Clinical cases of hepatitis E have been rare in Western Europe, Oceania, and North American countries; and the cases in these countries are often associated with travel to regions where hepatitis E is endemic. Recently, HEV has been identified in human hepatitis E patients in the United States, where HEV is considered nonendemic, with no association with international travel (13
). Subsequently, the HEV sequence has been identified in humans in Austria, Italy, Spain, Greece, and Japan (19
). When the sequences of these isolates are compared to those of two classical types of HEV (designated genotype 1 and genotype 2 for the Asian and Mexican genotypes of HEV isolates, respectively) (31
), the sequence identities are strikingly low (only 70 to 72%). In parallel, an HEV strain has been isolated from pigs in the United States (22
), and it has been found that the nucleotide sequence of this swine form of HEV is similar to the sequences of the human HEV strains isolated from countries where HEV is believed to be nonendemic. The U.S. human HEV strains and the HEV strain US swine, all of which were isolated in the United States, where HEV is considered nonendemic, show an overall sequence identity of 91%, whereas the identities of the nucleotide sequences of these strains compared to those of the genotype 1 and 2 strains are 73 to 74% (Table ).
No known clinical case of HEV in humans has been reported in Canada, and accordingly, Canada has been considered free of HEV, at least in humans. Recent studies, however, have revealed that a large proportion (38 to 88%, depending on the areas tested) of commercial pigs become seropositive for HEV by 6 months of age (36
), suggesting active circulation of HEV in Canadian swine herds. Indeed, a portion of the HEV genomic sequence has been identified from the feces of pigs, and the virus has been recovered from these pigs. This piece of evidence demonstrates that HEV is truly circulating in pigs and the virus is being shed in the feces of infected animals. The HEV-infected pigs are clinically normal and do not exhibit any abnormal symptoms as a result of the infection. Similar serological observations have been made for pigs in the United States, New Zealand, and Australia (3
). All of these countries are considered free of human HEV, but swine HEV appears to be prevalent. A recent study with a relatively large number of subjects (389 individuals in the United States who work with swine) indicates that swine veterinarians are at higher risk of HEV infection than healthy blood donors (17
). Independently from this study, swine farmers have also been shown to have significantly higher levels of antibodies to HEV (14
). Thus, it seems that HEV in pigs may cross species barriers and be able to infect humans. HEV appears to be ubiquitous in pig populations worldwide, as it has been demonstrated in the United States, Canada, New Zealand, Australia, Taiwan, Korea, China, Japan, Spain, India, and The Netherlands (1
). Taking these facts together, HEV is likely a zoonotic agent that is transmitted from pigs to humans.
Except for the genomes of the prototype swine HEV strain isolated in the United States and the swine HEV strain recently isolated in Japan, whose full-length genomic sequences have been determined, only small portions of the genomes from other swine HEV isolates have been sequenced to date; and the remainder of the genome is largely undetermined. Accordingly, little is known about the genomic nature of the swine form of HEV, while many strains of human HEV have been completely sequenced. Sequencing of the HEV genome is cumbersome. Difficulties include a lack of a virus cultivation system, a relatively short period of a viremic state in vivo upon infection, a low level of virus shedding in the feces, low degrees of sequence homology among HEV isolates, and high G+C contents in some parts of the genome. Naturally infected animals are healthy and have subclinical infections; therefore, it is difficult to identify HEV-positive animals in order to collect appropriate samples for evaluation. Despite these obstacles, we have determined the full-length genomic sequence of a swine HEV isolate directly from fecal specimens. The Arkell strain of HEV isolated from pigs in Ontario, Canada, appears to be genetically distant from the two classical types of HEV, with sequence identities to the genotype 1 and 2 sequences of only 73 and 74%, respectively. Instead, the Canadian Arkell virus sequence is rather closely related to that of the prototype swine HEV strain isolated in the United States and the swine HEV strain recently isolated in Japan, and the sequences of these isolates are some distance from those of both classical types (Fig. ). When the full-length Arkell virus genomic sequence is compared with those of genotype 3 HEV isolates, the sequence identities appear to be much lower than expected (only 87%). The phylogenetic studies indicate that the Canadian Arkell virus represents a distinct variant among the genotype 3 isolates (Fig. ). We have compared the sequences of the individual ORFs of the Arkell virus with the respective sequences of the ORFs of other swine HEVs, and very similar phylogenetic observations have been obtained (Fig. ). Partial sequences of swine HEV isolates have additionally been identified in New Zealand and Spain (6
