Hepatitis E virus infections in humans and animals.

Hepatitis E has traditionally been considered an endemic disease of developing countries. It generally spreads through contaminated water. However, seroprevalence studies have shown that hepatitis E virus (HEV) infections are not uncommon in industrialized countries. In addition, the number of autochthonous hepatitis E cases in these countries is increasing. Most HEV infections in developed countries can be traced to the ingestion of contaminated raw or undercooked pork meat or sausages. Several animal species, including pigs, are known reservoirs of HEV that transmit the virus to humans. HEVs are now recognized as an emerging zoonotic agent. In this review, we describe the general characteristics of HEVs isolated from humans and animals, the risk factors for human HEV infection, and the current status of human vaccine development.

Introduction

Hepatitis E virus (HEV), originally recognized as non-A and non-B hepatitis inducing agent, was recently classified into the genus Hepevirus and family Hepeviridae [1,2]. HEV is a non-enveloped virus with a diameter of 27-34 nm. It has a single-stranded and positive sense RNA genome that is approximately 7.3 kb in length. It also has a short 5' non-coding region (NCR), a 3' NCR, and 3 open reading frames (ORFs). ORF1, ORF2, and ORF3 encode nonstructural proteins including an RNA-dependent RNA polymerase, a capsid protein, and a phosphorylated small protein, respectively [3].

A number of HEVs have been isolated from humans and several animal species, but HEV has only 1 serotype. HEV isolates are classified into 4 major genotypes. The representative genotypes 1, 2, 3, and 4 are the Burma, Mexico, USA, and China strains, respectively [3-6]. Genotype 1 is most prevalent in Asia and Africa. Genotype 2 circulates in Mexico, Nigeria, and Chad. Genotype 3 is distributed globally in both industrialized and developing countries. Genotype 4 is found exclusively in Asia [7]. However, a recent study proposed classification of 6 HEV genotypes, including the 4 previously described as well as 2 additional genotypes isolated from wild boars [8]. In addition to HEVs isolated from humans and pigs, HEV variants have been identified in rats, ferrets, rabbits, bats, and chickens. While HEV genotypes 1 and 2 only infect humans, genotypes 3 and 4 have been isolated from human patients as well as from infected animals such as pigs. Therefore, HEV genotypes 3 and 4 are considered zoonotic viruses.

HEV infection occurs mainly through fecal-oral transmission. Waterborne outbreaks of HEV are often reported in endemic countries [9,10], where most infections occur in young adults aged 15-45 years [11,12]. HEV has a relatively low fatality rate, ranging 0.5-4%, but infections can induce acute liver failure in pregnant women, leading to 20-30% mortality [13-15]. Fulminant hepatitis and hepatic encephalopathy are the main causes of death [15].

Human HEVs

Clinical signs of acute HEV infection are similar to those of other viral hepatitis infections. HEV can cause jaundice, vomiting, appetite loss, fatigue, hepatalgia, and hepatomegaly [16,17]. Increases in liver enzymes such as alanine aminotransferase, aspartate transaminase, and gamma-glutamyl transpeptidase are also common [17,18]. HEV is typically diagnosed by detection of viral RNA in serum or fecal samples and testing for anti-HEV IgM or IgG antibodies in the patient sera [16]. Most HEV outbreaks are reported in developing countries in Asia, Africa, Central and South America, and the Middle East [19].

HEV infections usually occur from the consumption of contaminated or insufficiently treated drinking water [20-22]. Heavy rainfall and flooding seem to contribute to outbreaks of waterborne HEV infection in endemic regions [10,19]. Developed countries were previously assumed to be free of or non-endemic for HEV; sporadic cases of HEV in these countries have generally been associated with travel to endemic countries [23-25]. However, a considerable increase in acute HEV cases in patients who had never traveled to endemic regions prompted a study of the overall HEV infection status and the origin of infections in several developed countries [5,26]. Serological studies indicated that a considerable portion of people in developed countries had anti-HEV antibodies. For example, 19-21% of US blood donors had antibodies specific to HEV [27,28]. Studies conducted in several European counties such as England, Germany, Italy, and France demonstrated that the study populations also had relatively high rates (13-53%) of anti-HEV antibodies [29-32]. Similarly, seroprevalence studies in Asian countries such as Japan, South Korea, Hong Kong, Taiwan, and China have reported that approximately 6-43% of their populations had anti-HEV antibodies [33-37].

