Potential conflict of interest: Nothing to report.
Hepatitis E and HEV research in the authors' laboratories have received generous support from the Department of Biotechnology, India, and the Indian Council of Medical Research (to R.A.), and also The Wellcome Trust, UK, National Institutes of Health, and the Department of Biotechnology, India (to S.J.).
Hepatitis E refers to liver disease caused by the hepatitis E virus (HEV), a small, nonenveloped virus with a single-stranded RNA genome. The virus has four genotypes, but only one serotype. Genotypes 1 and 2 exclusively infect humans, whereas genotypes 3 and 4 also infect pigs and several other mammalian species. Though HEV does not grow well in cell culture, several aspects of its biology and pathogenesis have been worked out using animal models and cell transfection studies, and by analogy with other related viruses. HEV itself appears noncytopathic, and the liver injury during hepatitis E may be mediated by the host immune response. In areas with poor sanitation, HEV infection is common and presents as outbreaks and also as sporadic cases with acute self-limited hepatitis. The transmission is feco-oral, usually through contaminated drinking water. The disease often affects young adults and is particularly severe among pregnant women and persons with preexisting liver cirrhosis. In the developed world, the disease is being increasingly recognized. It occurs as occasional sporadic cases, most often among elderly men with coexisting illnesses. These appear to be related to zoonotic transmission. Chronic infection is known among immunosuppressed persons in these regions and may progress to liver cirrhosis. Serological tests for diagnosis of HEV exposure and recent infection, namely immunoglobulin (Ig)G and IgM anti-HEV, respectively, need further improvement in sensitivity and specificity, particularly when used in developed countries. Two recombinant protein vaccines have undergone successful human trials, but are not yet commercially available. Recent development of cell-culture methods for HEV should allow a better understanding of this enigmatic agent. (HEPATOLOGY 2011)
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Discovery of hepatitis A virus (HAV) and hepatitis B virus (HBV) in the 1970s led to the realization that some cases with viral hepatitis were not related to these infections. A large majority of such cases were parenterally acquired and were related to infection with hepatitis C virus (HCV). An enteric non-A, non-B agent was first suspected based on epidemiological investigations into an outbreak of viral hepatitis in 1978-1979 in Kashmir, India1 and retrospective analysis of a large waterborne outbreak in 1955-1956 in Delhi, India.2 This agent was initially known as the enterically transmitted non-A, non-B hepatitis virus. It was subsequently named the hepatitis E virus (HEV),3 based on its enteric transmission and association with hepatitis epidemics.
Infection with HEV, initially thought to be limited to residents of developing countries, has, in recent years, been found to have a wider geographic and host species distribution. The increasing identification of HEV infection among several animal species and humans, and of human disease in the developed world has led to a resurgence of interest in this infection.
ALT, alanine aminotransferase; FHF, fulminant hepatic failure; gRNA, genomic RNA; HAV, hepatitis A virus; HBC, hepatitis B virus; HCV, hepatits C virus; HEV, hepatitis E virus; Ig, immunoglobulin; kb, kilobases; VLPs, virus-like particles.
Biology and Pathogenesis
HEV is classified in the genus Hepevirus and family Hepeviridae.4 The family also includes closely related viruses that infect pigs (i.e., swine HEV), rabbits, rats, deer, and mongoose, which belong to the same genus as the human HEV, and the more distant avian HEV, which is associated with hepatitis-splenomegaly syndrome in chickens. Within the genus Hepevirus, at least four genotypes of HEV are recognized as species: Genotype 1 and 2 strains are restricted to humans, whereas genotypes 3 and 4 have a broader host range and are zoonotic (Fig. 1).4, 5 Interspecies transmission has been demonstrated for HEV genotypes 3 and 4. The human and swine HEV strains show extensive serological cross-reactivity with a single serotype.
