Hepatitis E Virus. Hepatitis E virus (HEV) is the causative agent of hepatitis E 1 and the. History. Virus DAVID A. ANDERSON

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1 177 Hepatitis E Virus DAVID A. ANDERSON Hepatitis E virus (HEV) is the causative agent of hepatitis E 1 and the type member of a distinct virus family. HEV is enterically transmitted and causes an acute and generally self-limiting infection of the liver but with a higher mortality in general than for infections with hepatitis A virus (HAV), which is transmitted via the same route. HEV is unique among the hepatitis viruses because of a high mortality during pregnancy. Clinical hepatitis E is of most importance in developing countries, where it represents the most common form of acute hepatitis that occurs in both sporadic and epidemic forms. Clinical hepatitis E is rare in developed countries, but zoonotic infections are presumed to be responsible for the rare cases that are locally acquired in these countries. Practical and accurate diagnostic assays are available, and an effective vaccine has been developed but is not yet available commercially. History Evidence for an enterically transmitted form of viral hepatitis distinct from viral hepatitis A came from serologic studies of waterborne epidemics of hepatitis in India in the late 1970s. Khuroo 2 and Wong and colleagues 3 showed that patients involved in such epidemics of hepatitis in the Kashmir region and in Delhi, India, respectively, lacked serologic evidence of recent HAV infection. In fact, all the patients were found to have immunoglobulin G (IgG) class but not immunoglobulin M (IgM) class antibodies to HAV, which indicated that they had been infected with HAV in the past and were presumably immune to reinfection. Therefore, the investigators concluded that another agent must have caused the hepatitis. Three years later, Balayan and co-workers 4 confirmed the existence of a new hepatitis virus with transmission of hepatitis to a volunteer from a patient involved in an outbreak of enterically transmitted non-a, non-b hepatitis in central Asia. The volunteer had preexisting antibody to HAV, developed a severe hepatitis, shed virus-like particles in his feces, and developed antibodies to the virus-like particles during convalescence. Balayan and co-workers 4 also inoculated cynomolgus monkeys with the new virus and again showed virus-like particles and an immune response to the particles in this primate species. The new form of non-a, non-b hepatitis came to be known as epidemic non-a, non-b hepatitis or enterically transmitted non-a, non-b hepatitis, and serologic crossreactivity between virus particles isolated around the world established that one class of viruses was responsible for most if not all cases. 5 Subsequently, the name of the disease was changed to hepatitis E to conform with the accepted nomenclature for the other types of viral hepatitis, and the virus was designated hepatitis E virus. The subsequent isolation of molecular clones of parts of the HEV genome by Reyes and colleagues 1 provided the basis for development of the first practical tools for diagnosis of the disease. Virus MOLECULAR VIROLOGY Virions of HEV are nonenveloped, icosahedral particles of around 32-nm diameter, without any obvious distinguishing morphology (Fig ). HEV has a buoyant density of 1.35 to 1.40 g/cm 3 in CsCl 4 and a sedimentation coefficient of 183 S. 5 The virus appears to be relatively stable to environmental and chemical agents. 6 HEV is not present in large amounts in clinical material (bile and feces), and yields from cell culture are generally very low, which has limited the opportunities for characterization of authentic viral particles. However, cell culture systems now reveal many molecular details of virus replication. Propagation of HEV was first seen in primary macaque hepatocytes, 7 but more robust cell culture systems have been reported in recent years. The first of these uses a subclone of hepatocyte-derived HepG2 cells, and although virus yields remain low, it has allowed some key questions of virus replication to be addressed 8-11 and has provided a system for the study of virus neutralization. 12 Growth and serial passage of a particular isolate of HEV has also been described in hepatocyte-derived PLC/PRF/5 cells 13 ; this system has allowed some preliminary characterization of progeny virus particles. 14 Hepatitis E virus has a single-stranded RNA genome of positive polarity that contains three open reading frames (ORFs), organized as 5 -ORF1-ORF3-ORF2-3 (Fig ), with ORF3 and ORF2 largely overlapping. The prototype infectious complementary DNA (cdna) clone of the SAR55 strain is 7204 nucleotide (nt). 