Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus

Masaharu Takahashi1, Tsutomu Nishizawa1, Haruko Miyajima2, Yuhko Gotanda2, Teruhiko Iita3, Fumio Tsuda4 and Hiroaki Okamoto1

1 Immunology Division and Division of Molecular Virology, Jichi Medical School, Tochigi-Ken 329-0498, Japan
2 Japanese Red Cross Saitama Blood Center, Saitama-Ken 338-0001, Japan
3 Institute of Swine Industry, Ibaraki Prefectural Livestock Research Center, Ibaraki-Ken 300-0508, Japan
4 Department of Medical Sciences, Toshiba General Hospital, Tokyo 140-8522, Japan

Correspondence
Hiroaki Okamoto
hokamoto{at}jichi.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Japanese patients with sporadic acute hepatitis E are infected with polyphyletic strains of hepatitis E virus (HEV). Hepatitis E is considered a zoonotic disease. Thus far in Japan, only three strains of swine HEV have been identified and an antibody study for HEV antibodies has not been done on Japanese pigs. To determine the prevalence of swine HEV infection in Japan and the extent of genetic variation among Japanese swine HEV strains, we tested serum samples obtained from 2500 pigs from 2 to 6 months of age at 25 commercial swine farms in Japan for the presence of IgG antibodies to HEV and swine HEV RNA. Anti-HEV antibodies were detected in 1448 pigs (58 %). One-hundred-and-thirteen (15 %) of the 750 3-month-old pigs and 24 (13 %) of the 180 4-month-old pigs were positive for swine HEV RNA. The nucleotide sequence of a 412 bp region within open reading frame 2 of the 137 swine HEV isolates was determined. Sequence analyses revealed that the 137 isolates shared 76·6–100 % nucleotide sequence identities and were classifiable into genotype III (93 %) or IV (7 %) and that the isolates from the same farm were >=97·1 % similar to each other. Phylogenetic analysis showed that the Japanese swine and human HEV isolates segregated into four clusters, with the highest nucleotide identity being 94·4–100 % between swine and human isolates in each cluster. These results indicate that swine HEV is widespread in the Japanese swine population and further support the hypothesis that swine serve as reservoirs for HEV infection.

The nucleotide sequences of the 137 swine HEV isolates reported have been assigned DDBJ/EMBL/GenBank accession nos AB094203AB094339.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis E virus (HEV) is an unclassified virus that is the major causative pathogen of enterically transmitted non-A, non-B hepatitis in many developing countries in Asia, Africa and Latin America (Purcell & Emerson, 2001a). There is a growing consensus that HEV-associated hepatitis also occurs among individuals in industrialized nations who have no history of travel to areas endemic for HEV (Harrison, 1999; Purcell & Emerson, 2001a; Schlauder & Mushahwar, 2001).

The genome of HEV is a single-stranded, positive-sense RNA of approximately 7·2 kb and contains a short 5' untranslated region (5'UTR), three open reading frames (ORF1, ORF2 and ORF3) and a short 3'UTR terminated by a poly(A) tract (Reyes et al., 1990; Tam et al., 1991). HEV sequences have tentatively been classified into four major genetic groups (genotypes I–IV) (Schlauder & Mushahwar, 2001). The majority of HEV infections in several countries in Asia and Africa are caused by genotype I and the majority of HEV infections in Mexico and Nigeria are caused by genotype II, while only isolated cases of infection with HEV of genotype III or IV have been described in the US, European countries, Argentina, Taiwan and China (Hsieh et al., 1999; Pina et al., 2000; Schlauder et al., 1998, 1999, 2000; Wang et al., 1999, 2000, 2001; Worm et al., 2000; Zanetti et al., 1999). In Japan, 13 % (11/87) of the cases of acute non-A, non-B, non-C hepatitis were caused by HEV infection (Mizuo et al., 2002) and multiple HEV strains of genotype III or IV have been isolated from Japanese patients with sporadic acute hepatitis E who have never been abroad (Mizuo et al., 2002; Takahashi et al., 2001, 2002a, b), indicating that heterogeneous HEV strains are circulating in Japan, where HEV infection had been considered to be non-endemic.

