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

Veterinary Microbiology 68 (1999) 95±105

Serological evidence for swine hepatitis E virus infection in Australian pig herds Jenalle D. Chandlera, Michaela A. Riddella, Fan Lia, Robert J. Loveb, David A. Andersona,* a

Macfarlane Burnet Centre for Medical Research, Yarra Bend Road, P.O. Box 254, Fairfield 3078, Vic., Australia b Department of Veterinary Clinical Sciences, University of Sydney, Werombi Road, Camden 2570, NSW, Australia

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. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis E virus; HEV; Pig; ELISA

1. 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. *

Corresponding author. Tel.: +61-3-9282-2239; fax: +61-3-9282-2100 E-mail address: [email protected] (D.A. Anderson) 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 0 6 5 - 6

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Fig. 1. Schematic representation of the HEV genome. PORF1, non-structural polyproteins; PORF2, structural (capsid) proteins; PORF3, immunogenic protein of unknown function.

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 HEVinfected 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

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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. 2. Materials and methods 2.1. 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 wildcaught pigs collected in the outback of the Northern Territory, and from a research herd located in Victoria (Table 1). 2.2. 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, 1997) and ELISA formats (Anderson et al., 1999). These assays are based on a recombinant protein which presents both linear and conformational epitopes. This `ORF2.1' protein represents the Table 1 Pig serum collections examined for IgG anti-HEV Herd (type)

Location (state)

Age (weeks)

Samples tested (n)

A (commercial) B (commercial) C (wild-caught) D (research) E (commercial)

NSW NSW NT Victoria NSW

F (SPF commercial)

NSW

random random random 20 7±9 11±12 14 16 19 9 13 17 21 28±30

20 20 59 32 36 34 20 20 20 25 25 25 25 25

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carboxy terminal 40% of the viral capsid protein and reacts with antibody against all known human HEV strains, including the Mexican strain which is the most divergent (Huang et al., 1992). In this region of the capsid protein, the swine strain is no more variant from Chinese HEV (used in our assays) than is the Mexican strain (93% amino acid homology). We, therefore, reasoned that the conserved epitopes in the recombinant protein are likely to be reactive with antibody to swine HEV. 2.2.1. Enzyme immunosorbent assay (ELISA) An in-house ELISA was used to detect HEV antibody reactivity in serum samples. The assay detects serum IgG levels to ORF2.1 using a recombinant GST-ORF2.1 antigen following a standard protocol (AMRAD, Australia) (Anderson et al., 1999), modified by replacement of secondary antibody with peroxidase-labelled goat anti-swine IgG (ICN, USA). In brief, sera diluted 1 : 300 were incubated with the GST-ORF2.1 bound to a microtitre well. Anti-swine conjugate diluted 1 : 10 000 was used to detect the specifically bound anti-HEV IgG with tetramethylbenzidine substrate. The assay cutoff was determined with reference to a negative pig population (see Section 3.1). 2.2.2. SDS-PAGE and western blotting Antibody to swine HEV was also detected by western immunoblotting, employing GST-fusion proteins produced from partially overlapping clones of the ORF 2 region, as described previously (Li et al., 1997). Differential reactivity to these proteins has been shown to correlate with maturation of the antibody response, with acute-phase reactivity directed against a broad range of epitopes whereas the dominant convalescent reactivity is directed against a conformational epitope, optimally presented in the ORF2.1 antigen (Li et al., 1997). Briefly, fusion proteins were subjected to 10% SDS-PAGE, proteins transferred to nitrocellulose, and the membranes were blocked with 3% casein in TBST (0.1M Tris pH 7.5, 0.1M NaCl, 0.3% Tween 20 (Sigma, USA)) and then probed with selected pig sera diluted 1 : 500 in TBST containing 1% casein. HRP-conjugated goat anti-swine IgG was used to counterstain the membranes and enzyme complexes were detected by enhanced chemiluminescence (ECL; Amersham, UK). 2.3. Statistical analysis Sample to cutoff ratios for age-specific sets of sera were statistically analysed by ANOVA using STATA (STATA 5.0 for Macintosh, STATA Corporation, USA). 3. 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