15 Full-length HEV sequences deposited in Genbank range in size up to 7251 nt plus the poly A tail. The 5 end of the genome has a 7-methylguanosine cap 16 that is essential for infectivity, 15 and the genome contains short, highly conserved 5 and 3 untranslated regions (UTRs) of 35 and 68 to 75 nt, respectively. Early studies described the presence of two subgenomic viral RNAs in the liver of infected macaques that were presumed to encode the ORF2 and ORF3 proteins separately. 17 However, the development of functional replicons of HEV RNA has recently allowed the identification of a single subgenomic RNA that functions as a bicistronic messenger RNA (mrna) for translation of both ORF2 and ORF3 proteins. 11 Translation of ORF1 yields a polyprotein (PORF1) of approximately 186 kd that contains sequence motifs consistent with methyltransferase, papain-like protease, RNA helicase, and RNA-dependent RNA polymerase (RDRP) activities. 17 The putative HEV protease may require other cofactors because expression of PORF1 alone in HepG2 cells or in an in vitro translation system failed to show any proteolytic processing into mature products. 18 The PORF3 is a basic protein with an isoelectric point around 12.5 and is the most variable protein between HEV strains. PORF3 appears to be initiated from the third in-frame AUG codon, 19 with a mass of around 11 to 13 kd. PORF3 is associated with the cytoskeleton and is phosphorylated at serine residue 80 by mitogen activated protein kinase. 20 A large number of host cell protein interactions have been reported for PORF3, but the specific biologic role of these interactions has not been determined. The role of PORF3 in viral replication remained unclear for many years, with conflicting results on whether the protein was essential for replication. This conflict was resolved with the discovery of a specific RNA structure that is essential for replication, the cis-reactive element (CRE) in the open reading frame for PORF3. 26 Mutations that abolished PORF3 synthesis without affecting the RNA structure of the CRE allowed replication in cell culture, 10 but translation of PORF3 was essential for infectivity in nonhuman primates. 26 PORF3 may play a role in virus release to allow amplification in the liver, 10 with some recent data that provided evidence for PORF3 associated with HEV virions from cell culture 14 consistent with this putative role. Phosphorylation does not appear to be essential for these functions. 26 The capsid protein (PORF2) is translated as a 660 amino acid (aa) protein. A large proportion of the nascent protein is modified by 2411

2 2412 Part III Infectious Diseases and Their Etiologic Agents Figure Antibody-coated hepatitis E virus particles detected in feces of a patient with hepatitis E in Pakistan (immune electron microscopy). (Modified from Ticehurst J, Popkin TJ, Bryan JP, et al. Association of hepatitis E virus with an outbreak of hepatitis in Pakistan: Serologic responses and pattern of virus excretion. J Med Virol. 1992;36:84-92.) N-glycosylation after heterologous expression in mammalian cells, 27 but this glycosylated form of the protein is highly unstable. 28 When PORF2 is expressed in insect cells, it is cleaved at a predominant site between aa 111 and 112 and at various sites within the C-terminus of the protein. At least some of these truncated forms of PORF2 have the ability to self assemble into virus-like particles (VLPs) or subviral particles (SVPs), and cryoelectron microscopy has revealed structural details of such particles. 33,34 Conversely, a protein the size of full-length, nonglycosylated PORF2 has been detected in HEV replicon cell lines. 35 Further studies of native virus particles are necessary to determine whether the native capsid protein is full-length or truncated, and glycosylated or nonglycosylated, but yields from cell culture remain too low for detailed studies. Expression of truncated PORF2 (aa 112 to 636) in the baculovirus system leads to the formation of HEV VLPs. 29,36,37 These VLPs are smaller than the intact virus particle (27 versus 32 nm), but cryoelectron microscopy 33,34,38 suggests that HEV VLPs are assembled as a T = 1, icosahedral particle that contains 30 dimeric subunits of 50 kd PORF2, with the potential to form an intact virion of the correct size with a T = 3 arrangement of 90 dimeric subunits. 33 PORF2 dimerization appears to be the result of noncovalent interactions in the C-terminal part of the protein. 34,39,40 CLASSIFICATION Hepatitis E virus was tentatively assigned to the Caliciviridae family for some years on the basis of its particle structure and overall genome organization. However, detailed analysis of the viral genome showed that it had many features that were inconsistent with classification in any existing virus family, 41 and HEV has now been assigned to the new genus Hepevirus, family Hepeviridae. 