Correlation between positivity for immunoglobulin A antibodies and viraemia of swine hepatitis E virus observed among farm pigs in Japan

Masaharu Takahashi1, Tsutomu Nishizawa1, Toshinori Tanaka1, Bira Tsatsralt-Od1, Jun Inoue1,2 and Hiroaki Okamoto1

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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To evaluate the usefulness of detection of antibodies to hepatitis E virus (HEV) to screen for viraemic pigs, serum samples obtained from 1425 1–6-month-old pigs in Japan were tested for swine HEV RNA and IgG, IgM and IgA classes of anti-HEV antibody. Fifty-five (5 %) of the 1071 2–5-month-old pigs were positive for swine HEV RNA, but none of 218 1-month-old pigs or 136 6-month-old pigs had detectable HEV RNA. The prevalence of anti-HEV IgG among the viraemic pigs (67 %, 37/55) was similar to that among the non-viraemic pigs (55 %, 757/1370) and the prevalence of anti-HEV IgM among the viraemic pigs and non-viraemic pigs was 7 and 3 %, respectively. However, anti-HEV IgA was detected significantly more frequently among viraemic pigs than among non-viraemic pigs (55 vs 10 %, P<0·0001). These results suggest that anti-HEV IgA is more useful than anti-HEV IgM to screen for viraemic pigs.

The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequence data reported in this paper are AB194476–AB194530.

Supplementary tables and phylogenetic trees are available in JGV Online.


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Hepatitis E virus (HEV), the causative agent of hepatitis E, is classified as the sole member of the genus Hepevirus in the family Hepeviridae. Its genome is a single-stranded, positive-sense RNA of approximately 7·2 kb, with three partially overlapping open reading frames (ORF1, -2 and -3) (Tam et al., 1991; Huang et al., 1992; Wang et al., 2000). Although only one serotype has been recognized, extensive genomic diversity has been noted among HEV isolates, and HEV sequences have been classified into four major genotypes, 1–4 (Schlauder & Mushahwar, 2001). Transmission of HEV in developing countries occurs primarily via the faecal–oral route through contaminated water supplies (Purcell & Emerson, 2001). Recent studies have indicated that zoonosis is involved in the transmission of HEV, especially in industrialized countries (Meng et al., 1997, 1998b, 1999; Erker et al., 1999; Harrison, 1999; Meng, 2000, 2003; Smith, 2001; Tei et al., 2003; Yazaki et al., 2003). Increasing lines of evidence indicate that pigs are animal reservoirs of HEV and that hepatitis E may be transmitted zoonotically from viraemic animals to humans (Meng et al., 1997; Harrison, 1999; Meng, 2000, 2003; Smith, 2001; Nishizawa et al., 2003; Okamoto et al., 2003; Takahashi et al., 2003a). Numerous HEV strains of genotypes 3 and 4 have been isolated from pigs in both developing and industrialized countries (Hsieh et al., 1999; Pina et al., 2000; Garkavenko et al., 2001; van der Poel et al., 2001; Arankalle et al., 2002; Huang et al., 2002a; Pei & Yoo, 2002; Wu et al., 2002; Choi et al., 2003). However, the extent of genomic variability and geographical distribution of swine HEV strains is not fully understood in Japan and there have been little or no data on the prevalence of IgM and IgA antibodies against swine HEV (anti-HEV) among domestic pigs. In the present study, we aimed to understand further the genomic heterogeneity of swine HEV strains throughout Japan and to elucidate whether detection of particular classes of anti-HEV antibodies is useful as a tool to screen for viraemic pigs.

Serum samples were obtained from 1425 pigs (mean age±SD, 3·5±1·6 months, range 1–6 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 8–360 (71·3±89·1) pigs at 1–20 (4·6±5·5) farms in 2001 and 2002 and were kept below –20 °C until testing.


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Table 1. Age-dependent prevalence of HEV RNA in the sera of farm pigs in 20 prefectures in Japan

–, Not tested or not applicable.

 
The serum samples obtained from all 1425 pigs were tested for HEV RNA by RT-PCR with the ORF2 primers as described previously (Mizuo et al., 2002). Although none of the 218 1-month-old pigs or 136 6-month-old pigs had detectable HEV RNA, HEV RNA was detectable in the swine serum samples from 11 (6 %) of the 2-month-old pigs, 32 (10 %) of the 3-month-old pigs, 10 (6 %) of the 4-month-old pigs and two (0·5 %) of the 5-month-old pigs (Table 1). HEV RNA was detected in pigs from 31 of the 92 farms tested. According to prefecture, pigs from 14 of the 20 prefectures had detectable HEV RNA; in the remaining six prefectures where no viraemic pigs were detected, the number of pigs aged 2–4 months in this study ranged from only five to 18. Therefore, in total, 55 viraemic pigs were identified in the current study. The swine HEV isolates recovered from the infected pigs were named as shown in Table 1.

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 76·4–100 % 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 79·9–98·8 (89·1±2·8) and 79·6–96·1 (86·9±2·7) %, respectively, at the nucleotide level, but they differed by 17·2–27·9 % 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 87·9–93·0 (91·1±2·0) and 83·5–89·3 (86·5±1·2) % similar to reported genotype 4 HEV isolates of Japanese and non-Japanese origin, respectively, but differed by 19·2–26·6 % 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 0–11·9 % different from each other and 10·7–16·5 % 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 0–20·1 % from each other and by 4·3–20·4 % 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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Fig. 1. Phylogenetic tree constructed by the neighbour-joining method based on the partial nucleotide sequence (412 nt; nt 5987–6398 of the HE-JA10 genome, GenBank accession no. AB089824) of the ORF2 region of 59 human and swine HEV isolates, using a chicken HEV isolate (AY535004) as an outgroup. Four prototype human HEV isolates of each of genotypes 1–4, whose entire sequences are known, and the 55 Japanese swine HEV isolates obtained in the present study were included. After the slash, the name of the country other than Japan where the HEV strain was isolated is shown. Bootstrap values of >70 % are indicated for the major nodes as a percentage of the data obtained from 1000 resamplings (Felsenstein, 1985).