Zoonotic spread of HEV is suspected, as swine and human HEV strains are closely related and cross-species infection between swine and humans has been documented (Erker et al., 1999; Hsieh et al., 1999; Huang et al., 2002a; Meng et al., 1997, 1998). Recently, it was shown that swine veterinarians in the US (Meng et al., 2002) and other pig handlers in China, Taiwan and Thailand (Hsieh et al., 1999; Meng et al., 1999) are at increased risk of zoonotic HEV infection, suggesting that swine are animal reservoirs for HEV infection. In Japan, three swine HEV strains with high similarity to Japanese isolates of human HEV have been isolated from farm pigs (Okamoto et al., 2001), providing evidence for circulation of swine HEV in Japanese pigs. However, no antibody study has been performed in Japanese swine and genetic analysis of swine HEV strains in Japan has been limited. Therefore, in the present study, we tested serum samples from 2500 pigs at 25 swine farms in Japan for relative titres of the IgG class of HEV antibodies (anti-HEV) and for the presence of HEV RNA, and determined the nucleotide sequence of the swine HEV strains isolated from the infected pigs, in order to investigate the prevalence of swine HEV infection among pigs of different ages in Japan and the extent of genetic variation among Japanese swine HEV strains. We also sought to define the genetic relatedness of Japanese swine and human HEV strains.


   METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sera from farm pigs.
Serum samples were obtained from 2500 pigs at 25 commercial farms located (from north to south) in Hokkaido, in Aomori and Akita on mainland Honshu and in Miyazaki and Kagoshima on Kyushu Island of Japan (see Table 1). At each swine farm, serum samples were collected from 20 pigs of 2 months of age, 30 pigs of 3 months, 20 pigs of 4 months, 20 pigs of 5 months and 10 pigs of 6 months of age between 2000 and 2002 and were kept below -20 °C until testing: 69 % of the swine serum samples were collected in 2002. In the present study, pigs of 55–65, 85–95, 115–125, 145–155, or 175–185 days after birth were regarded as pigs of 2, 3, 4, 5 or 6 months of age, respectively. None of the pigs came in contact with or were in proximity to imported pigs. All 25 herds investigated were maintained in similar conditions. In addition, control sera from 118 pigs (aged 1–6 months old) raised under conditions similar to those for specific-pathogen-free pigs at an HEV-free herd of swine in Japan (Institute of Swine Industry) were used to determine the cut-off value for the swine anti-HEV IgG assay.


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Table 1. Age-dependent prevalence and A450 values of anti-HEV IgG in the sera of farm pigs in 25 herds in Japan

 
ELISA for detecting anti-HEV antibodies.
To detect swine anti-HEV IgG, ELISA was performed using purified recombinant ORF2 protein of the HE-J1 strain (genotype IV) that had been expressed in the pupae of silkworm, according to the method described previously by Mizuo et al. (2002) with slight modifications. In brief, wells of microplates (Microlon 600; Greiner Labortechnik) were coated with 50 µl of the recombinant ORF2 protein (5 µg ml-1 in PBS) and incubated at room temperature for 4 h. One-hundred µl saline containing 40 % (v/v) calf serum (Gibco BRL) was added. The microplates were incubated at room temperature for 1 h with shaking. The blocking buffer was discarded and each well was washed with saline. Fifty µl of each sample was added to each well at a dilution of 1 : 100 in saline containing 40 % calf serum. The microplates were incubated at room temperature for 1 h with gentle agitation and then washed with washing buffer (0·05 % Tween 20 in saline). Fifty µl PBS containing 25 % (v/v) foetal bovine serum (Medical & Biological Laboratories) and peroxidase-conjugated rabbit IgG fraction to swine IgG (whole molecule) (ICN/Cappel) was added to each well. The microplates were incubated at room temperature for 1 h with gentle agitation and then washed. Fifty µl TMB soluble reagent (ScyTek Laboratories) was added to each well as a substrate. The plate was incubated at room temperature for 10 min in the dark, then 50 µl TMB stop buffer (ScyTek Laboratories) was added to each well. The absorbance (A) of each sample was read at 450 nm. Test samples with A450 values greater than or equal to the cut-off value were considered to be positive for anti-HEV IgG.