42 A related virus of chickens (avian HEV) 43,44 is the only other family member known at this time. GEOGRAPHIC DISTRIBUTION AND GENETIC VARIATION Although rare cases of sporadic HEV have been reported from many developed countries, the major disease burden of HEV is in developing countries, consistent with its enteric transmission. The Indian subcon- tinent, Egypt, and parts of China are recognized as highly endemic areas, with HEV the most common cause of acute hepatitis in these countries. HEV has been responsible for major epidemics in refugee settings in Darfur, Sudan, over the past several years Sporadic HEV has been reported in South Africa, Chad, Tunisia, and Morocco and is likely to be prevalent throughout the African continent. The prevalence of HEV in South America has been the subject of only isolated studies, but studies in Cuba have shown quite high prevalence of clinical HEV infection. 51,52 Epidemic HEV has also been reported in Mexico. 53 In many developing countries, the incidence and prevalence of HEV infection has not been examined, and countries with poor sanitation should be assumed to have a high risk for endemic HEV infection and disease. The distribution of HEV within endemic countries can be highly variable, providing ideal conditions for epidemics in conditions of population displacement when combined with poor sanitary conditions, such as in refugee camps or in army recruits. Strains of HEV recovered from within one geographic region generally are genetically similar and characteristic of that region and differ from strains indigenous to other regions However, all human strains recovered to date appear to belong to the same serotype. 5 Comparison of HEV sequences provides the basis for classification of strains into genotypes, with around 75% nucleotide identities between genotypes (Fig ) Genotype 1 includes the prototype Burmese HEV 1,17 and related strains (including most Chinese strains), and genotype 2 is represented by the Mexican strain 65 and has also been detected in Africa, 66,67 where it cocirculates with genotype Genotype 3 includes the prototype swine HEV strain discovered in the United States 69 and closely related strains isolated from patients infected in the United States. 58 Genotype 4 includes isolates from a minority of patients in China (T1 strain) 61 and both patients and swine in Taiwan. 70,71 The strong relationship between swine and human HEV isolates in the United States, 58,69 Taiwan, 70,71 Korea, 72 and Japan 73,74 is 5' UTR 5' AAAn3' MT Papain-like protease Viral (+) RNA PORF3 Regulatory protein? Helicase RDRP PORF2 Capsid protein 3' UTR Translation PORF1 Replicative polyprotein ORF2.1 epitope Neutralizing epitope Figure Genome organization and major antigenic domains of HEV. Genome of around 7200 nucleotides contains 5 7-methylguanosine cap and highly conserved 5 and 3 untranslated regions (UTRs) of 35 and 68 to 75 nt, respectively. Three open reading frames (ORFs), organized as 5 -ORF1-ORF3-ORF2-3, encode viral proteins. PORF1 polyprotein contains replicative proteins. PORF2 is the major capsid protein, and the function of PORF3 is unknown but dispensable for replication in vitro. Both PORF2 and PORF3 are translated from single bicistronic, subgenomic RNA. Linear antigenic domains have been identified with peptide scanning throughout each of the three proteins; within PORF2, a conformational, immunodominant epitope (ORF2.1 epitope) is found between aa 394 to 457, and a conformational, neutralizing epitope is found between aa 578 to 607. (Modified from Anderson DA, Shrestha IL. Hepatitis E virus. In: Richman DD, Whitley RJ, Hayden FG, eds. Clinical Virology. Washington, DC: ASM Press; 2008: )

3 177 Hepatitis E Virus 2413 AKL 90 India Burma Myanmar Nepal Hyderabad, India U22532 India Vietnam D11092 China D11093 China Hetian, China KS2-87 China SAR 55 Pakistan Egypt 93 Egypt 94 Morocco Greece 1 Italy Greece 2 Swine HEV USA US 1 US 2 Swine HEV NZ Mexico Ch t11 China Ch t21 China HEVT 1 China Avian HEV USA 5 5 Changes Figure Genetic diversity of hepatitis E virus strains. Five major genotypes have been identified. Genotype 1 is found in Asia, the Middle East, and North Africa. Genotype 2 has been found in Mexico and Nigeria. Genotype 3 has been recovered from humans in North and South America, Europe, and Japan and from swine in North America, Europe, Asia, and New Zealand. Genotype 4 has been recovered from humans and swine in Asia. Genotype 5 has been recovered from chickens in North America and Australia. (Modified from Haqshenas G, Shivaprasad HL, Woolcock PR, et al. Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis- splenomegaly syndrome in the United States. J Gen Virol. 2001;82: ) consistent with presumed zoonotic exposure and shows the importance of viral genotypes in helping to understand the epidemiology of HEV infection, especially in nonendemic countries. Avian HEV shares only around 50% nucleotide sequence identity but also shows some antigenic cross-reactivity with human and swine strains. 