However, the prevalence of anti-HEV antibodies can vary depending on factors such as age, diet, type of employment, and environment. The seroprevalence studies in developed countries indicate that many HEV infections could be subclinical without development of acute hepatitis. Anti-HEV antibody levels commonly increase with age regardless of residence in endemic or non-endemic countries. Elderly people aged more than 50-60 years show higher seroprevalence of HEV infections than younger people. Similarly, swine farmers and veterinarians have much higher HEV-specific antibodies than control groups who do not have regular contact with pigs [35,38-40]. Employment in slaughterhouses is one of the highest risk jobs for HEV exposure, with the risk of infection increasing by as much as 1.5-3.5-fold [38]. Therefore, consumption of poorly sanitized water and frequent exposure to pigs may be the main risk factors for HEV infections in humans [41].

Animal HEVs

Swine HEV infection was first detected by the identification of HEV RNA in swine serum and fecal samples in 1995 [42]. However, the term "swine HEV" was first used 2 years later [43]. The infected pigs show no clinical signs but viremia and anti-HEV antibodies are detected in serum samples from infected pigs. The first isolated swine HEV showed a close similarity to human HEV, with 79-80% and 90-92% homologies in nucleotide and amino acid sequences, respectively. Subsequent serological studies have shown considerable levels of HEV infection in pigs in both HEV endemic and non-endemic countries [44,45]. Although there is significant variation between herds, anti-HEV antibodies were detected in about 20-100% of growing and adult pigs. In Korea, the overall prevalence of anti-swine HEV antibodies was approximately 15% [46]. Similarly, HEV RNA has been detected in 17.5% of fecal samples [47]. Serological studies and detection of HEV RNA in serum and fecal samples show that most swine HEV infections appear to occur at 2-3 months of age. HEV genotypes 3 and 4 have been isolated from pigs and have very similar genetic sequences to human HEV isolates [46,48,49]. These genotype isolates were tested to identify cross-species infection in pigs and humans. HEV genotype 3 isolated from pigs could infect human surrogate rhesus monkeys and a chimpanzee [50]. In a reverse experiment, genotype 3 human HEV isolate could infect pigs. Similarly, pigs and rhesus monkeys were infected by genotype 4 human HEV and genotype 4 swine HEV, respectively [51,52]. These experiments provide strong evidence that genotypes 3 and 4 of swine HEV could be the primary sources for human infections. A recent study reported a chimeric virus containing the capsid gene of genotype 4 human HEV in the backbone of a genotype 3 swine HEV. The chimeric HEV could infect both human cells and pigs [53]. These data underscore the potential for the emergence of a chimeric virus in nature with both human and swine HEV gene sequences. In addition, wild boars have been reported to be widely infected with genotypes 3 and 4 HEV [54]. Their genomic sequences are also very similar to those of the corresponding human HEV isolates. Therefore, both domestic pigs and wild boars are major reservoirs of HEV genotypes 3 and 4 that could infect humans.

Avian HEV was isolated in the USA from chickens with hepatitis-splenomegaly [55]. A subsequent study indicated that about 71% of chicken flocks and 30% of chickens in the USA were positive for HEV antibodies [56]. A similar pattern of HEV seroprevalence was observed in Korea, with seropositivity rates of 57% in chicken flocks and 28% in chickens [57]. These studies suggest that avian HEV is enzootic in several countries. Avian HEV has about 50% sequence identity with human and swine HEVs [58]. It is currently classified into 3 genotypes that cluster by geographical region: genotype 1 (Australia), genotype 2 (USA), and genotype 3 (Europe) [59,60]. Several B-cell epitopes have been identified in the capsid protein of avian HEV [61]. At least 2 of these epitopes are common to the human HEV capsid protein, and 1 epitope is common to avian and swine HEVs.

A novel HEV was recently isolated from rabbits in China [62]. The overall full-length genetic sequence shared 74%, 73%, 78-79%, 74-75%, and 46-47% identity with genotypes 1, 2, 3, 4, and avian HEV, respectively. Other rabbit HEVs isolated from the USA and France also show high levels of homology with genotype 3 HEV [63]. Both this close genetic similarity between rabbit HEV and human HEV genotype 3 and reports of rabbit HEV replication in human cell lines imply that rabbit HEV may be another zoonotic agent [64,65]. Like other genotype 3 HEVs, rabbit HEV isolates show high genetic heterogeneity. Sera collected from HEV-positive rabbits recognized the capsid proteins of human, swine, rat, and avian HEVs. In addition, antibodies produced against the capsid proteins of human, swine, rat, and avian HEV reacted with the rabbit HEV capsid protein [64]. The cross-reactivity of rabbit HEV antibody with other HEVs prompted further cross-species infection experiments. When pigs were intravenously inoculated with rabbit and rat HEV, half of the pigs inoculated with rabbit HEV showed evidence of infection [64]. In contrast, pigs inoculated with rat HEV did not show any evidence of infection. These data indicate that rabbit HEV has antigens closely related to those of other HEV strains and may infect both pigs and humans.