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study we investigated whether HEV was present in Australian pig populations using this `in-house' ELISA, and by analysing the dynamics of anti-HEV antibody responses by western immunoblotting in comparison with well characterised responses in patients and macaques (Li et al., 1997) 3.1. Pilot study of pig IgG reactivity to recombinant HEV ORF2.1 antigen The ORF2.1 ELISA has been optimised for detection of human IgG anti-HEV, with blood donor sera from a non-endemic region being used to establish baseline reactivity (Anderson et al., 1999). Preliminary studies were, therefore, performed to establish baseline reactivity for pig sera, with the primary aim of determining a `negative' population, using sera from two commercial pig herds (Herds A and B), wild-caught pigs from the Northern Territory (Herd C), and a small research herd in Victoria (Herd D). Except for herd D, where all animals were 20 weeks of age, no information was available on the ages of animals. As shown in Fig. 2, sera from herds A, B and C demonstrated widely varying levels of reactivity, whereas herd D demonstrated low and relatively uniform reactivities consistent with the husbandry practices for this research herd which might be expected to prevent the introduction of HEV infection. Analysis of the reactivities for Herd D (n = 32) showed an approximately normal distribution (not shown) as seen for negative human samples in this assay, whereas those for Herds A, B and C had significant numbers of `outlying', highly reactive samples. We, therefore, considered Herd D to represent an HEV-negative pig population, and the mean + 3 SD of this population was used as the cutoff for subsequent studies. In practice, the OD for a single reference serum on each plate (serving as negative control) was multiplied by a factor of 1.4, calculated to be equivalent to the cutoff.

Fig. 2. Pig IgG reactivity to HEV ORF2.1 antigen. Sera from pigs in various Australian domestic herds (Herds A, B and D) or wild pigs (Herd C) were assessed for anti-HEV by ELISA, and assay ODs are shown. Herd D showed uniformly low reactivity consistent with an uninfected herd, and was used to establish the assay cutoff.

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Table 2 HEV Seroprevalence in various pig herds Herd

Herd size (n)

No. swine with -HEV (%)

A (commercial) B (commercial) C (wild-caught) F (research)

20 20 59 32

6 (30) 6 (30) 15 (17) 0 (0)

On this basis, significant levels of anti-HEV were found in 30% of animals from Herds A and B (Table 2). In comparison, 17% of wild-caught pigs were reactive, which may suggest that HEV infection is more prevalent in commercial animals than those found in the wild. However, it should be noted that many serological assays for human HEV detect high levels of reactivity during the acute phase of infection, but levels of detectable antibody rapidly decline to baseline levels during convalescence. Because we do not know the ages of animals in Herds A, B and C, we cannot yet determine whether the moderate rate of reactivity observed is due to a low infection rate, or the loss of detectable antibody as seen in human HEV with first-generation IgG assays. To assess this question we examined age-specific serum panels from two separate, large commercial herds. 3.2. Age-specific seroprevalence and seroreactivity to HEV in commercial pig herds The anti-HEV antibody responses in animals of different ages was examined by using age-specific serum panels from a standard commercial herd (Herd E) and an SPF commercial herd (Herd F) from NSW (Fig. 3). Fig. 3(A) illustrates the percentage of

Fig. 3. Age-specific IgG reactivity to HEV in two commercial pig herds. The percentage of samples positive for IgG anti-HEV at each age is shown. (A) Standard commercial herd; (B) SPF commercial herd.

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specimens with positive anti-HEV IgG activity from Herd E. Following a decline between Weeks 7 and 9, presumably due to loss of maternal (colostrum) IgG, a steady increase in reactivity to HEV ORF2.1 was observed, from 6% of samples taken from pigs of 9 weeks age, to 95% of animals by the age of 16 weeks. However, by week 19, the rate of positivity has decreased to 80%. The specimens examined from the SPF herd demonstrated a largely similar pattern of reactivity as for Herd D (Fig. 3(B)). By week 13, 80% of samples were strongly reactive to HEV ORF2.1. This infection rate increased to 92% by Weeks 28±30. These results suggest that HEV infection within the Australian pig population is most likely almost universal, as might be expected for an enteric infection in high density farming. Accordingly, we hypothesised that the lower infection rates observed in Herds A, B and C may be due to a decline of anti-HEV IgG reactivity following acute infection, as is seen in humans, leading to a loss of reactivity in some older convalescent animals. We, therefore, examined the reactivities of the age-specific serum panels in more detail, comparing the sample/cutoff ratios for each age group (Fig. 4). These ratios were also compared by ANOVA. For herd E (Fig. 4(A)), it can be seen that the levels of antibody (which are directly proportional to the sample/cutoff ratio; results not shown), as well as rate of positivity, increase from Weeks 9 to 16. Most significantly, the mean level of

Fig. 4. Levels of IgG reactivity in pigs of different ages. The sample/cutoff ratio for individual sera at each age is shown. (A) Standard commercial herd; (B) SPF commercial herd, as for Fig. 3. Mean reactivities for each age are shown as solid bars.