44 Avian HEV has not yet been formally classified; it is currently considered as genotype 5 in the Hepevirus genus (see Fig ) but might also be considered to represent a separate genus, as in the case of the mammalian and avian hepatitis B viruses. ANTIGENIC COMPOSITION The capsid (PORF2) protein is the major, if not only, structural protein of HEV and represents the most relevant antigen for diagnosis and prevention. Mammalian genotypes of HEV exist as a single serotype with cross-genotype serologic protection. Immunization of macaques with recombinant PORF2 proteins of 55 kd (soluble, 112 to 607 aa) or 62 kd (VLP, 112 to 636 aa) expressed with a baculovirus system confers immunity to both homologous and heterologous virus challenge, 37,75-77 which suggests that major protective epitopes are common among HEV genotypes. A major neutralizing epitope has been identified in the region of amino acids 458 to 607 in PORF2 and shows equivalent reactivity with all genotypes. 78 The enzyme-linked immunosorbent assay (ELISA) based on this antigen should prove useful in clinical vaccine development. A distinct immunodominant, conformational PORF2 epitope has also been identified in the region of amino acids 394 to 457, 79 although its formation is dependent on expression of the longer HEV fragment from 394 to approximately 660 because of the requirement for dimerization. This ORF2.1 epitope is highly conserved between HEV strains and represents as much as 60% of the total HEV-specific antibody repertoire, 79 properties that have contributed to development of improved commercial diagnostic tests In contrast to these conserved epitopes, type-specific epitopes in PORF2 and PORF3 were first reported by Yarbough and colleagues 83 in comparisons of the Burmese and Mexican strains of HEV. Antibody responses to these type-specific epitopes also vary between patients infected with the same types, which compromises their performance in diagnostic assays. However, pools of type-specific peptide epitopes (PORF2 and PORF3) are used in a number of commercially available diagnostic assays, and a mosaic protein that encodes multiple peptide epitopes 84,85 has also shown some utility. Cell culture neutralization assays have been reported but are difficult to perform. 12,86 However, these have provided further evidence for the existence of a single serotype with cross-neutralization of genotype 1 virus seen with sera from animals infected with each of the four genotypes. 12,86 EPIDEMIOLOGY Developing Countries In endemic countries, sporadic HEV infection is often the most common form of viral hepatitis on an annual basis (Fig ). HEV is responsible for at least 25% of sporadic hepatitis between epidemics in northern India, 87 with other estimates as high as 70% in Kathmandu, Nepal. 88 Major epidemics also occur with a period of around 7 to 10 years. For example, HEV was responsible for 16 of 17 epidemics of enterically transmitted hepatitis in India. 89 In general, epidemics are associated with the wet season (summer in most endemic countries). Large outbreaks of HEV were seen after flooding that coincided with the conflict and displacement of 1.8 million civilians in the Darfur region of Sudan. 45,46 From July to December 2004, 2621 hepatitis E cases were recorded in the Mornay refugee camp of 78,800 inhabitants (an attack rate of 3.3%), with an overall case-fatality rate of 1.7%. In many studies, the prevalence of anti-hev in infants and children has been much lower than expected for a virus transmitted via the fecal-oral route The greatest incremental increase in the prevalence of anti-hev has generally been found among young adults, the age group at the highest risk of clinical disease (Figs and 177-6) In older adults from regions in which the virus is endemic, the prevalence of anti-hev is relatively constant (10% to 40%), with little or no difference in the prevalence between men and women Although such a pattern of age-specific anti-hev might suggest a cohort effect representing the disappearance of HEV from endemic regions, as was seen for HAV previously, 95 similar age-specific anti-hev patterns have been reported for sera collected 10 years apart from the same population in an area that is highly endemic for HEV (see Fig ) Thus, HEV appears to have epidemiologic characteristics that are quite different from those of most viruses, such as HAV, that are transmitted via the fecal-oral route. In many developing countries with endemic HEV, most of the population has been exposed to HAV during childhood (see Fig ), when clinical attack rates are low. As such, clinical hepatitis in young and older adults is more likely to be hepatitis E than hepatitis A. However, in countries with endemic HEV and intermediate rates of HAV exposure (such as Cuba), sporadic cases and outbreaks can result from contamination of water supplies with both enterically transmitted viruses, resulting in dual infection. 52