Detection of HEV RNA.
RT-PCR was performed for detection of HEV RNA. Total RNA was extracted from 100 µl of swine serum, reverse-transcribed and subjected to nested PCR with ORF2 primers, as described previously (Mizuo et al., 2002). Briefly, a part of the ORF2 sequence was amplified using the primer pair HE044 [sense: 5'-CAAGGHTGGCGYTCKGTTGAGAC-3' (H=A, T or C; Y=T or C; and K=G or T)] and HE040 [antisense: 5'-CCCTTRTCCTGCTGAGCRTTCTC-3' (R=A or G)] in the first round for 35 cycles and HE110-2 [sense: mixture of three sequences, 5'-GYTCKGTTGAGACCTCYGGGGT-3', 5'-GYTCKGTTGAGACCACGGGYGT-3' and 5'-GYTCKGTTGAGACCTCTGGTGT-3' (common nucleotides underlined)] and HE041 [antisense: 5'-TTMACWGTCRGCTCGCCATTGGC-3' (M=A or C; W=A or T)] in the second round for 25 cycles. The size of the amplification product of the first-round PCR was 506 bp and that of the second-round PCR was 458 bp. The amplification products were electrophoresed on a 1·5 % (w/v) NuSieve 3 : 1 agarose gel (FMC BioProducts), stained with ethidium bromide and photographed under UV light. To avoid contamination during PCR procedures, the guidelines of Kwok & Higuchi (1989) were strictly followed. Four negative controls and two positive controls were included for every 24 test samples. Results were recorded only when false-positive results were not obtained for the negative controls and HEV RNA was detected in the positive controls. The negative control was water, which was processed in the same manner as the serum samples. The positive control was serum from a Nepali patient with sporadic acute hepatitis E, infected with HEV of genotype I (Shrestha, 1987), at a dilution of 1 : 1000 in anti-HEV-negative human sera obtained from healthy individuals.

Sequence analysis of PCR products.
The amplification products were sequenced directly on both strands, using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Sequence analysis was performed using Genetyx-Mac version 11.2.2 (Software Development) and ODEN version 1.1.1 from the DNA Data Bank of Japan (DDBJ; National Institute of Genetics, Mishima, Japan) (Ina, 1994). Sequence alignments were generated by CLUSTAL W (version 1.8) (Thompson et al., 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 299 nucleotides). Bootstrap values were determined on 1000 resamplings of the data sets (Felsenstein, 1985). The final tree was obtained using the TreeView program (version 1.6.6) (Page, 1996).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Prevalence of anti-HEV in Japanese pigs
To determine the cut-off value in the swine anti-HEV IgG assay, 118 control swine sera that were exclusively negative for HEV RNA were used as a panel. The A450 values ranged from 0·033 to 0·253 and the value of 0·366, which was calculated as 6 SD above the mean value (0·083), was used as the tentative cut-off value. Using this cut-off value, serum samples obtained from the 2500 pigs were tested for anti-HEV IgG. The sera from 1448 pigs (58 %) were positive for anti-HEV, with the prevalence by swine farm ranging from 34 % to 75 % (Table 1). The prevalence of swine anti-HEV differed remarkably by age, being 7 % among the 2-month-old pigs, 40 % among the 3-month-old pigs, 87 % among the 4-month-old pigs and of note, 90 % among both the 5-month-old and 6-month-old pigs. When restricted to pigs of 4–6 months of age, swine anti-HEV was detected in 89 % of the pigs (1110/1250); its prevalence ranged from 66 % to 100 % in different herds. In 17 of the 25 herds, 90–100 % of the 4-month-old pigs had swine anti-HEV. Furthermore, in 24 of the 25 herds, 90–100 % of the 5-month-old and/or 6-month-old pigs had detectable anti-HEV.

Table 1 also indicates the mean A450 values of swine anti-HEV according to age (months after birth) of the pigs tested. Swine anti-HEV was detected at the highest A450 value (1·869±0·996) in 4-month-old pigs and the titre then decreased gradually, detectable at the mean A450 value of 1·637 in 5-month-old pigs and at 1·244 in 6-month-old pigs.

