Serological evidence for swine hepatitis E virus infection in Australian pig herds

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Abstract

Hepatitis E virus (HEV) is an enterically transmitted human pathogen, with some similarities to caliciviruses. A variant of HEV was recently identified in pigs in the USA, infecting almost 100% of animals in commercial herds. Phylogenetic analysis suggests that this is a true ‘swine HEV’ distinct from the human virus, but the swine virus may also infect man. Using an in-house ELISA based on a highly conserved, recombinant HEV protein, we have examined collections of sera from Australian pigs for evidence of HEV infection in local pig herds. Sera from one research herd (n = 32) were uniformly non-reactive, and this was used to establish an assay cut-off (= mean + 3 SD of reference pig serum reactivities). Screening of sera from other herds demonstrates that swine HEV is present in Australia, with reactivity observed in 30% (12/40) of random samples from two piggeries, 92–95% of pigs by the age of 16 weeks in two other piggeries (n = 45), and 17% (15/59) of wild-caught pigs. Further studies are required to examine whether HEV causes disease in pigs and to determine the risk of swine HEV transmission to man.

Introduction

Hepatitis E virus (HEV) was first recognised as an enterically transmitted human pathogen, being the major cause of epidemic acute viral hepatitis in many developing countries. HEV infection is a self-limiting disease that does not progress to chronicity. Complete recovery is normally achieved within 2 months, with a 0.1–1% mortality rate from fulminant hepatitis. An increase in disease severity is observed in pregnant women, with a mortality rate of up to 25% during the third trimester. Hepatitis E virus is most prevalent in Africa, Asia and Central America, whereas in developed nations infection has primarily been recognised in travellers returning from endemic areas.

Cloning and sequencing of the HEV genome has identified a single-stranded positive sense RNA virus of approximately 7.5 kb in length (Reyes et al., 1990, Tam et al., 1991). The virion is non-enveloped and spherical, 27–34 nm in size with an indefinite surface structure. Electron microscopy suggests icosahedral symmetry, and physiochemical properties and overall genome organisation resemble those of the Caliciviridae family (Cubitt et al., 1995). Three open reading frames (ORFs) have been identified within the HEV genome (Fig. 1). The ORF 1 encodes non-structural proteins, whereas the major structural protein or capsid protein is encoded by ORF 2. The function of the immunogenic protein encoded by ORF3 is unknown.

During the 1980s, some evidence was presented on experimental infection of pigs with human HEV (Balayan et al., 1990), and serological evidence for HEV infection in pigs from areas endemic for human HEV has also been reported (Clayson et al., 1995). More recently, however, a variant of HEV was identified in commercial pig herds in the USA, where human HEV infection is not thought to be widespread (Meng et al., 1997). Phylogenetic analysis suggests that this is a true ‘swine HEV’ distinct from the human virus. Homology between the swine and human strains in the capsid protein region was found to be 79 and 90% with respect to nucleotide and deduced amino acids, respectively. Evidence for infection with swine HEV was found in almost 100% of pigs greater than 3 months of age from all USA herds tested (with the exception of a specific pathogen free [SPF] herd) (Meng et al., 1997). These results indicate that the virus causes almost universal infection of young pigs. No evidence was found for clinical disease in HEV-infected pigs, although histology detected mild inflammation consistent with subclinical hepatitis. However, it should be noted that children rarely develop clinical hepatitis following infection with the human HEV.

In 1997, the isolation of HEV RNA (strain HEV US-1) from a patient with acute clinical hepatitis in the USA was reported (Kwo et al., 1997). This patient had not travelled to areas where HEV is endemic. The strain of HEV isolated was significantly divergent from other human isolates (76.8–77.5% range in nucleotide identities), and phylogenetic analysis of the HEV US-1 and swine viruses suggests that they may represent related isolates of a new strain of HEV (Schlauder et al., 1998). The discovery of this novel strain of HEV has raised many important questions regarding the virus, including whether HEV may be a significant zoonosis with the swine population as one of its hosts.

As HEV causes an acute infection with a limited excretion period, we considered it possible that Australian quarantine procedures might have coincidentally prevented the establishment of endemic swine HEV in this country. However, following examination of pig sera for IgG reactivity to HEV antigens, we report evidence which suggests that swine HEV infection is common in Australian commercial pig herds and wild pigs.

Section snippets

Serum samples

Swine serum samples were obtained from three standard commercial herds and an SPF commercial herd from rural New South Wales (including age-specific panels), from wild-caught pigs collected in the outback of the Northern Territory, and from a research herd located in Victoria (Table 1).

Detection of HEV antibody response

We have developed sensitive and specific enzyme immunoassays for the detection of antibody to human HEV in both western immunoblot (Li et al., 1994, Li et al., 1997) and ELISA formats (Anderson et al., 1999).

Results

HEV infection in humans is most commonly diagnosed by the detection of IgG or IgM anti-HEV. While commercial HEV ELISAs based on recombinant proteins are available, our ongoing studies suggest that an ELISA based on the ORF2.1 fragment offers more efficient and quantitative detection of IgG to diverse strains of HEV, by virtue of a highly conserved, conformational epitope unique to ORF2.1 (Anderson et al., 1999). In this study we investigated whether HEV was present in Australian pig

Discussion

The discovery of a strain of HEV within the USA pig population represented a significant progression in understanding the worldwide distribution of HEV (Meng et al., 1997). The isolation of a new strain within a non-endemic community raises many questions regarding possible hosts, routes of transmission, and the true seroprevalence in developed nations, which has been confusing (Mast et al., 1997, Mast et al., 1998, Thomas et al., 1997). The detection of these HEV isolates stimulated our

Conclusions

Swine HEV infection appears to be widespread in Australian commercial piggeries and in wild pigs. Assays based on a highly conserved epitope within human HEV strains are reactive with infected pig sera, but further improvements may be expected with the use of homologous viral sequences. The effects of swine HEV on animal production and its’ possible role in human disease remain to be established but are reasons for concern.

Acknowledgements

These studies were supported in part by Project Grant No. 950876 (to DAA) from the National Health and Medical Research Council and the Research Fund of the Macfarlane Burnet Centre for Medical Research. We are grateful to Wayne Brown, Frank Dunshea, John Walker and Bart Currie for gifting pig serum panels, to AMRAD Biotech for HEV ELISA plates, and to Tony Shannon, Robert Dixon and Joseph Torresi for helpful discussions.

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