4 2414 Part III Infectious Diseases and Their Etiologic Agents 22.7% India (New Delhi) 5.3% A Non A-E E B 10.7% C 8.0% 17.5% Saudi Arabia (Jedda, Mecca) 13.4% 5.1% Non A-E A E B D C 4.1% 53.3% 13.8% 46.1% 75 cases (adults) 217 cases (adults) Figure Importance of hepatitis E virus (HEV) in etiology of viral hepatitis in regions where the virus is endemic. HEV is the most important cause of sporadic hepatitis among adults (and the second most important cause among children) in Asia and the second most important cause among adults (after hepatitis B virus) in the Middle East and North Africa. Hepatitis of unknown etiology is important in both regions. (Adapted from Das K, Agarwal A, Andrew R, et al. Role of hepatitis E and other hepatotropic virus in aetiology of sporadic acute viral hepatitis: a hospitalbased study from urban Delhi. Eur J Epidemiol. 2000;16: ; and Ghabrah TM, Strickland GT, Tsarev S, et al. Acute viral hepatitis in Saudi Arabia: seroepidemiological analysis, risk factors, clinical manifestation, and evidence for a sixth hepatitis agent. Clin Infect Dis. 1995;21: ) Developed Countries In developed countries, sporadic clinical HEV is rare, and epidemics have not been reported. Clinical HEV infection in nonendemic, developed countries is often associated with recent travel to endemic areas, but indigenous cases are also detected. Swine HEV strains have a worldwide distribution, and infection in pigs from both commercial farms and wild populations is almost ubiquitous. 70,72,73,96-99 Swine HEV has been shown to cross species, 100 and the close genetic relationship between swine HEV and strains isolated from some patients in the United States, 58,69 Taiwan, 70,71 Korea, 72 and Japan 73,74 is consistent with a zoonotic source of these indigenous infections, although swine may not be the only reservoir. As such, HEV infection should be considered a possibility in patients with acute hepatitis who do not have a relevant travel history or markers of other hepatitis viruses. The true rate of subclinical HEV infections in developed countries remains controversial. Serologic assays based on truncated PORF2 protein (aa 112 to 660) expressed with the baculovirus system have yielded seroprevalence rates of more than 20% in blood donors from Baltimore, 101 similar to the rates observed in Saudi Arabia with pre- Percent positive Anti-HAV Anti-HEV >45 Months Age groups Years Figure Age-specific prevalences of antibodies to hepatitis A virus and hepatitis E virus in a population in Pune, India. Antibodies were measured with enzyme-linked immunosorbent assay (ELISA). Infection with hepatitis A virus occurred at an earlier age and in a higher proportion of the population than infection with hepatitis E virus. (Modified from Arankalle VA, Tsarev SA, Chadha MS, et al. Age-specific prevalence of antibodies to hepatitis A and E viruses in Pune, India, 1982 and J Infect Dis. 1995;171: ) sumed high rates of endemic infection. 102 These assays may therefore overestimate exposure rates in nonendemic populations. Conversely, with an ELISA based on the ORF2.1 protein (aa 394 to 660) expressed in Escherichia coli, rates of less than 2% were found in Australian blood donors compared with more than 35% in highly endemic Nepal. 103 This assay also showed significant differences in exposure rates between urban (low-risk) and indigenous rural (high-risk) populations in Malaysia. 104 The ORF2.1-based assays may therefore provide a better estimate of exposure to HEV. The avian HEV appears to share some antigenic cross-reactivity with human HEV strains, 43,105 and rats have also been implicated as a potential reservoir of HEV. 106 Exposure to these HEV-like viruses and crossreactive antibody may contribute to the high seroprevalence estimates in developed countries with some assays. Further studies are needed to resolve the true rate of HEV exposure in these populations. MODES OF TRANSMISSION Hepatitis E is transmitted via the fecal-oral route. Consumption of water contaminated with human fecal waste is by far the most common route of transmission for clinical cases in countries where HEV is endemic, although foodborne outbreaks had been reported in China before the advent of specific serologic assays for HEV infection. Number of cases Age groups Figure Age-specific clinical attack rate of hepatitis in a large, waterborne epidemic of hepatitis in Delhi, India, 1955 to Epidemic was caused by hepatitis E virus. (Modified from Viswanathan R. Epidemiology. Indian J Med Res. 1957;45:1-29. Copyright 1957 Indian Council of Medical Research.)