Prevalence of HEV RNA in Japanese pigs
The serum samples obtained from all 750 3-month-old pigs and all 250 6-month-old pigs were tested for HEV RNA by RT-PCR using ORF2 primers (Table 2). Although all 6-month-old pigs were negative for HEV RNA, HEV RNA was detectable in the swine serum of 113 (15 %) of 3-month-old pigs and in 16 (64 %) of the 25 farms, with the prevalence by swine farm ranging from 3 % (Farms 5 and 23) to 77 % (Farm 25). Among the 750 3-month-old pigs tested, 51 (7 %) were positive for HEV RNA but negative for anti-HEV IgG and 239 (32 %) were positive for anti-HEV IgG but negative for HEV RNA; 62 pigs (8 %) were positive for both. In the remaining nine farms where no HEV-viraemic pigs of 3 months of age were identified, the serum samples from 2- and 4-month-old pigs were additionally tested for HEV RNA. None of the 180 pigs of 2 months of age on the nine farms had detectable HEV RNA. In contrast, six of the nine farms had viraemic pigs of 4 months of age, with the prevalence ranging from 5 % to 55 %. Consequently, 137 viraemic pigs were identified in the current study: they were confirmed to be reproducibly positive for HEV RNA. The swine HEV isolates recovered from the infected pigs were named as shown in Table 2.


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Table 2. Age-dependent prevalence of HEV RNA in the sera of farm pigs in 25 herds in Japan

-, Not tested.

 
Genetic heterogeneity of swine HEV recovered from viraemic pigs in Japan
The amplification products of ORF2 (412 nucleotides; primer sequences at both ends excluded) from 137 viraemic pigs were sequenced and compared (Table 3). The nucleotide sequence identity among swine HEV isolates in the same herd ranged from 97·1 to 100 %, contrasting with that among swine HEV isolates in different herds (76·6–98·1 %). Compared with the inter-farm nucleotide sequence identity, the intra-farm nucleotide sequence identity was remarkably high in each farm, indicating the homogeneous nature and uniqueness of swine HEV isolates in each of the independent swine farms. Among the 137 swine HEV isolates obtained from the viraemic pigs, 128 isolates (93 %) were close to the prototype human HEV isolate of genotype III (US1) (Schlauder et al., 1998), with an identity of 83·0–93·4 % at the nucleotide level, but they differed by 18·2–24·9 % from known HEV isolates of other genotypes (I, II and IV). The remaining nine isolates were 85·9–87·1 % similar to the prototype human HEV isolate of genotype IV (T1) (Wang et al., 2000), but were only 78·1–82·5 % similar to known HEV isolates of the other three genotypes. These results indicate that 128 and nine Japanese swine HEV isolates obtained in the present study were classifiable into genotype III and genotype IV, respectively. Of the 22 herds from which HEV RNA was isolated, 20 herds (91 %) had pigs infected with swine HEV of genotype III and only two herds had pigs infected with swine HEV of genotype IV.


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Table 3. Intra- and inter-farm nucleotide sequence identities and genotypic grouping of swine HEV isolates obtained in the present study

 
Based on pairwise comparison of the 412-nucleotide ORF2 sequence, the swine isolates obtained in the present study were further segregated into four clusters (IIIjp, IIIus and IIIsp within genotype III and IVjp within genotype IV; tentatively named only in this paper): the letters ‘jp’ stand for ‘presumably Japan-indigenous’, the letters ‘us' for ‘US-like’ and the letters ‘sp’ for ‘close to Spanish isolates' (see Fig. 2). Cluster IIIjp comprised 73 swine HEV isolates, cluster IIIus comprised 38 isolates, cluster IIIsp comprised 17 isolates, and cluster IVjp comprised nine isolates (Table 4). Seven farms had pigs infected with swine HEV isolates of cluster IIIjp, 10 farms had pigs infected with cluster IIIus HEV, three farms had pigs infected with cluster IIIsp HEV and two farms had pigs infected with cluster IVjp HEV (Table 3).



