Cocirculation of genotype 3 and 4 HEV strains among humans and animals provides opportunities for cross-species transmission and may underlie the occurrences of non-travel-associated hepatitis E in industrialized countries, including the United States (38
). To effect programs for the surveillance of HEV infection in humans and animals, several molecular and serological approaches have been developed and applied. Molecular techniques such as RT-PCR generate data that identify active HEV infection by revealing the presence of the HEV genome in the tissues, body fluid, and excreta of the infected hosts. Viremia and fecal HEV shedding are mostly transient, however, and persistent HEV infection is rare, so the likelihood of identifying active infection in a person or an animal in cross-sectional investigations is small. Thus, in the present study, only 3 of 457 animals determined to be DASA-reactive were found to carry HEV RNA in their blood.
Serological EIAs are widely used for detection of anti-HEV, a marker of prevalent HEV infection (29
). Most of these EIAs employ for antibody capture antigens that are derived from HEV genotypes 1 and 2 and so may not be sufficiently sensitive to detect heterotypic antibodies generated in human and animal hosts infected by genotype 3 (19
) or genotype 4 (2
). Moreover, some indirect EIAs tend to generate false-positive reactivities (52
) and therefore require, in order to validate the specificity of their reactivities, the extra steps of neutralization, immunoblotting, or prior production of genus-specific positive controls (1
). To circumvent such problems inherent to indirect EIAs, DASAs have been developed. A principal advantage of DASAs is that they enable transgenus antibody detection. Another advantage is that sensitivity is potentiated by the detection of total rather than class-specific antibodies. DASAs have particular capability to detect IgM. Being decavalent, IgM substantially amplifies DASA OD readings, because for each pentameric immunoglobulin complex from which one Fab end has bound to the capture antigen, nine other Fab ends are potentially free to bind to the reporter antigen. Thus, in the course of testing the porcine seroconversion panels, we observed peaks of DASA s/co values in the immediate few weeks after experimental infection (), reflecting the possible detection of anti-HEV-IgM that were being transiently generated during acute infection.
The DASA we developed has several unique features. First, it incorporates antigens representing all four HEV genotypes, so allowing for the detection of antibodies generated in the host regardless of the genotype of the infecting HEV. Other DASAs developed for anti-HEV detection (14
) mostly utilize antigenic preparations derived from one genotype only. Second, the highly immunodominant p166 antigens (37
) are used to capture antibody, as well as to report its solid-phase capture, thereby conferring both sensitivity and specificity to anti-HEV detection. The fine specificity is exemplified in the low background OD readings generated from the blood-donor samples (), thereby lending DASA the capability to test samples without predilution. Testing samples undiluted preserves sensitivity by conserving the original concentration of the analyte and facilitates ease of use for field studies. Third, the antigen mixture utilized for antibody capture was His tagged, whereas the mixture for antibody detection was GST tagged. Such a design was conceived to enhance the specificity of the DASA by minimizing solid-phase capture of antibody against His or GST that might be carried in the test sample.
Owing to the absence of gold standards for HEV serology, evaluating the performance characteristics of the DASA described here required various assemblages of test samples and reagents. Considering that as much as a fifth of the U.S. general population may be anti-HEV positive (30
), it would be inappropriate, for the purpose of establishing the DASA cutoff, to include OD reactivities generated from every U.S. blood donor serum sample acquired. Therefore, only those of blood donor samples that were unreactive in the DS assay and unreactive or weakly reactive in DASA were included. The established cutoff value was then used as basis to determine the diagnostic specificity of DASA. Next, to determine diagnostic sensitivity, we incorporated another panel, constituted from sera derived from people who were undergoing acute hepatitis E. Although such a panel would not represent prevalent HEV infection, the presence of HEV RNA assured that its constituents originated from truly HEV-infected hosts. Nonetheless, such a panel preferentially favors the detection of IgM, so the ability of DASA to identify monomeric immunoglobulins may not have been fully assessed. An evaluation using sera obtained from five species of laboratory animals that had been immunized with HEV proteins provided proof of concept that DASA can detect anti-HEV generated across different genera and species. Lastly, the added usage of the WHO reference reagent permitted the analytic sensitivity of our DASA to be determined; this reagent was also used as a standard against which the content of anti-HEV in animal sera could be measured.