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antibody then declines between Weeks 16 and 19 (p < 0.05), even though it can be assumed that almost all the animals in this herd have been infected. This pattern is consistent with exposure of most animals shortly after weaning at 7 weeks, with largely uniform antibody responses thereafter and an eventual waning of detectable antibody. In contrast, no such trend in the mean levels of antibody was seen for Herd F (Fig. 4(B)). However, the detection of individual, highly reactive sera within each age group suggests that infection in this SPF herd is more sporadic, although all animals may eventually be infected. On the basis of these cross-sectional studies, we believe that many animals will eventually lose reactivity in the current ORF2.1 ELISA, which may account for the intermediate prevalence observed in Herds A, B and C. Further, longtitudinal studies are required to address this issue directly. 3.3. Fine specificity of anti-HEV responses The apparent decrease in reactivity observed in one age-specific panel (Herd E) suggests that during convalescence, the antibody response in pigs may be maturing to a narrow range of epitopes with a lower overall level of reactivity. This has previously been demonstrated for HEV-infected macaques using a series of partially overlapping ORF2 recombinant antigens, representing N-terminal truncations and extensions relative to ORF2.1 in the western blot format (Li et al., 1997). We, therefore, used the same methodology to compare presumed `acute' and `convalescent' pig sera. A number of serum samples exhibiting significant anti-HEV IgG reactivity were initially screened by western blotting for activity against two of the recombinant proteins: ORF2.1 and the larger, overlapping C2 (results not shown). Human acute (<3 months postinfection) and convalescent serum samples (>3 months postinfection) were used as controls. IgG from the human acute-phase control reacted to both antigens, whereas antibody from the human convalescent serum demonstrated activity only to ORF2.1. The various pig serum samples reacted either to both proteins or to ORF2.1 only, consistent with different stages of infection, with a general trend that older pigs from both Herds E and F had a predominance of ORF2.1 reactivity (results not shown). Several samples that were evidently from animals in either the acute or convalescent phases were then assayed against the entire panel of overlapping ORF2 antigens. IgG from an `acute-phase' pig (13 weeks, Herd F) was highly reactive against the whole series of ORF2 fragments (Fig. 5A). Conversely, anti-HEV IgG in `convalescent' pig sera (28± 30 weeks, Herd F) reacted only to ORF2.1 (Fig. 5(B)). IgG from convalescent swine sera is, thus, reacting specifically to the conformational ORF2.1 epitope, as shown by the lack of reactivity against 2.1 ÿ 1 and 2.1 + 1 (ORF2.1 ÿ 20 aa or + 20 aa, respectively). This demonstrates that the conformational ORF2.1 epitope is important in the pig response to HEV, as in humans and macaques infected with human strains of HEV (Li et al., 1997). The possible loss of reactivity in most animals over time, suggested by the waning levels in Herd E between 16 and 19 weeks, implies that the current ORF2.1 employed in the immunoassays may not be ideal for the detection of past infections with pig HEV. Efforts

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Fig. 5. Western immunoblot of presumed acute- and convalescent -phase pig sera against a panel of partially overlapping recombinant ORF2 antigens, including the conformational ORF2.1 antigen. GST-fused proteins were electrophoresed on a 10% acrylamide gel, the proteins were transferred to nitrocellulose and the membrane was blocked with casein and probed with pig sera. Immune complexes were detected by HRP-streptavidin (molecular weight markers, lane MW) and by HRP-conjugated anti-pig IgG, with enhanced chemiluminescence. (A) Immunoblot with presumed acute-phase pig serum. ORF2 protein fragments are illustrated above the corresponding lanes. (B) Immunoblot with presumed convalescent-phase pig serum. Note that convalescentphase antibodies react preferentially with the conformational epitope unique to ORF2.1.

are thus required to make swine HEV-specific reagents with the characteristics of ORF2.1 (i.e., conformational epitopes in an easily produced protein). 4. 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.,

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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, 1998; Thomas et al., 1997). The detection of these HEV isolates stimulated our interest in whether HEV was present within the Australian pig population. Antibody responses to the highly conserved HEV ORF2.1 antigen were detected in both wild-caught and commercial pigs within Australia. Although definite patterns of infection and antibody responses cannot be established from these results, we have strong evidence that HEV is common in the Australian pig population. In addition, within the limitations of a cross-sectional study, it appears that the dynamics and specificity of the pig IgG response to swine HEV is very similar to that seen in humans infected with human HEV. Specifically, the conformational epitope presented by ORF2.1 appears to be significant, as demonstrated by the preferential reactivity of this protein with sera from older pigs in Herds E and F. Our preliminary observations of HEV reactivity among Australian pig herds is reason for some concern, as epidemics of HEV infection among susceptible pigs might lead to considerable economic losses. This may be especially true among pregnant pigs, by analogy with the human virus. In addition, it is not known whether `subclinical' HEV infection may have adverse effects on growth rates in juvenile pigs. Another issue raised from the discovery of swine HEV questions the possible routes of transmission of virus between pigs and man and vice versa: although Balayan et al. (1990) demonstrated transmission of human HEV to pigs, and virus closely resembling the swine HEV strain has been isolated from patients (Schlauder et al., 1998), other studies have demonstrated experimental infection of pigs with swine HEV but not with two separate strains of human HEV (Meng et al., 1998). Further studies of pig HEV infection in Australia and worldwide are clearly required. 5. 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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