). Phylogenetic studies with the available partial sequence of the ORF1 region indicate that the New Zealand swine HEV isolate is closely related to the European (Italy) human HEV isolate (88% identity) (Fig. ) (32
). When the New Zealand swine HEV isolate is compared to the Canadian strain, it is found that they share 85% identity. The Spanish swine HEV strain has 83.9% similarity to the New Zealand swine HEV in a region of 304 nucleotides in ORF2. The Spanish human HEV isolate and two Spanish swine HEV isolates share similarities of 92.1 and 94.0%, suggesting a possible relatedness of the swine and human HEV isolates in the same geographic region. While further information is required, it seems that genetic variations exist within genotype 3 HEV strains and that swine HEV isolates may have infected humans in the same geographic region. A recent study with a collection of swine HEV isolates recovered from six states in the United States also demonstrates the sequence heterogeneity of swine HEV isolates (10
Canadian swine HEV strain Arkell is distantly related from HEV strain US swine, while HEV strain US swine is more closely related to the U.S. human HEV isolates. In Canada, approximately 2% of the general population and 2.4% of Canadian external affairs employees have been found to be seropositive for HEV (4
), and a higher rate (7.4%) of positivity for antibodies to HEV has been shown among Indochinese immigrants. These cases are likely associated with travel to and from areas where HEV is endemic. Despite the absence of clear evidence of the active circulation of a human form of HEV in Canada, it is tempting to speculate that if HEV strains were isolated from humans residing in Ontario, Canada, they would be more closely related to the Arkell HEV strain than to the U.S. HEV strains. Recently, several new swine and human HEV sequences have been identified in China, where HEV is endemic, and in Taiwan, where HEV is not endemic. Sequence comparisons indicate that these HEV isolates are distant from the classical type of HEV but share rather high degrees of similarity to each other. The newly isolated Chinese HEV strain appears to be distinct from all three known genotypes and has been suggested to be the fourth genotype (31
). Accordingly, swine HEV strains isolated in Taiwan are clustered within genotype 4, while swine HEV strains isolated from imported U.S. pigs remain genotype 3 (33
). In contrast, swine HEV strains in India have been identified to be genotype 4, whereas human HEV strains in the same region remain genotype 1 (1
). Further studies are required to verify the geographic relationship between human and swine HEV strains.
Pigs naturally infected with swine HEV appear to be asymptomatic and healthy. Since a large proportion of the human population is reported to be seropositive for HEV and most of these individuals are asymptomatic, swine HEV infection seems mild. Swine is considered an attractive donor animal species for xenotransplantation, and in this regard, swine HEV is of concern as a potential xenogeneic agent that can be transmitted from pigs to humans via organ transplantation, especially under immunosuppressive conditions (35
). All other known swine HEV isolates reported to date have been identified in commercial swine herds, and our study is the first report on the isolation of swine HEV from specific-pathogen-free pigs. Our study indicates that swine HEV may be more widely distributed in pigs than was previously thought and that it is distributed not only in pig production units but also in animals raised in a closed environment for specific purposes. It raises a concern that potential donor pigs for organ transplantation should be closely monitored for the presence of swine HEV, even if they are housed under isolated conditions. The sensitivities and the specificities of the enzyme-linked immunosorbent assay and reverse transcription-PCR used for detection of HEV may need to be further elaborated, and the present study may be helpful in improving such techniques.