The HEV virions are icosahedral, nonenveloped, spherical particles of 27-34 nm, with a single capsid protein and a linear, positive-sense RNA genome of approximately 7.2 kb (Fig. 2). The genome has short 5′ and 3′ untranslated regions, a 5′-methylguanine cap, a 3′ poly(A) stretch, and three overlapping open reading frames: orf1, orf2, and orf3 (Fig. 2).6 It also has conserved sequences close to the 5′ end of orf1, which may fold into stem-loop and RNA hairpin structures.7 These and the junction region between orf1 and orf2/orf3, which contains regulatory elements, are, together, important for replication of the HEV genome.8
The ORF1 nonstructural polyprotein contains domains with methyltransferase, putative papain-like cysteine protease, RNA helicase, and RNA-dependent RNA polymerase activities, which are important for viral replication (Fig. 2).8 The implication of the recently identified “macro domain” within the ORF1 polyprotein that encodes a poly(ADP-ribose)-binding polypeptide is unclear.9 The ORF2 protein consists of three linear domains and forms homodimers, which act as capsomeres and form the viral capsid (Fig. 2).10 Truncated versions of the ORF2 protein expressed in insect cell or bacterial systems assemble into empty virus-like particles (VLPs), which have been used as candidate vaccines.11, 12 The ORF3 protein is required for HEV replication in the host, but not in vitro; in addition, it has pleiotropic effects on host cell pathways and plays a role in viral egress from infected cells.13
The understanding about the replication cycle of HEV is based largely on analogy to other positive-strand RNA viruses. The cellular receptor and mode of entry of HEV into the cell are not known, but heparan sulfate proteoglycans are required for HEV attachment and infection of target cells.14 It is proposed that after uncoating, the positive-strand viral RNA is translated into nonstructural (i.e., ORF1) proteins, which, in turn, help produce a negative-strand RNA intermediate. The latter serves as a template to produce several positive-strand genomic RNAs (gRNA) and a subgenomic RNA, which is translated into the ORF2 and ORF3 proteins. The ORF2 capsid protein packages the gRNA into new virions, which egress through an unexplained pathway that utilizes the ORF3 protein and cellular lipids.15
Inefficient in vitro propagation of HEV has been a bottleneck in virological studies. Genotype 3 and 4 viruses from human specimens with high HEV titers were recently propagated in human liver and lung epithelial cells.16 Another genotype 3 virus was recently adapted to grow in HepG2 (i.e., human liver) cells.17 Reliable culture systems and the ability to generate virions from transfected infectious molecular clones should pave the way for much-needed virological studies on HEV.
Animal Models and Pathogenesis.
Nonhuman primates, such as chimpanzees and various macaque species, have played a major role in the discovery of HEV, subsequent molecular and pathogenetic studies, and vaccine development.18 The discovery of swine HEV has provided specific pathogen-free pigs as an alternate animal model for genotype 3 and 4 HEV. The recent discovery of rat and rabbit strains of HEV may allow the development of a reliable small animal model.19, 20
Studies in two human volunteers, patients with epidemic hepatitis E and experimentally infected primates have provided a composite picture of pathogenesis, including viral replication and shedding, antibody responses, and liver damage during hepatitis E (Fig. 3). Viremia and fecal shedding begin 1-2 weeks before and last 2-4 weeks after the onset of symptoms. The immune response is marked by an initial increase in anti-HEV immunoglobulin (Ig}M, followed closely by an IgG response; whereas IgM titers wane off in 4-6 months, IgG persists for longer periods,21 but with questionable efficacy. In experimentally infected nonhuman primates, HEV RNA is observed in serum, bile, and feces before the elevation of aminotransferases; the HEV antigens first appear in hepatocytes around day 7 postinfection, followed by rapid spread to 70%-90% of hepatocytes.
It appears that HEV, like other hepatitis viruses, is not directly cytopathic, and liver injury results from the host immune response. Pathogenetic events leading to increased mortality after HEV infection during pregnancy are not fully understood; endotoxin-mediated hepatocyte injury and elevated T-helper type 2 responses may have some role.22
Distinct epidemiological patterns are identified in regions where the disease is highly endemic and where it is not; these differ in routes of transmission, affected population groups, and disease characteristics (Table 1).
