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
Correspondence
Hiroaki Okamoto
hokamoto{at}jichi.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequence data reported in this paper are AB194476AB194530.
Supplementary tables and phylogenetic trees are available in JGV Online.
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MAIN TEXT |
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Serum samples were obtained from 1425 pigs (mean age±SD, 3·5±1·6 months, range 16 months) at 92 commercial farms in 20 prefectures including Hokkaido and Okinawa, which are the northernmost and southernmost prefectures of Japan, respectively (see Table 1): there were no overlapping serum samples or swine herds between the previous studies in seven prefectures (Okamoto et al., 2001
; Takahashi et al., 2003a
, b
; Tanaka et al., 2004
) and the present study. In each prefecture, serum samples were collected from 8360 (71·3±89·1) pigs at 120 (4·6±5·5) farms in 2001 and 2002 and were kept below 20 °C until testing.
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The amplification products of ORF2 (412 nt; primer sequences at both ends excluded) of the HEV isolates from the 55 viraemic pigs were sequenced directly on both strands as described previously (Okamoto et al., 2001) and sequence analysis was performed by using Genetyx-Mac version 12.2.6 (Genetyx) and ODEN (version 1.1.1) from the DDBJ (Ina, 1994
). Phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987
) based on the partial nucleotide sequence of the ORF2 region (412 or 298 nt). The 55 swine HEV isolates obtained from the viraemic pigs were 76·4100 % identical to each other and segregated into two phylogenetic groups (Fig. 1
). Among the 55 swine HEV isolates obtained in the present study, 52 (95 %) were close to the reported genotype 3 HEV isolates of Japanese and non-Japanese origin, with an identity of 79·998·8 (89·1±2·8) and 79·696·1 (86·9±2·7) %, respectively, at the nucleotide level, but they differed by 17·227·9 % from known HEV isolates of the other three genotypes (1, 2 and 4) (see Supplementary Table S1, available in JGV Online). The remaining three isolates were 87·993·0 (91·1±2·0) and 83·589·3 (86·5±1·2) % similar to reported genotype 4 HEV isolates of Japanese and non-Japanese origin, respectively, but differed by 19·226·6 % from known HEV isolates of the other three genotypes. These results indicate that 52 and three Japanese swine HEV isolates obtained in the present study are classifiable into genotype 3 and genotype 4, respectively, and that these swine genotype 3 and genotype 4 HEV isolates are closer to known Japanese HEV isolates than to HEV isolates of non-Japanese origin of the respective genotype, as illustrated in the Supplementary Figure (available in JGV Online). In Japan, a total of 212 swine genotype 3 HEV isolates and 13 swine genotype 4 HEV isolates have been identified to date, including those obtained in the present study. These swine genotype 4 HEV isolates are 011·9 % different from each other and 10·716·5 % different from swine genotype 4 HEV isolates reported outside Japan (China, India, Indonesia and Taiwan). More remarkably, Japanese swine HEV isolates of genotype 3 differed by 020·1 % from each other and by 4·320·4 % from those of non-Japanese origin (Canada, the Netherlands, Spain, Taiwan and the USA), indicating that Japanese pigs are infected with HEV strains of two distinct genotypes that are markedly heterogeneous.