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Fig. 2. Phylogenetic tree constructed by the neighbour-joining method based on the partial nucleotide sequence (299 nucleotides; nt 5968–6266 of the HE-JI4 genome) of the ORF2 region of 119 human and swine HEV isolates. Twenty-seven reported human and swine HEV isolates of genotypes I–IV whose entire or almost entire sequence is known, 70 reported isolates of genotypes III and IV (accession nos are indicated in parentheses) for which the ORF2 sequence of 300–1497 nucleotides has been determined and 22 representative Japanese swine HEV isolates (one from each of the 22 herds) obtained in the present study were included. For simplicity, the abbreviated names (swUS01–swUS27) are used for the 27 US swine isolates reported by Huang et al. (2002a). All Japanese human and swine HEV strains are indicated in bold for visual clarity. Vertical bars indicate clusters of Japanese human and swine HEV isolates. Bootstrap values of >70 % are indicated for the major nodes as a percentage of the data obtained from 1000 resamplings.

 

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Table 4. Comparison of the 412-nucleotide ORF2 sequences of 137 Japanese swine HEV isolates obtained in the present study with each other and with 42 human and swine HEV isolates whose entire or 412-nucleotide sequence is known

 
The intra-cluster nucleotide sequence identity was 88·6–100 % in cluster IIIjp, 90·0–100 % in cluster IIIus, 93·4–100 % in cluster IIIsp and 97·3–100 % in cluster IVjp, while the inter-cluster nucleotide identity was only 76·6–88·3 % (Table 4). When compared with known Japanese isolates of human HEV at the nucleotide level, the Japanese swine HEV isolates were highly homologous to human isolates in the same cluster, with 89·0–98·8 % identity in cluster IIIjp, 90·0–94·4 % identity in cluster IIIus, 89·3–90·8 % identity in cluster IIIsp and 87·9–100 % identity in cluster IVjp. On comparison with HEV isolates of non-Japanese origin whose entire or almost entire sequence is known, the cluster IIIus isolates were closely related to human HEV (US1 and US2) and swine HEV (swUS) of US origin (Erker et al., 1999; Meng et al., 1998; Schlauder et al., 1998), with 89·8–94·2 % nucleotide sequence identity, although the cluster IIIjp and cluster IIIsp isolates were only 82·0–89·8 % similar to these US human and swine strains. The cluster IVjp isolates were closest to the T1 isolate of genotype IV, which had been recovered from a sporadic case of acute hepatitis in China (Wang et al., 2000), although they had a nucleotide sequence identity of <90 % in the ORF2 sequence with the T1 isolate. When compared with HEV isolates of non-Japanese origin whose partial sequence is known, the cluster IIIsp isolates were most closely related to the Spanish HEV isolates (VH1, VH2 and E11) (Pina et al., 2000), with 84·6–87·6 % nucleotide identity in the ORF2 sequence of 304 nucleotides, among the human and swine HEV isolates thus far identified.

Phylogenetic analyses with reported swine and human HEV isolates
The phylogenetic tree constructed based on the partial ORF2 sequence of 412 nucleotides confirmed that the 137 swine HEV isolates obtained in the present study belonged to genotype III or IV and that they were segregated into three clusters (IIIjp, IIIus and IIIsp) within genotype III and one cluster (IVjp) within genotype IV (Fig. 1). The cluster IIIjp swine isolates grouped with several Japanese isolates of human HEV (cluster IIIjp human isolates: JRA1, HE-JA5, HE-JA6, HE-JA9 and HE-JA11); cluster IIIus swine isolates grouped with several other Japanese isolates of human HEV (cluster IIIus human isolates: JKN-Sap, JMY-Haw, HE-JI3, HE-JA4, HE-JA7, HE-JA8 and HE-JA10); cluster IIIsp swine isolates grouped with a different Japanese isolate of human HEV (cluster IIIsp human isolate: HEV-Sendai); and cluster IVjp swine isolates grouped with the remaining Japanese isolates of human HEV (cluster IVjp human isolates: JKK-Sap, JAK-Sai, HE-JI4, HE-JA1, HE-JA2 and HE-JA3).



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Fig. 1. Phylogenetic tree constructed by the neighbour-joining method based on the partial nucleotide sequence of the ORF2 region (412 nucleotides; nt 5916–6327 of the HE-JI4 genome) of 179 human and swine HEV isolates. Twenty-seven reported human and swine HEV isolates of genotypes I–IV whose entire or almost entire sequence is known (see Table 4 for the names of the isolates and relevant accession nos), 15 reported isolates of genotypes III and IV (accession nos are indicated in parentheses) whose partial sequence of 412 nucleotides is available and the 137 Japanese swine HEV isolates obtained in the present study were included. Japanese swine HEV isolates with a nucleotide sequence identity of >99·5 % are shown with a solidus; for example, ‘swJ1-1/2/3’ means swJ1-1, swJ1-2 and swJ1-3. Vertical bars indicate clusters of Japanese human and swine HEV isolates. Bootstrap values of >70 % are indicated for the major nodes as a percentage of the data obtained from 1000 resamplings. For visual clarity, Japanese isolates of human HEV are indicated in bold.