Highly Disease-Endemic Regions.
HEV is endemic to tropical and subtropical countries in Asia, Africa, and Central America. In these areas, infection is most often transmitted through the fecal-oral route, usually through contaminated water. Less frequent routes of transmission include contaminated food, transfusion of infected blood products, and materno-fetal transmission.
Outbreaks of hepatitis E have been reported from the Indian subcontinent, China, Southeast and Central Asia, the Middle East, and northern and western Africa.1, 2, 23, 24 Two small outbreaks were recorded in Mexico during 1986-1987, but none have been reported thereafter. The epidemics are usually related to contamination of drinking water with human excreta. These vary from small unimodal outbreaks lasting a few weeks to multipeaked epidemics lasting many months with several thousand cases.2, 23 Water contamination is often related to heavy rainfall and floods,1, 2 diminution of water flow in rivers increasing the concentration of contaminants,23, 25 or leaky water pipes passing through sewage-contaminated soil. Occasional, small foodborne outbreaks have been reported.
During outbreaks, 1%-15% of the population may be affected. Young adults are most often affected. Infection in children is more often asymptomatic. Men usually outnumber women, possibly because of greater exposure to contaminated water.
During the outbreaks, pregnant women have a higher disease attack rate and are more likely to develop fulminant hepatic failure (FHF) and die. In the 1978-1979 Kashmir outbreak, 8.8%, 19.4%, and 18.6% of pregnant women in the first, second, and third trimesters, respectively, had icteric disease, compared to 2.1% of nonpregnant women and 2.8% of men.26 Furthermore, pregnant cases developed FHF more often (22%) than nonpregnant women (0%) or men (3%). Once FHF appears, the case-fatality rate may be similar in pregnant women with hepatitis E or other causes of liver injury.27 Immunological or hormonal factors may be responsible for this specific predilection among pregnant women.22, 28, 29
In high-endemic areas, HEV infection accounts for a substantial proportion of acute sporadic hepatitis. These patients resemble those of epidemic hepatitis E in age distribution, severity, and duration of illness, greater severity among pregnant women, and absence of chronic sequelae. The transmission in sporadic cases is most likely through contaminated water or food.
Person-to-person transmission of HEV appears uncommon.30, 31 Secondary attack rates among household contacts of hepatitis E cases are only 0.7%-2.2%, as compared to 50%-75% for HAV, another enterically transmitted virus. Multiple cases in a household are often the result of a shared water source, rather than person-to-person spread.
Materno-fetal32 and transfusion-related transmission33 of HEV are well documented. However, the contribution of these routes may be small.
HEV isolates from epidemic and sporadic cases in these regions mainly belong to genotypes 1 and 2; genotype 3 and 4 virus has been isolated from some sporadic cases (Fig. 1).
Regions With Lower Endemicity.
In the United States, Western Europe, and developed countries of the Asia-Pacific, hepatitis E is quite infrequent. Initially, most such cases were considered as related to travel to high-endemic areas. However, in recent years, an increasing number of sporadic cases, and occasional small foodborne outbreaks related to autochthonous (i.e., locally acquired) hepatitis E, has been reported from these regions.34
Several observations suggest that autochthonous cases in these areas are caused by zoonotic spread of infection from wild or domestic animals.35 First, the HEV isolates from such cases have belonged to genotypes 3 or 4, which also infect animals.34, 36 Second, these human isolates have been experimentally transmitted to pigs, and swine HEV to nonhuman primates.37, 38 Finally, a cluster of Japanese cases who had recently consumed inadequately cooked deer meat has been reported.39 The genomic sequences of HEV isolated from these cases were identical to those from the leftover frozen meat, establishing foodborne transmission. These isolates also had a 99.7% genomic sequence homology with those obtained from a wild boar and another wild deer.40
Commercially available pig liver in these areas has also been shown to contain genotype 3 or 4 HEV.41 This finding, along with the association of sporadic hepatitis E with eating uncooked or undercooked pig livers, suggests that consumption of such meat accounts for at least some autochthonous cases. Contaminated shellfish may be another potential source.42
Reservoirs of Infection.