 
To detect swine anti-HEV IgG, an in-house ELISA was performed as described previously (Takahashi et al., 2003a), using purified recombinant ORF2 protein of the HE-J1 strain (genotype 4) that had been expressed in the pupae of silkworm (Mizuo et al., 2002), with the modifications described below. The ELISA microplates (Greiner Bio-One) coated with the recombinant ORF2 protein were prepared as described previously (Takahashi et al., 2005). Each sample was added to each well of the microplate at a dilution of 1 : 100 in 10 mM Tris-buffered saline containing 40 % Block Ace (Dainippon Pharmaceutical), 0·18 % Tween 20 and a mock protein (A280=0·1) that had been obtained from the pupae of silkworm infected with non-recombinant baculovirus. As enzyme-labelled antibodies, peroxidase-conjugated rabbit IgG fraction to swine IgG (whole molecule) (MP Biomedicals) was used for the swine anti-HEV IgG assay, peroxidase-labelled goat IgG specific for the pig IgM-µ chain (Bethyl Laboratories) for the swine anti-HEV IgM assay and purified goat IgG against porcine IgA (Serotec) for the swine anti-HEV IgA assay. The A450 of each sample was read. To determine the cut-off values in the swine anti-HEV IgG, anti-HEV IgM and anti-HEV IgA assays, 118 control swine serum samples that were exclusively negative for HEV RNA (Takahashi et al., 2003a) were used as a panel. An A450 value of 0·274, 0·335 and 0·303 (mean+6SD) was used as the tentative cut-off value for the swine anti-HEV IgG, IgM and IgA assays, respectively. Test samples with A450 values equal to or greater than the cut-off value were considered positive for anti-HEV IgG, anti-HEV IgM or anti-HEV IgA. The specificity of the anti-HEV assays was verified by absorption test with the same recombinant ORF2 protein (50 µg ml–1 final concentration for anti-HEV IgG or anti-HEV IgA assay; 150 µg ml–1 final concentration for anti-HEV IgM assay) that was used as the antigen probe. Briefly, prior to testing, the serum sample was diluted to 1 : 100, 1 : 300, 1 : 1000 or 1 : 3000 to adjust its A450 value to below 1·5. If the A450 value of the tested sample decreased by >=50 % in the anti-HEV IgM assay or by >=70 % in the anti-HEV IgA or IgG assay after absorption with the recombinant ORF2 protein, the sample was considered to be positive for anti-HEV.

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 3–6 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 2–5 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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Table 2. Age-dependent prevalence and A450 values of anti-HEV IgG, IgM and IgA in the serum samples of farm pigs in relation to HEV viraemia

 
The reported results of the anti-HEV IgG assay in pigs in various parts of the world indicate that anti-HEV IgG is present among pigs in HEV-endemic countries, such as China, India, Indonesia, Nepal and Thailand, as well as among pigs in non-endemic countries, such as Australia, Canada, Korea, New Zealand, Taiwan and the USA (Clayson et al., 1995; Meng et al., 1997, 1999; Chandler et al., 1999; Hsieh et al., 1999; Garkavenko et al., 2001; Arankalle et al., 2002; Wang et al., 2002; Choi et al., 2003; Wibawa et al., 2004). However, there have been little or no data on the prevalence of swine anti-HEV IgM and anti-HEV IgA among domestic pigs. In the present study, the serum samples obtained from 1425 pigs were tested not only for anti-HEV IgG, but also for anti-HEV IgM and IgA to elucidate whether the ability to detect anti-HEV antibodies is correlated with viraemia among pigs in terms of immunoglobulin classes. Among the 1425 1–6-month-old pigs, anti-HEV IgG was detectable in 37 viraemic pigs (67 %) and 757 non-viraemic pigs (55 %) (P=0·0785) and anti-HEV IgM was detectable in only four viraemic pigs (7 %) and 37 non-viraemic pigs (3 %) (P=0·0462); although the latter showed a significant difference, anti-HEV IgM was detected in a very small percentage of the pigs and the result was not considered to be useful. Our results suggest that swine anti-HEV IgG and anti-HEV IgM assays are not useful as tools to screen for viraemic pigs. In support of our findings, it has been reported that anti-HEV IgM appeared earlier than anti-HEV IgG, but was detectable for only 1–2 weeks in domestic pigs that had been infected experimentally with swine or human HEV and that the A450 values of anti-HEV IgM were relatively low (below 0·5) (Meng et al., 1997, 1998a). In the current study, the A450 value of anti-HEV IgM ranged from 0·058 to 0·817 in the 2-month-old viraemic pigs, from 0·065 to 0·682 in the 3-month-old viraemic pigs and from 0·086 to 0·604 in the 4-month-old viraemic pigs; none of the 5- or 6-month-old pigs were positive for anti-HEV IgM. Evidence of clinical disease or elevation of liver enzymes or bilirubin was not found in pigs that had been infected experimentally with swine or human HEV (Halbur et al., 2001; Meng et al., 1998b). Hence, the lack of clinical disease in infected pigs may be associated with an impaired or reduced immune response of anti-HEV IgM.

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.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Ministry of Health, Labour and Welfare of Japan and from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


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Received 21 January 2005; accepted 2 March 2005.