 
Fig. 2 shows the phylogenetic tree constructed based on the common 299 nucleotides within the ORF2 sequence of human and swine HEV strains of Japanese and non-Japanese origin. Phylogenetic analysis indicated that 17 Japanese human and swine isolates of cluster IIIus were segregated into a cluster, with a nucleotide identity of 89·3–99·7 %, although 11 US human and swine isolates were interspersed among the cluster IIIus human and swine HEV isolates of Japanese origin. The clusters IIIjp and IIIsp were clearly separate from the clusters consisting of US, Taiwanese and Spanish strains and from the cluster IIIus strains in the branch of genotype III, although the IIIsp strains were bifurcated from the common branch with the Spanish strains (VH1, VH2 and E11).

The human and swine HEV isolates of cluster IVjp belonged to genotype IV, but they were clearly separate from known genotype IV isolates from China and Taiwan (Fig. 2). Recently, Arankalle et al. (2002) reported 12 swine isolates of genotype IV in Western India, which shared only 78·8–85·5 % nucleotide identity with eight human and swine isolates of cluster IVjp in the partial ORF2 sequence of 241–263 nucleotides. The phylogenetic tree constructed by the neighbour-joining method based on the common 241 nucleotide sequence of ORF2 revealed that Indian isolates belong to a cluster of genotype IV that includes all the swine HEV isolates from India, two Taiwanese swine HEV isolates (TW32SW and TW74SW) and three human HEV isolates from China and Taiwan (181Ch, 210Ch and TW5483E). These results suggest that cluster IVjp Japanese human and swine isolates belong to a new subgroup of genotype IV, being separate from other subgroups into which the Chinese, Taiwanese and Indian isolates are classifiable.


   DISCUSSION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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The reported data on the anti-HEV IgG assay in pigs in various parts of the world indicate that anti-HEV IgG is present in pigs in HEV-endemic countries such as China, Nepal and Thailand, as well as in pigs in non-endemic countries such as Australia, Canada, Germany, New Zealand, Taiwan and the USA (Chandler et al., 1999; Clayson et al., 1995; Garkavenko et al., 2001; Hartmann et al., 1998; Hsieh et al., 1999; Meng et al., 1999), with the prevalence ranging from 4·1 % in Ontario, Canada, to 79·0 % in the midwestern USA (Meng et al., 1999). Thus, HEV is considered to be enzoonotic in pigs, even though hepatitis E is common or rare in the resident human population. In the present study conducted in Japan where clinical HEV infection rarely occurs, swine anti-HEV IgG was highly prevalent among Japanese pigs of 2–6 months of age (58 % or 1448/2500) in the 25 herds of swine located on Hokkaido Island, mainland Honshu and Kyushu Island of Japan. The prevalence of swine anti-HEV IgG differed by herd, ranging from 34 % to 75 %, but the prevalence among pigs of 5 or 6 months of age was remarkably high at 80–100 % in all herds. Consistent with previous reports (Meng et al., 1997, 1999), the prevalence of anti-HEV IgG in pigs increased with age, being 7 % among 2-month-old pigs and 40 % among 3-month-old pigs in the present study. Of note, 1110 (89 %) of the 1250 pigs of 4–6 months of age were positive for swine anti-HEV IgG, where the highest mean A450 value of 1·869±0·996 (SD) was seen among the 4-month-old pigs. The mean A450 value of anti-HEV IgG decreased gradually in 5- and 6-month-old pigs, reflecting transient infection of swine HEV at an early growing stage of the piglets.