The reservoirs of HEV responsible for maintaining the disease in hyperendemic populations remain unclear. Protracted viremia and prolonged fecal shedding of HEV have been suggested; however, viral shedding in feces appears short-lasted.43 Frequent detection of HEV genomic sequences in sewage suggests a role for an environmental reservoir.44
In an experimental model, HEV-infected animals that lacked biochemical evidence of liver injury excreted large amounts of infectious HEV.45 In high-endemic areas, similar fecal shedding of HEV by persons with subclinical infection could maintain a circulating pool of infectious individuals who could periodically contaminate the water supplies.
The importance of an animal reservoir in high-endemic regions remains unresolved. High prevalence of anti-HEV antibodies in several animal species and isolation of HEV genomic sequences from pigs support its existence. However, whereas HEV isolates from sporadic human cases and animals in China and Vietnam have both belonged to genotype 4, in India, these have belonged to genotypes 1 and 4, respectively (Fig. 1).46, 47 Furthermore, genotype 1 HEV, which is responsible for the majority of cases in hyperendemic countries, has not been isolated from pigs and has failed to infect pigs in experimental studies.48 Thus, zoonotic transmission appears unlikely for the widely prevalent genotype 1 HEV infections in high-endemic areas.
Anti-HEV Seroprevalence Data.
Anti-HEV IgG antibodies represent a marker of previous exposure to HEV. However, wide variations in sensitivity and specificity rates of various anti-HEV IgG assays makes the interpretation of seroepidemiological studies of HEV infection difficult. Furthermore, the duration of persistence of these antibodies remains uncertain. In one study, nearly half of those who had been affected during a hepatitis E outbreak had detectable anti-HEV 14 years later.21 However, in another study, IgG anti-HEV levels had declined significantly within 14 months.49
Prevalence rates for anti-HEV antibodies are generally higher in areas where clinical hepatitis E is common. However, somewhat inexplicably, age-specific seroprevalence rates of anti-HEV are much lower than those for anti-HAV in several high-endemicity countries.50 In contrast, in Egypt, anti-HEV prevalence rates among adults exceed 70%, though disease outbreaks do not occur.51 These findings are not explained by variations in performance of various anti-HEV assays.
In developed countries, anti-HEV antibody prevalence rates vary from 1% to above 20%.35, 52 These appear too high, given that hepatitis E disease is infrequent in these areas, and may reflect exposure to infected animals, previous subclinical HEV infection, serologic cross-reactivity with other agents, and/or false-positive serologic tests. In particular, in a study of nearly 18,000 sera collected during the Third National Health and Nutrition Examination Survey (NHANES III) in the United States, the IgG anti-HEV seropositivity rate was 21%,53 in marked contrast to the infrequency of symptomatic hepatitis E in the United States. Quite significantly, seropositivity was associated with history of eating liver or organ meat more than once per month, suggesting a role for foodborne zoonotic transmission. Other risk factors included male gender, non-Hispanic white ethnicity, residence in certain geographical parts, and having a pet at home. HEV seroprevalence rates are higher among veterinarians and swine farm workers, implicating contact with pigs.
Highly Disease-Endemic Areas.
In hyperendemic areas, the most common clinical presentation is as acute icteric hepatitis, indistinguishable from other forms of viral hepatitis. The incubation period is 2-10 weeks, with an average of 6-7 weeks. The illness usually has two distinct phases. The initial preicteric phase is characterized by fever, anorexia, dysguesia, vomiting, bowel alterations, and abdominal pain and lasts for a few days. The onset of the icteric phase (i.e., jaundice) is marked by the disappearance of prodromal symptoms; it is usually self-limited and improves in a few weeks. Examination findings include jaundice, hepatomegaly, and often a soft splenomegaly. Some patients experience a prolonged cholestatic illness with troublesome itching, though usually with good ultimate outcome.