5 177 Hepatitis E Virus 2415 Most human isolates of HEV in countries where the virus is not considered endemic are found to be more closely related to swine HEV strains (Meng 107 provides a comprehensive review of zoonotic HEV infection). Clearly defined cases of zoonotic HEV infection in Japan have been associated with consumption of HEV-infected pig or deer meat, and swine HEV has been detected in both wild pigs and deer in Japan. 73,99, However, in most nonendemic countries, the true rate of zoonotic infection remains unclear because of limited testing. Hepatitis E is not commonly transmitted via person-to-person contact (in contrast to hepatitis A), so household transmission is therefore not a significant risk factor. Nosocomial infections with HEV are probably rare, but transfusion-associated HEV has been reported in Japan 111 and is likely to be more common in countries in which the virus is endemic, with viremia of up to 45 to 112 days detected with polymerase chain reaction (PCR) in naturally infected patients. 112,113 HOST RANGE: EXPERIMENTAL TRANSMISSION TO ANIMALS Human strains of HEV can readily infect and cause disease in many nonhuman primates, with cynomolgus macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta) widely used for experimental purposes and New World monkeys (owl monkeys, squirrel monkeys, and tamarins) also susceptible. The swine HEV was first detected in pigs in the United States 69 and subsequently shown to be endemic in swine worldwide, and in wild deer in Japan. 73,99, Swine HEV has been transmitted to macaques, and human HEV (genotype 3) to swine. 100,107 Swine do not appear to have clinical or biochemical disease develop after infection with either swine or human isolates of HEV. 114 Rodents and sheep have been reported to be susceptible to experimental infection, 115,116 but these studies need confirmation. The course of infection in experimentally infected primates is similar to that in humans. 117 The incubation period to peak liver enzyme levels is generally 3 to 8 weeks but can be quite variable, depending on the dose of virus administered. 31, Peak viremia and peak shedding of virus in the feces occur during the incubation period and early acute phase of disease. The detection of HEV antigens in the liver parallels the detection of viremia in the serum and feces, and histologic changes in the liver generally parallel biochemical evidence of hepatitis. 121 As in humans, hepatitis E in nonhuman primates is acute and self-limiting. Unlike experimental hepatitis caused by the other human hepatitis viruses, experimental hepatitis E is dose dependent: high doses of virus are associated with histologic and biochemical evidence of hepatitis, whereas lower doses of virus (1000 infectious doses) are more likely to be associated with a normal histologic appearance and normal serum liver enzyme values. 122 Infection with HEV protects nonhuman primates from hepatitis E after reexposure to the virus Evidence for fetal wastage was detected in one study but not in a second study of experimental HEV infection of pregnant rhesus monkeys. 126,127 HEPATITIS E VIRUS INFECTION IN ANIMALS Although a number of species including rats, sheep, and domestic cattle have been reported to have serologic evidence of exposure to HEV, unequivocal evidence for HEV infection in animals has relied on the detection of virus with PCR. This detection was first achieved in the landmark studies by Meng and colleagues 69 in which an HEV strain isolated from young domestic swine in the United States was experimentally transmissible to other swine. This virus was genetically distinct from all previously recovered human strains and probably represents the first recognized nonhuman strain of HEV. Interestingly, two isolates of HEV from patients with human hepatitis E in Tennessee and Minnesota were shown to be closely related to swine HEV, and one of these was experimentally transmitted to swine. 