Evidence is accumulating that hepatitis E is a zoonosis (Balayan, 1997; Harrison, 1999; Meng, 2000) and cross-species infection of HEV has been documented (Erker et al., 1999; Meng et al., 1998). Swine HEV strains were found in pigs from different geographic regions in the US and they showed nucleotide sequence identities of 89–96 % with human HEV strains of genotype III (US1 and US2) (Meng et al., 1997, 1998; Huang et al., 2002a). Similarly, Taiwanese swine and human isolates of HEV were closely related, with 97 % identity at the nucleotide level, forming a monophyletic group (Hsieh et al., 1999). Wu et al. (2002) also showed that HEV strains present in Taiwanese herd pigs were closely related genetically to human HEV found in hepatitis patients in Taiwan, but were distantly related to HEVs of other genotypes. Pina et al. (2000) reported the genetic identification of novel HEV strains from the sera of hepatitis patients (VH1 and VH2 in Fig. 2) and from sewage samples of animal origin from a slaughterhouse (E11) in Spain. The E11 strain was genetically distinct from known HEV strains worldwide, but was most closely related to the Spanish VH1 and VH2 strains of human HEV, with 92–94 % nucleotide sequence identities. Recently, Wang et al. (2002) reported that Chinese swine HEV strains most closely resembled viruses isolated from Chinese patients with sporadic acute hepatitis. These results suggest that in particular geographical regions, the HEV strains isolated from pigs and humans are closely related.

In Japan, only three swine HEV strains of genotype III had been isolated from farm pigs in a limited geographical area, which were up to 89 % similar to known Japanese isolates of human HEV (Okamoto et al., 2001). In the current study, a large number of farm pigs (2500 pigs) in 25 herds in Japan were tested. Reflecting the high prevalence of swine HEV infection among Japanese pigs, 137 HEV RNA-positive pigs of 3 or 4 months of age were identified, while none of the 6-month-old pigs had detectable HEV RNA. Considering food safety, it is fortunate that HEV viraemia was not detected in any pig of 6 months of age ready for sale. Furthermore, a recent study by Kasorndorkbua et al. (2002) has shown that the risk of contracting swine HEV infection through consuming uncooked virus-contaminated pork is extremely small, although pork is usually well cooked before ingestion. None of the 2-month-old-pigs tested in nine out of 25 farms had detectable HEV RNA. Maternal antibody from seropositive sows, which is reported to wane at about 8 weeks of age (Meng et al., 1997), might provide protection to 2-month-old pigs (or ‘55–65-day-old pigs' in the present study) from HEV infection. However, it has been reported that HEV RNA is detected among 2-month-old pigs at a frequency of 4·5 % (3/67) (Wu et al., 2002) or 3 % (2/73) (Okamoto et al., 2001). Therefore, we cannot rule out the possibility that some of the 2-month-old-pigs are HEV-viraemic in the remaining 16 farms where HEV RNA-positive pigs of 3 months of age were identified.

The 137 swine HEV isolates obtained in the present study were genetically heterogeneous and they segregated into four phylogenetic clusters within two major genotypes, III and IV (tentatively designated as clusters IIIjp, IIIus and IIIsp within genotype III and cluster IVjp within genotype IV), comparable with those of Japanese isolates of human HEV. Of particular interest, a pair of Japanese swine and human HEV isolates showed the highest nucleotide sequence identity in each of the four clusters: the highest identity at the nucleotide level was 98·8 % between swJ18-2 and HE-JA9 in cluster IIIjp, 94·4 % between swJ2-3 and HE-JA10 in cluster IIIus, 98·3 % between swJ791 and HEV-Sendai in cluster IIIsp, and 100 % between swJ13-1 and HE-JA1 in cluster IVjp. It is noteworthy that the Japanese swine HEV isolate of swJ13-1 was isolated from a viraemic pig from a herd of swine in Hokkaido and that the HE-JA1 isolate was obtained from a patient living in Hokkaido who had never been abroad (Mizuo et al., 2002), although the actual date of blood sampling from the infected pig and that from the patient were 5 years apart. With regard to the geographic distribution of the four clusters of Japanese swine HEV strains, clusters IIIsp and IVjp were restricted to swine farms in Hokkaido. On the other hand, cluster IIIjp swine HEV strains were not found in farms in Hokkaido, but tended to be prevalent in farms on Kyushu Island: five of the six farms on Kyushu Island had pigs infected with cluster IIIjp swine HEV. These results indicate that heterogeneous HEV strains are circulating in the Japanese swine population and further support the hypothesis that sporadic hepatitis E in Japan is a zoonosis. To draw a definitive conclusion on the region-dependent distribution of swine HEV genotype/clusters in Japan, additional studies with more viraemic pigs are required.