HEV infection may be largely asymptomatic, because most residents of high-endemic regions who have anti-HEV antibodies do not recall earlier acute hepatitis. During hepatitis E outbreaks, laboratory testing of asymptomatic persons has revealed frequent anicteric hepatitis, with elevated liver enzymes and HEV viremia, but normal serum bilirubin.
In hyperendemic areas, HEV superinfection may occur in persons with preexisting, known or asymptomatic, chronic liver disease of any etiology; such patients can present as acute-on-chronic liver disease and liver decompensation.54 They are at a higher risk of poor outcome.
Among hospitalized patients with hepatitis E, case-fatality rates have been 0.5%-4%. This may reflect a selection bias, because rates in population surveys during outbreaks are much lower (0.07%-0.6%).23, 24
As indicated previously, the disease is characterized by a high attack rate and higher rates of occurrence of FHF and death among pregnant women.26, 55 Infants with vertically acquired HEV infection can develop icteric hepatitis, anicteric hepatitis, or hyperbilirubinemia; prematurity, hypothermia, and hypoglycemia are common and mortality rates approach 50%.56
Determinants of disease severity are poorly understood. In animal studies, severity of liver injury has depended on viral inoculum, with lower doses leading to subclinical infection.45 In humans, Fulminant hepatitis E has been associated with higher viral titers than uncomplicated disease.29
Areas With Lower Disease Prevalence.
Clinical presentations in these areas include icteric hepatitis, anicteric illness with nonspecific symptoms, and asymptomatic transaminase elevation.35 Hepatitis E is often recognized as the cause only after serological test results are available. It is possible that many cases in these regions remain undiagnosed, because tests for HEV infection are either not available or not routinely done. For instance, patients in whom liver injury was thought to be drug related have been found to have HEV infection.
Patients in these areas are somewhat older, are predominantly male with a higher frequency of underlying liver disease or alcohol use and a higher frequency of nonspecific symptoms, and pregnant women do not show severe disease.57, 58 The mortality rate is somewhat higher in these areas, probably because of older age and coexistent illnesses. Variations in modes of transmission, disease epidemiology, or prevalent virus genotypes may account for these differences.
Chronic Hepatitis E.
HEV infection was previously believed to be always self-limited. Persistent HEV infection was first reported in 2008 in 8 French solid-organ transplant recipients who were receiving immunosuppressive drugs, had recently developed transaminase elevation, and had infection with genotype 3 HEV.59 These patients had evidence of more marked immunosuppression than those with organ transplantation and resolving HEV infection. Chronic HEV viremia has also been reported in patients with hematological diseases,60 human immunodeficiency virus infection,61 or those receiving anticancer chemotherapy.60 Liver histology in such patients shows portal hepatitis with dense lymphocytic infiltrate, piecemeal necrosis, and fibrosis; in some cases, serial liver biopsies showed the development of liver fibrosis,62 suggesting the possibility of progression to cirrhosis.
Persistent infection has not been reported with genotype 1 or 2 HEV, or among otherwise healthy persons, or from highly endemic regions.
Infrequent, Nonhepatic Manifestations of Hepatitis E.
Several nonhepatic manifestations have been described with HEV infection, mostly as case reports or small case series (Table 2), based usually on the detection of anti-HEV IgM, rather than HEV RNA.
Table 1. Features of HEV Infection in Highly Endemic and Nonendemic Areas
Highly Endemic Areas
Numbers in parentheses indicate low frequency.