Rat has been suspected to be a host of HEV based on a high prevalence of anti-HEV antibody [66]. Recently, rat HEV was isolated from wild Norway rats in Germany [67]. The comparison of partial genomic sequences demonstrated 59.9% and 49.9% homology to human and avian HEV isolates, respectively. However, rats may act as carriers to transmit HEV infection to pigs. Wild rats and Norway rats captured around a pig farm contained genotype 3 HEV that was genetically identical to swine HEV found at the same farm [68,69]. These studies support the theory of HEV transmission from rats to pigs. Rat HEVs recently reported in Vietnam and Indonesia have genetic sequences that are similar within their geographical areas but very distinct from the first reported rat HEV in Germany [70,71]. Therefore, rats may host both their own HEV and genotype 3-related HEVs. However, a recent report showed that rats are susceptible only to rat HEV but not to genotype 1, 3, and 4 HEVs [72]. It has also been demonstrated that rat HEV cannot infect rhesus monkeys, suggesting that rat HEV may not be a zoonotic agent [73]. However, more evidence is required to determine if rats may be carriers of HEVs that infect humans and pigs.

Several studies have shown that a wide variety of animals can act as HEV hosts. In addition to pigs, wild boars, deer, and rats, novel HEV strains have also been found in ferrets and bats [74,75]. Ferret HEV is genetically close to rat HEV but distinct from other HEVs. Genetic sequence analysis indicated that bat HEV is also distinct from previously reported HEVs. A serological study revealed that about 16% of goats had anti-HEV antibodies; however, goat HEV RNA could not be isolated from the sera [76].

Zoonosis

Accumulating data indicate that human HEV infections are mediated by the consumption of uncooked or undercooked animal meat or foods made with pig organs such as liver. In Japan, considerable cases of fulminant hepatitis E have been reported after consumption of meats and entrails of pigs, wild boars, and deer [77-79]. A study reported that the full genome of an HEV isolated from wild boars had a nearly identical sequence (99.7% identity) to that of HEV isolated from wild deer and patients who contracted HEV after eating raw deer meat [80]. These data imply that pigs, wild boar, and wild deer are an important source of HEV infection in humans. Recently, European countries have also reported sporadic cases of HEV infection. Consumption of raw seafood, pork liver sausage, and exposure to wild boars are proposed as major risk factors associated with HEV infection in Italy [25]. One of the risk factors for HEV infection in France is the ingestion of raw pork liver sausage, which has been reported to contain infectious HEV particles [81,82]. In addition, HEV was detected in muscle samples in experimentally infected pigs [83]. In Korea, there has been a single instance of fulminant HEV infection in a patient who consumed raw bile juice from wild boar [84]. The genetic sequence of HEV isolated from the patient matched genotype 4 swine HEV. These data clearly suggest swine HEV is mainly transmitted to humans through the consumption of under-cooked or raw pork meat and liver products.

Eating raw shellfish is another risk factor for human HEV infections. About 9% of oysters collected from the coastal regions of Korea harbored HEV whose sequence matched genotype 3 swine HEV [85]. These and other data indicate that raw shellfish cultivated in sewage-contaminated waters may be a significant source of HEV infection in humans [86].

Vaccine Development

The HEV capsid protein encoded by ORF2 is a structural unit that is assembled into virus particles. The capsid proteins of several HEV isolates contain neutralizing antigenic epitopes [87-89]. Therefore, several experimental HEV vaccines for humans have been developed using the capsid protein expressed by Escherichia coli, baculovirus, or plasmid DNA [90-92]. One of these vaccine candidates, "Hecolin," was recently approved by the Chinese government after successful phase III clinical trials and is now available for use in China [93-95].

Conclusion

Accumulating research data indicate that HEV is not an old disease restricted to developing countries. It is now generally accepted that HEV is endemic in both developing and industrialized countries and that HEV is a serious public health threat worldwide. Animal HEVs appear to cause most HEV infections in developed countries. Therefore, effective animal HEV vaccines should be developed to prevent cross-species HEV infection to humans.