58,69,128 Because swine HEV and the two human United States isolates were genetically very similar (including sharing specific nucleotide insertions) and because they came from the same geographic region, all three of the viruses are possibly of swine origin. Although no direct epidemiologic linkage could be drawn between these first identified cases in the United States and exposure to swine, clearly defined cases of zoonotic HEV infection in Japan have been associated with consumption of HEV-infected pig or deer meat. 73,74,99, Conversely, zoonotic infection likely plays only a minor role in circulation of HEV in countries where the virus is endemic in human populations. In India, genotype 1 circulates in humans, whereas genotype 4 circulates in swine in the same region. 129,130 However, differences have been observed in the magnitude of antibody responses among patients with acute hepatitis E during the epidemic versus interepidemic season in Nepal, which could possibly represent exposure to zoonotic strains (genotypes 3 and 4) with somewhat lower antibody reactivity to the human genotype 1 proteins used in the assay. 131 The transmission of animal viruses to humans via transplantation of animal organs and tissues to humans is a potential threat. The prospects for such transmissions have been greatly increased with improvements in procedures to control immediate and delayed rejection of animal organs by the human immune system. With the control of rejection a possibility, growing interest is seen in the xenotransplantation of animal organs and tissues because of the shortage of suitable clinical materials of human origin. The discovery of swine HEV has added another virus to the list of viruses that must be excluded from swine herds before tissues and organs from those herds can be considered for xenotransplantation (Meng 107 provides a more extensive review of HEV risk in xenotransplantation). Pathogenesis INCUBATION PERIOD The incubation period from exposure to the onset of clinical disease is approximately 28 to 40 days, based on analysis of waterborne epidemics in which the time of exposure was identified. 132 In experimental HEV transmission studies in humans, liver enzyme values peaked 42 to 46 days after ingestion of the virus, 4 and in experimental infections of macaques with intravenous challenge and high doses of virus, disease can be evident as early as 2 weeks after exposure 122,133,134 and viremia is detectable with reverse transcriptase-polymerase chain reaction (RT-PCR) as early as 9 days after exposure. 122,134 VIRAL REPLICATION Although some progress has been made in understanding the replication cycle of HEV with use of animal models, cell culture systems, and infectious cdna clones, most aspects of viral replication in vivo can only be inferred (Fig ). After ingestion of HEV, the virus may be absorbed directly through the gastrointestinal mucosa into the circulation to reach the liver or after one or more rounds of amplification in enterocytes. However, no evidence has been obtained for replication of HEV at this site, in contrast to HAV for which replication in enterocytederived Caco2 cells is well established. 135 HEV is likely to interact with one or more specific receptors/coreceptors leading to penetration and uncoating of the virus, and the input viral RNA then serves as mrna for PORF1. The PORF1 polyprotein is then cleaved by viral (and perhaps cellular) proteases to yield the mature replicative proteins, but the viral protease activity has not been directly demonstrated. The viral RNA-dependent RNA polymerase (RDRP) then copies the input viral genome to yield negative-strand RNA, which in turn serves as a template for the transcription of further positive-strand RNA molecules, including new genomes and the subgenomic, bicistronic RNA encoding ORF2 and ORF3 proteins. Genomic RNA assembles together with PORF2, although the precise form involved in particle assembly (fulllength or truncated, glycosylated or nonglycosylated) is unclear. PORF3 may also associate with the virus particle during assembly, 14 although it is dispensable for replication in vitro. The virus is then released from the hepatocyte via unknown mechanisms.

