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
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ABSTRACT |
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The nucleotide sequences of the 137 swine HEV isolates reported have been assigned DDBJ/EMBL/GenBank accession nos AB094203AB094339.
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INTRODUCTION |
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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 IIV) (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.
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METHODS |
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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
).
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RESULTS |
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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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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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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 78·885·5 % nucleotide identity with eight human and swine isolates of cluster IVjp in the partial ORF2 sequence of 241263 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.
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DISCUSSION |
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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 8996 % 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 9294 % 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 5565-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.
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ACKNOWLEDGEMENTS |
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Received 22 October 2002;
accepted 18 November 2002.