The source of the swine HEV variants in the 137 viraemic pigs studied is unclear. However, the presence of closely related swine HEV isolates with >=97·1 % nucleotide sequence identities within the same herd, but being clearly separate from those in the other herds, suggests that a certain independent swine strain(s) made an inroad into each herd in the past and that a predominant strain(s) unique to each herd has been maintained over a long period in the herds investigated. As illustrated in the phylogenetic tree constructed based on the partial ORF2 sequence (Fig. 2), clusters IIIjp and IIIsp within genotype III and cluster IVjp within genotype IV included Japanese swine and human HEV isolates, but not those from other countries, suggesting that these swine and human isolates classifiable into cluster IIIjp, IIIsp or IVjp had already been domestic and were widespread in the past in Japan. On the other hand, the cluster IIIus Japanese swine and human HEV isolates were interspersed among US swine and human strains (Fig. 2). Therefore, we cannot rule out the possibility of an outside source for the cluster IIIus HEV strains: they may spread unnoticed among pigs and possibly across countries through trading, as suggested in Taiwan (Wu et al., 2002). To elucidate this issue, global molecular epidemiological studies on swine and human HEV strains of various genotypes including genotype III are required.

The potential zoonotic infection of HEV is also supported by a report that veterinarians working with swine are at higher risk for HEV infection than normal blood donors in the US and other countries (Meng et al., 2002). In Taiwan, individuals who worked for pork companies but had no direct contact with pigs were twice as likely to have anti-HEV IgG compared with controls, and pig handlers were three times more likely to be HEV-seropositive than controls (Hsieh et al., 1999). In our previous study, we found that a high prevalence of HEV-associated hepatitis among sporadic acute hepatitis cases of non-A, non-B, non-C aetiology was significantly associated with males, higher age (>=40 years) and living in the northern part of Japan (Mizuo et al., 2002). As described above, swine HEV infection is widespread, not only in the northern part but also in the southern part of Japan. However, this cannot explain why sporadic acute hepatitis E occurs in Japan, with higher prevalence among males, among those over 40 years of age and among patients living in the northern part of Japan. Nearly all patients with hepatitis E in our previous studies did not report contact with swine before the onset of the disease (Mizuo et al., 2002; Takahashi et al., 2002b). Therefore, although our present results raise further public health concerns for HEV zoonosis, extended studies are required to examine whether circulation of HEV in swine and humans continues independently or whether cross-species infection takes place, as such data will have a major impact on the strategy for control of hepatitis E. Should cross-species infection take place, epidemiological studies are needed to clarify the possible transmission routes or factors that may be implicated in the cross-species infection of swine HEV to humans in a large cohort. Furthermore, there are a number of animal species other than pigs such as rats, mice, dogs, cows, sheep and goats that could also potentially serve as reservoirs (Favorov et al., 1998, 2000; Kabrane-Lazizi et al., 1999; Tien et al., 1997) and strains of HEV antigenically and genetically related to human HEV have been identified from chickens (Haqshenas et al., 2001; Huang et al., 2002b; Payne et al., 1999). The reported high prevalence of anti-HEV in a number of animal species other than pigs may suggest that multiple sources of exposure to HEV exist in the general population in industrialized countries who are not at apparent risk for exposure to HEV (Purcell & Emerson, 2001b).

In conclusion, swine HEV is highly prevalent and widespread in Japan, where clinical HEV infection is rare, and swine HEV isolates that segregated into four distinct clusters within two major genotypes (III and IV), comparable with those of Japanese isolates of human HEV, were identified from 137 viraemic pigs of 3 or 4 months of age. Our study may raise further public health concerns for HEV zoonosis. However, further efforts to clarify whether the domestic spread of HEV infection in industrialized countries occurs through zoonosis are warranted: direct evidence of HEV infection from swine to humans in clinical cases of hepatitis E is required in future studies.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We are grateful to Professor M. Mayumi for advice and encouragement during this study.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 22 October 2002; accepted 18 November 2002.