the third cluster within genotype 3 (shaded box in Fig. 2b). In the UK, sporadic cases of hepatitis E (Ijaz et al., 2005; McCrudden et al., 2000; Wang et al., 2001) and the existence of a close genetic relationship between human and swine HEV strains (Banks et al., 2004) have been reported. As swine are one of the major reservoirs of HEV (Meng, 2003; Smith, 2001; Takahashi et al., 2003), it is conceivable that UK-isolate-like HEV isolates represented by the HE-JA04-1911, swJ8-5 and swJ12-4 isolates entered Japan via importation of pigs from the UK. This speculation is supported by the historical evidence that the Japanese government started to import several kinds of Yorkshire and Berkshire pigs from the UK in 1900 to introduce high-quality domestic pigs for food [http://www.pig-pins.or.jp/youton/shiryo.html (in Japanese)]. Evidence that swine HEV can be imported through international trading of pigs was reported in Taiwan (Wu et al., 2002). However, in addition to the UK isolates, a Greek isolate (Gr2) also falls into the third divergent cluster within the genotype 3 (Fig. 2a). Therefore, it is possible that, as more genotype 3 HEV isolates are identified worldwide, this third cluster may expand to include isolates from other geographical regions.
In conclusion, the present study indicates that genotype 3 HEV strains are markedly heterogeneous with an intra-genotype divergence of up to 19.3 % over the entire genome and that the indigenization of UK-isolate-like HEV strains of genotype 3 represented by the human and swine isolates of HE-JA04-1911, swJ8-5 and swJ12-4 in Japan may be associated with the importation of pigs for food from the UK since 1900. Further studies are needed to provide more concrete data on the origin of these divergent genotype 3 Japanese swine and human HEV strains.
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ACKNOWLEDGEMENTS |
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Received 6 February 2006;
accepted 20 March 2006.
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