Highly frequent, both sporadic and endemic cases
Infrequent sporadic cases
Primarily human; possibly environment
Suspected to be zoonotic (pigs, boars, and deer)
Primary routes of transmission
Fecal-oral; mainly through contaminated water
Ingestion of undercooked meat, possibly contact with animals
The rise in serum aminotransferases, such as alanine aminotransferase (ALT) and aspartate aminotransferse, is a sensitive, though nonspecific, indicator of liver injury. Specific diagnosis of hepatitis E is based primarily on serological tests for anti-HEV antibodies. In endemic areas, detection of IgM anti-HEV suggests current infection, whereas IgG anti-HEV indicates past exposure. In nonendemic areas, IgG anti-HEV has also been used for the diagnosis of acute infection; recent reports of high anti-HEV seroprevalence rates, however, indicate that this may not be a correct approach. Currently available enzyme immunoassays are based on immunodominant parts of the ORF2 and ORF3 proteins; their sensitivity and specificity rates appear inadequate and thus assays need improvement.
HEV nucleic acid detection using amplification techniques not only provides detection of HEV infection, but also allows identification of viral genotype and genomic sequences of the infecting viral isolate. However, because viremia and viral shedding are both brief, these assays may lack sensitivity.
Because the illness is self-limiting, most patients need no specific treatment. Patients with acute or acute-on-chronic liver failure need admission to an intensive care unit, measures to control cerebral edema, and may need liver transplantation.63 No data are available on the effect of termination of pregnancy on liver function. Coagulopathy increases the risk of postpartum hemorrhage; prophylactic injection of uterine-muscle–constricting drugs immediately after delivery, and fresh frozen plasma infusions if bleeding supervenes, are helpful.
Pegylated interferon alpha-2a/alpha-2b64 or ribavirin65 for 3-12 months have been tried in persons with chronic HEV infection, with moderate success in achieving an absence of detectable serum HEV RNA for 3-6 months after stopping drugs. Only short case series are available, and controlled trials with longer follow-up are needed. Withdrawal or reduction in dose of immunosuppressive drugs has also led to the disappearance of HEV viremia and should be tried before considering antiviral treatment.
No data are available on the role of antiviral drugs in acute HEV infection associated with acute or acute-on-chronic liver failure.
Hepatitis E can be prevented by the provision of safe drinking water, proper disposal of human feces, and education about personal hygiene. During outbreaks, boiling and chlorination of water would be useful. Sanitary handling and proper cooking of pig and deer meat is recommended in areas with zoonotic transmission.
Two candidate vaccines against hepatitis E have undergone clinical testing. The first contained a 56-kDa truncated ORF2 protein of HEV (amino acids 112-607), expressed using a baculovirus expression system, which assembles into highly immunogenic VLPs. In a trial among 2,000 volunteer Nepalese soldiers, 3 doses of an alum-adjuvanted preparation of this protein (20 μg each at 0, 1, and 6 months) achieved 100% seroconversion and protective efficacy of up to 95.5% during a 2-year follow-up.11 The second vaccine consisted of a truncated ORF2 protein, p239 (amino acids 368-606), which is expressed in Escherichia coli and forms 23-nm VLPs. In a large clinical trial in southern China, administration of 3 doses (30 μg each) showed a protective efficacy of 100% during a 13-month follow-up.12 Though several questions remain, the successful clinical testing of these vaccines is a major step forward in the future control of hepatitis E. Unfortunately, neither vaccine has yet been licensed for marketing, possibly because the industry is not assured of a sufficient market.
Hepatitis E, though mainly a disease of the resource-poor regions of the world, has also been identified as occasional autochthonous cases in developed countries. The global presence of HEV, its ability to cause sporadic infections as well as large outbreaks, and its ability to cause chronic hepatitis in immunocompromised persons are all causes for concern. The disease has a complex epidemiology with both waterborne human-to-human and zoonotic transmission, and limited treatment options, and its pathogenetic mechanisms are poorly understood. Thus, hepatitis E deserves serious attention from physicians and researchers alike. Recent successful clinical testing of two recombinant vaccines augurs well for the future.
The authors thank Prasida Holla and Imran Ahmad for composing the figures.