HEV-infected animals potentially serve as reservoirs of infection to other animals and to humans. Since the discovery of the first porcine HEV strain (39
), evidence has mounted to indicate that HEV is epizootic in swine (38
). The present study reveals a DASA reactivity rate of 41% among farmed pigs, which is consistent with anti-HEV seropositivity rates determined in other studies (ranging between 15 and 90%). Because our study samples were collected from adult pigs, it is not unexpected to find just one sample to carry HEV RNA, since the majority of pigs in the United States are infected between 2 and 4 months of age (39
). The HEV seroprevalence in feral pigs, in contrast to farmed ones, has not been reported in the United States. We found that 3% of the feral pigs sampled were DASA reactive, and these seropositive pigs were collected in the vicinity of domestic swine farms (data not published). The seropositivity proportion is substantially lower than in farmed pigs, which not only suggests that HEV is less prevalent among feral than farmed pigs but implicates the pig-farming environment as potentially fostering HEV spread among swine. Such disparity might be due to geographical differences in the endemicity of HEV in farmed pigs and the proximity of farmed to feral pigs, which would facilitate cross-transmission of microbial agents (7
). Phylogenetic analyses indicated that all of the three newly identified porcine HEV strains belonged to genotype 3 and were most closely related to human and porcine HEV strains in the United States than to those known to circulate elsewhere. These findings suggest the potential in the United States for HEV transmission between farmed and feral swine, as well as cross-species transmission from them to humans (and vice versa).
Subclinical human infection with attenuated HEV strains such as porcine HEV might explain the high anti-HEV seropositivity rates among people living in developed countries (33
). Nonetheless, much of the U.S. population, being predominantly urban (59
), is neither likely to come into frequent contact with swine or their excreta nor disposed to adopt eating habits that would entail ingestion of raw or inadequately cooked pork, other pig meat, or offal including liver (6
). These considerations suggest that even if swine are maintenance hosts for HEV, they may not necessarily serve as the only reservoir of HEV infection (47
Accordingly, our studies extended to determine whether HEV can infect animals other than swine. Given the enormous diversity of the animal kingdom, exhaustive sampling was not possible. Emphasis was placed on investigating animals (pigs, cattle, deer, bison, horses, cats, coyotes, dogs, rats, psittacines, raccoons, skunks, and squirrels) which share habitats or are in frequent contact with humans or whose meat and offal may be eaten by humans. Testing samples from the more exotic animals (alpacas, addaxes, badgers, bats, bear, colobuses, ferret, fox, kangaroo, mountain lions, macaws, muntjac, ostrich, otters, wallabies, and warthog) was primarily to explore the possible broader tropism of HEV, but samples available from them were limited.
Among the nonporcine samples tested, anti-HEV positivity was found only among cattle, bison, dogs and Norway rats, the rates being 15, 4.6, 0.9, and 0.6%, respectively, with HEV RNA amplified from none. Since the samples from cattle, bison, dogs, and rats yielded weak OD readings (), the possibility that their DASA reactivities were not specific to anti-HEV cannot be excluded. It is also possible that the viruses infecting these species may be more distantly related, antigenically and genetically, to genotypes 1 to 4 of mammalian HEV, thus resulting in weaker reactivity.
The near absence of DASA reactivity among the rats sampled was unexpected. Previous HEV seroprevalence studies of rodents in the United States have yielded contradictory results. An early study of feral rats trapped in Louisiana, Maryland, and Hawaii found anti-HEV-seropositivity rates ranging between 44 and 90%, the seropositive species being Rattus norvegicus
, R. rattus
, and R. exulans
). In a subsequent investigation of 26 species of wild U.S. rodents, the highest seropositivity rate (60%) was found in Rattus
, and rodents caught in urban areas displayed a significantly higher rate than those in rural areas (12
). Norway rats sampled in Los Angeles yielded a 14% seropositivity rate (53
). Yet another study, conducted in North Carolina, found none of house mice (Mus musculus domesticus
) and Norway rats trapped in pig farms to be seropositive (64
). However, an investigation conducted in Baltimore found anti-HEV among 75% of Norway rats sampled (10
). Our 0.2% seropositivity rate was obtained among Norway rats that also originated from Baltimore. The poor concordances in anti-HEV seropositivity rates obtained from these various studies likely reflect variability in the performance of the serological assays applied, although geographical and sampling variations may contribute. A recent publication from Germany reported the detection of HEV RNA in two Norway rats, but the nucleotide sequences generated were only 60% related to human HEV strains (25
). The ability of such divergent HEV strains to effect cross-species transmission to humans seems remote. Nonetheless, the extensive sequence divergence between rat HEV and genotypes 1 to 4 of mammalian HEVs may also explain the lower anti-HEV-seropositivity rate among rats because of the limited antigenic cross-reactivity to rat HEV, since the DASA is based on mammalian HEV genotypes 1 to 4. Nonetheless, considering that peridomestic rodent infestation of human dwellings is rife (40
), definitive evidence of HEV infection in rodents would need to be sought (57
The development of a DASA for the detection of anti-HEV is described here. Its performance characteristics were validated to be sufficiently specific and sensitive for trans-genus HEV seroprevalence studies. When the assay was applied to nearly 5,000 sera collected from animals representing 35 genera sampled from the United States, the rate and extent of reactivity were substantial only among the porcine samples, in which HEV-specific nucleotide sequences also were detected. Owing to the limited numbers of samples available from the more exotic species of animals, the status of HEV infection in them would require further studies when more samples become available. The findings thus far obtained are consistent with limited enzooticity of HEV in the United States. Animals other than swine are unlikely maintenance hosts to HEV and so would not play appreciable roles as reservoirs of its transmission.