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By using the cut-off value of 0·274, serum samples obtained from the 1425 pigs in Japan were tested for the presence of swine anti-HEV IgG. The serum samples from 794 pigs (56 %) were positive for swine anti-HEV IgG, with the prevalence by prefecture ranging from 34 to 100 %. The prevalence of swine anti-HEV IgG differed remarkably by age, being 10 % among the 1-month-old pigs, 17 % among the 2-month-old pigs, 67 % among the 3-month-old pigs, 83 % among the 4-month-old pigs, 73 % among the 5-month-old pigs and 74 % among the 6-month-old pigs. When restricted to pigs of 36 months of age, swine anti-HEV IgG was detected in 73 % of the pigs (739/1009) (see Supplementary Table S2). By using the cut-off values described above, the serum samples obtained from the 1425 pigs in the present study were further tested for the presence of swine anti-HEV IgM and anti-HEV IgA (Table 2). Swine anti-HEV IgM was detected in the serum samples from 41 pigs (3 % or 41/1425), including four viraemic pigs. In contrast, swine anti-HEV IgA was detected in the serum samples from 169 pigs (12 % or 169/1425), including 30 viraemic pigs. When the age-dependent prevalence of anti-HEV antibodies was compared between pigs that were or were not viraemic, the prevalence of anti-HEV IgM did not differ significantly between the viraemic and non-viraemic pigs in any of the three age groups of 2, 3 and 4 months; the prevalence of anti-HEV IgG also did not differ statistically between the viraemic and non-viraemic pigs in any of the four age groups of 2, 3, 4 and 5 months. Of interest, however, was the fact that anti-HEV IgA was detected significantly more frequently among viraemic pigs than among non-viraemic pigs in the age group of 2 months (36 vs 9 %, P=0·0043) and in the age group of 3 months (63 vs 20 %, P<0·0001). Among serum samples obtained from all 1425 pigs, the prevalence of anti-HEV antibodies among viraemic pigs and that among non-viraemic pigs did not differ significantly in the anti-HEV IgG assay (67 vs 55 %), although they did differ significantly in the anti-HEV IgM and anti-HEV IgA assays (7 vs 3 %, P=0·0462; 55 vs 10 %, P<0·0001, respectively); however, the proportion of viraemic pigs that were positive for anti-HEV IgM was extremely low at 7 %. Although the prevalence of anti-HEV IgA among viraemic pigs was comparable with that of anti-HEV IgG among viraemic pigs, the accuracy of screening for positivity or negativity of HEV viraemia among pigs of 25 months of age based on the anti-HEV antibody status was significantly higher in the anti-HEV IgA assay than in the anti-HEV IgG assay [85 % (909/1071) vs 39 % (417/1071), P<0·0001].
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Of interest, anti-HEV IgA was detected significantly more frequently among viraemic pigs than among non-viraemic pigs (55 vs 10 %, P<0·0001). In humans, the IgA anti-HEV test has been utilized as an additional confirmatory test for recent HEV infection (Chau et al., 1993; Tokita et al., 2003
). Recently, we found that detection of anti-HEV IgA alone or with anti-HEV IgM is useful for serological diagnosis of hepatitis E with increased specificity and longer duration of positivity than that by RNA detection (Takahashi et al., 2005
). IgA anti-HEV was detectable in two out of four patients with subclinical HEV infection in the absence of alanine aminotransferase elevation, who were exclusively negative for anti-HEV IgM (Mitsui et al., 2004
), suggesting that detection of anti-HEV IgA is useful for serological diagnosis of acute HEV infection in the absence of anti-HEV IgM.
In conclusion, our present study indicates that HEV is highly prevalent among domestic pigs in swine farms distributed from Hokkaido to Okinawa in Japan and that markedly heterogeneous swine HEV strains of genotypes 3 and 4 are circulating in Japan, some of which are highly similar to HEV strains isolated from patients with domestically acquired hepatitis E in the same geographical region. In addition, the current study suggests that some pigs do not have the ability to generate and maintain a detectable antibody level of swine anti-HEV IgM after HEV infection, and that the anti-HEV IgA assay is more useful than the anti-HEV IgM assay as a tool to screen for viraemic pigs. Previous seroepidemiological studies revealed that anti-HEV IgG antibodies are present in numerous animal species other than pigs, including rodents, chickens, dogs, cats, cows, sheep and goats (Smith, 2001; Huang et al., 2002b
, 2004
; Usui et al., 2004
). However, HEV or HEV-like viruses have not yet been isolated from most of these animal species. Whether detection of anti-HEV IgA in pigs and other animals that may be natural reservoirs of HEV is useful as a tool to screen for viraemia deserves further analysis.
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ACKNOWLEDGEMENTS |
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Received 21 January 2005;
accepted 2 March 2005.