1 Immunology Division and Division of Molecular Virology, Jichi Medical School, Tochigi-Ken 329-0498, Japan
2 Department of Internal Medicine, Kin-ikyo Chuo Hospital, Hokkaido 007-0870, Japan
3 Japanese Red Cross Saitama Blood Center, Saitama-Ken 338-0001, Japan
Correspondence
Hiroaki Okamoto
hokamoto{at}jichi.ac.jp
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ABSTRACT |
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The entire nucleotide sequences of the swJ13-1 and HE-JA1 isolates reported herein have been assigned DDBJ/EMBL/GenBank accession nos AB097811 and AB097812, respectively.
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MAIN TEXT |
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For full-length sequencing, total RNA was extracted from 300 µl swine serum (swJ13-1) or 400 µl human serum (HE-JA1) using TRIzol-LS reagent (Invitrogen). The RNA preparation obtained was reverse-transcribed and subjected to nested PCR using the primers listed in Table 1. The central 7 kb sequence of each of the swJ13-1 and HE-JA1 genomes was divided into six overlapping sections and amplified as follows: nt 371270 (1234 nt) (primer sequences excluded) was amplified with primers HE090 and HE012 in the first round and HE092 and HE014 in the second round (HE090-HE012 and HE092-HE014); nt 11503142 (1993 nt) with primers HE011-HE136 and HE134-HE027; nt 31173899 (783 nt) with primers HE047-HE048 and HE049-HE050; nt 38895325 (1437 nt) with primers HE030-HE008 and HE031-HE009; nt 52516007 (757 nt) with primers HE067-HE036 and HE059-HE006; and nt 59857142 (1158 nt) with primers HE044-#166 (5'-AAGGATCCGTCGACATCGAT-3'), representing a part of primer SSP-T (described below), and HE112-HE064. The extreme 5'-end sequence (nt 167) was determined by a modified rapid amplification of cDNA ends (RACE) technique called RNA ligase-mediated RACE (RLM-RACE) with the First Choice RLM-RACE kit (Ambion), as described previously (Okamoto et al., 2001
). Briefly, the extracted RNA that was treated with calf intestinal phosphatase and then with tobacco acid pyrophosphatase was ligated to an RNA adapter supplied in the kit and this was used as a template to synthesize cDNA with an HEV-specific antisense primer, HE035, and SuperScript II RNase H- reverse transcriptase (Invitrogen). The cDNA was then amplified by nested PCR with TaKaRa Ex Taq (TaKaRa Shuzo) and the following primers: two RNA adaptor primers supplied in the kit were used as forward primers and HE034 and HE033 were used as reverse primers in the first and second round, respectively. Amplification of the extreme 3'-end sequence [nt 71177240 excluding the poly(A) tail] was attempted by the RACE method with a 20-mer primer (#167, 5'-CCGTCGACATCGATAATACG-3') representing a part of a 41-mer oligonucleotide containing (T)15 [SSP-T, 5'-AAGGATCCGTCGACATCGATAATACG(T)15-3'] and an HEV-specific sense primer (HE024) on cDNAs that had been reverse-transcribed from poly(A)+ RNAs with the 41-mer oligonucleotide with (T)15, 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. 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 the final tree was obtained using the TreeView program, version 1.6.6 (Page, 1996
).
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The Japanese swine HEV isolate, swJ13-1, was isolated in August 2002 from a serum sample obtained from a 4-month-old pig raised in a swine farm near Sapporo in Hokkaido. On the other hand, the Japanese human HEV isolate, HE-JA1, was recovered from a 55-year-old man who lived in Sapporo City in Hokkaido and who had contracted sporadic acute hepatitis E in December 1997. The infected patient in the current study had no history of travel to areas endemic for HEV, no known contact with persons who had travelled to endemic areas and no contact with farm pigs. There are accumulating lines of evidence that a number of animal species other than pigs, such as rats, mice, dogs, cows, sheep and goats, may act as natural reservoirs of HEV (Favorov et al., 1998, 2000
; Kabrane-Lazizi et al., 1999
; Purcell & Emerson, 2001b
; Tien et al., 1997
). Therefore, it is tempting to speculate that cross-species transmission occurs from pigs to other animals and that these animals act as vehicles to transfer HEV from pigs to humans, since HEV viraemia was detected at a high frequency of 15 % (113/750) among 3-month-old pigs and antibodies to HEV were highly prevalent among 4- to 6-month-old pigs in Japan (89 % or 1110/1250) in our previous study (Takahashi et al., 2003
). Recently, He et al. (2002)
reported that rodents were infected with a genotype I HEV with 9596 % identities in the 405 nt ORF2 sequence with human HEV strains circulating in Nepal. Therefore, such studies should be expanded to the domestic and wild animals present in industrialized countries that have a higher chance of contact with humans to elucidate the infectious source(s) and route(s) for acquiring sporadic hepatitis E in low endemic countries, including Japan.
In conclusion, the finding that a pair of swine and human HEV strains of genotype IV with 99 % identity over the entire genome and with 99·8, 100 and 100 % identities in the amino acid sequences of ORFs 13, respectively, was present in a restricted area in Hokkaido where clinical HEV infection occurs most frequently in Japan (Mizuo et al., 2002), further supports the notion that HEV is a potential zoonotic virus. However, no direct evidence of HEV infection from swine to humans has been obtained. Further clinical and ecological studies on HEV infection in humans and candidate animals to clarify how domestic HEV strains circulating in animal species are transmitted to humans are warranted.
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ACKNOWLEDGEMENTS |
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Received 19 December 2002;
accepted 20 January 2003.