Symptomatic human infection with HEV in the United States and other developed countries is rare. Most of the few cases reported in these countries were in travellers returning from endemic regions (8
), although in some cases in Western European countries this risk factor was absent (16
). The recent identification of a case of acute hepatitis E in the United States in a patient with no history of travel to endemic regions (20
) reiterates that absence of travel to an endemic region does not necessarily exclude the diagnosis of acute hepatitis E infection. The need to explain the source of infection acquired in industrialized countries and to understand the ecology and epidemiology of HEV has led to a hypothesis that an animal reservoir may exist (33
). In fact, antibodies reactive with HEV antigens have been detected in a number of animal species, including pigs, sheep, and rats (5
). Unfortunately, either virus was not recovered from these species or virus recovered was not sequenced. Our discovery of the swine HEV as a ubiquitous agent in the swine population of the midwestern United States lends credence to the possibility of an animal reservoir.
In the present study, a standardized infectious pool of swine HEV was prepared and characterized. In our previous studies, we found only subclinical infections in naturally infected young pigs (23
) or young pigs experimentally infected with an unquantified but relatively low dose of virus (24
). In the present study, pigs infected with 104.5
also did not have clinical or biochemical evidence of hepatitis, although HEV was detected in bile, indicating that swine HEV replicated in the liver. In previous studies, cynomolgus macaques infected with a human strain of HEV developed biochemical evidence of significant hepatitis (ALT ≥100 U/liter) when the infecting dose was around 103.5
50% monkey infectious doses or higher but had little or no biochemical evidence of hepatitis when inoculated with lower doses (40
). The rhesus macaques infected with 104.5
of swine HEV had mild focal necroinflammatory changes in liver biopsy specimens and a slight elevation of ALT and ICD levels in serum, which could indicate mild hepatitis and virus replication in the liver. The chimpanzee infected with 106
GEs of swine HEV was clinically normal, although fecal excretion of swine HEV and seroconversion to anti-HEV IgG were detected. This inoculum has not yet been titrated for infectivity; however, based on the ratio of GE to infectivity of the original pool of swine HEV in swine feces (106
), the infectious dose may have been the same as that given to the rhesus monkeys. Additional studies must be performed to determine if a larger dose will induce clinical disease. The patterns of appearance of viremia, anti-HEV, and fecal excretion of virus in primates infected with swine HEV were similar to those observed in infected pigs. Infection of primates with swine HEV demonstrated that swine HEV can cross species barriers, at least under experimental conditions, suggesting the possibility of human infection with swine HEV. The extremely high prevalence of swine HEV in pigs (23
) and its ability to cross species barriers may put swine practitioners, swine producers, and other pig handlers at possible risk of zoonotic infection by the virus.
Balayan et al. (5
) previously reported that Russian domestic swine were experimentally infected with a Central Asian strain of HEV isolated from a naturally infected patient. However, the virus infecting the pigs in this experiment was not sequenced to confirm its identity; thus, it is not clear whether the pigs were infected with the virus in the inoculum or were fortuitously infected by a swine virus circulating among the pigs at the time. We were unable to infect crossbred SPF pigs with strains of human HEV representing two major genotypes (24
), Mex-14 (Mexican) and Sar-55 (Pakistan), even though the Sar-55 strain is from the same geographic region as the strain used by Balayan et al. and even though high doses of infectious virus (105
monkey infectious doses) were administered (24
). Similarly, Platt et al. (28
) failed to infect SPF pigs experimentally with the Mexican strain (Mex-14) of human HEV. Since the swine HEV shares only about 75% nucleotide sequence homology with either of the two human strains of HEV tested, our failure to infect swine with these strains (24
) was likely due to a difference in susceptibility of pigs to different HEV strains. In contrast, when we inoculated SPF pigs with the US-2 strain of human HEV, the inoculated pigs became infected and, in one case, virus even spread to the uninoculated pig housed in the same room. Since the virus recovered from the infected pig had a unique sequence identical to that of the virus in the inoculum, these data provided conclusive experimental evidence for cross-species infection by HEV. Like pigs infected with swine HEV, swine infected with the US-2 strain of human HEV (either by inoculation or by contact) remained clinically normal, and levels of liver enzymes in serum were not significantly elevated. The rapid seroconversion of pigs infected with the US-2 strain of human HEV further suggested that the US-2 strain is already competent to replicate in swine and may be of swine origin. These data strongly suggest that a swine virus strain or one very similar to swine HEV infects and causes hepatitis in rare cases in humans in the United States and perhaps in other countries. The results suggested that pigs could well be an animal reservoir for HEV in the United States.
We have amplified and sequenced nearly the full-length genome of the swine HEV. Sequence analyses revealed that, in all three ORFs and the 3′ NCR, swine HEV and the two U.S. strains of HEV are very closely related. However, both swine HEV and the U.S. strains diverged extensively from other strains of HEV, especially in the HVR. Phylogenetic analyses have shown that genotypes of HEV generally have unique geographic distributions. Swine HEV and the closely related U.S. strains were all isolated in the United States. In general, the many Asian strains isolated are related both genetically and geographically. The African strains appear to be similar to but distinguishable from Asian strains. The Mexican strain is the only strain that forms a single branch. However, exceptions to this geographic distribution of HEV strains are the two unique strains of HEV, G-20 and G-9, recovered from patients in China (14
). These two Chinese strains do not appear to group with the other Asian strains. The observed genetic diversities among different strains of HEV may affect the development of HEV vaccines. Although the experimental HEV vaccines are very promising (31
), their efficacy must be evaluated for protection against these novel and divergent strains.
The data presented in this study suggested that swine may serve as an animal reservoir for HEV. Young pigs infected naturally (23
) or experimentally (24
) with swine HEV appear not to have clinical symptoms, and primates showed negligible or minimal evidence of hepatitis. Therefore, subclinical infection of humans with swine HEV could explain the relatively high prevalence of anti-HEV in apparently healthy individuals in the United States and other industrialized countries (16
). However, anti-HEV was also found in healthy individuals in the United States who seem to have had no contact with swine or other farm animals (22
). Thus, it is likely that other animal species may also serve as reservoirs for HEV. These putative animal reservoirs of HEV could be a source of contamination in regions where HEV is endemic and may also be responsible for the anti-HEV detected in healthy individuals in nonendemic regions.
Recently, xenotransplantation has become the focus of intensive research for a solution to the shortage of organ donors for transplantations (4
). Swine are relatively easy to breed and maintain; therefore, xenotransplantation with pig organs has received considerable attention (4
). However, xenozoonoses, the transmission of pathogens from pigs to human recipients, is of major concern in xenotransplantation (25
). Many swine viruses are known to infect humans (9
). Our results proved that swine HEV also has the ability to cross species barriers and therefore might infect humans. Although swine HEV appeared to be nonpathogenic for pigs and primates in experimental infections, it might become pathogenic in immunosuppressed xenotransplantation recipients. Therefore, it is important to develop sensitive and easy-to-perform assays to screen for swine HEV in donor pigs used for xenotransplantation. In addition, adequate diagnostic reagents are needed not only for screening of xenotransplantation donors but for epidemiologic studies as well. With the recent diagnosis in the United States of acute hepatitis E that was not associated with travel to endemic regions and the recovery from such a case of HEV that is closely related to swine HEV, HEV should now be considered as a possible etiologic agent in persons with acute hepatitis in the United States.