6 2416 Part III Infectious Diseases and Their Etiologic Agents 1 epithelium before infection of the liver, as for HAV. Low amounts of HEV can be detected in serum during the late incubation period and for 2 to 6 weeks after the onset of illness. HEV is excreted in feces; virus has not been reported in other excretions. BC 9 8 Viral RNA (positive) 3 4 A small amount of HEV is found in plasma during infection, consistent with release of progeny virus through the basolateral domains of hepatocytes leading to spread through the liver, but most of the virus is likely to be excreted through the biliary system to complete the transmission cycle, 126,134 consistent with release of virus through the apical domain of hepatocytes. Interestingly, however, HAV shows preferential release through the basolateral domain of hepatocytes in vitro, contrary to the similar expectation of its apical release into the biliary system, which suggests an indirect route for excretion of that virus. 136 Hepatitis E virus replication has not been observed in tissues other than liver, but a low level of replication is likely to occur in enteric 7 2 Polyprotein PORF1 Replicative proteins Negative RNA Positive RNA(s) 5 PORF3 6 Structural protein(s) PORF2 Nucleus Figure Putative replication cycle of hepatitis E virus. After oral ingestion, HEV particles reach the liver where they attach to an unidentified receptor on the basolateral domain of hepatocytes, leading to virus penetration (step 1) and uncoating of the genome (step 2) within the cell. Translation of the input genome (step 3) yields the PORF1 polyprotein, which is cleaved at unknown sites to yield the replicative proteins, which copy the input genome to yield full-length negative strand RNAs (step 4), followed by subgenomic positive strand (messenger) RNAs and full-length positive strand RNAs (new viral genomes; step 5). These subgenomic RNAs are translated to yield further molecules of PORF1, PORF2 (capsid), and PORF3 (regulatory) proteins (step 6). PORF2 and new viral genomes assemble into virions (step 7); whether virions contain full-length or truncated PORF2 products is not known, and the role of membranes and PORF3 in assembly is unclear. Release of progeny virus through basolateral domains of the infected cell (step 8) may result in viremia and infection of distal hepatocytes or infection of neighboring hepatocytes, but transmission to new hosts through the environment is achieved via release of progeny through the apical domain (step 9) to the bile canaliculi (BC) and ultimately the feces. (Modified from Anderson DA, Shrestha IL. Hepatitis E virus. In: Richman DD, Whitley RJ, Hayden FG, eds. Clinical Virology. Washington, DC: ASM Press; 2008: ) PATHOLOGY Histopathologic studies of liver specimens obtained from patients in epidemics have shown that infection with HEV can produce morphologic changes in the liver that comprise both cholestatic and classical acute hepatitis, but these features are not unique or diagnostic in hepatitis E. Typical histopathologic changes include lobular disarray with enlargement of portal tracts, Kupffer cell proliferation, focal hepatocyte necrosis and bridging necrosis, ballooning of hepatocytes and acidophilic degeneration of hepatocytes, and mononuclear cell infiltration. Within hepatocytes, dilation occurs of the cisternae of the endoplasmic reticulum with an increase in the number and size of lysosomes within the cytoplasm. Condensation of the matrix within mitochondria together with dilation of the outer mitochondrial membrane has also been reported. Cholestatic hepatitis E is characterized by bile stasis in canaliculi and gland-like transformation of hepatocytes, degeneration of hepatocytes, and intralobular and portal tract infiltrates of lymphocytes and polymorphonuclear leukocytes. A prominent feature is the presence of cholestasis and glandular transformation of the liver cell plates, with the cholestatic changes persisting until clinical recovery occurs. These histopathologic changes gradually resolve over a period of 3 to 6 months. Patients with fulminant hepatitis E infection have necrosis of parenchyma with collapse of liver lobules; swelling of hepatocytes, which have a foamy appearance; arrangement of hepatocytes into an acinar pattern; proliferation of small bile ductules; phlebitis of portal and central veins; and portal inflammation with lymphocytic and polymorphonuclear leukocyte infiltration. 138,140,141 HEV replication has also been found also in bile epithelial cells in rhesus monkeys and in many extrahepatic sites in swine, 142,143 but the significance of these extrahepatic sites is unclear. The average severity of HEV infections is somewhat greater than that of hepatitis A infections, with a mortality rate of around 1%, compared with 0.2% in hepatitis A. However, HEV infection during pregnancy is associated with a high mortality rate from fulminant hepatitis, around 25% in the third trimester. 141,144,145 The mortality rate appears to increase with each succeeding trimester of pregnancy IMMUNE RESPONSE The patterns of IgG-class and IgM-class antibody responses during HEV infection are shown in Figure These responses are typical of those detected with the 55 to 63 kd ORF2 antigens expressed with a baculovirus system and the ORF2.1 protein expressed in E. coli, which can detect specific IgG and IgM responses in most patients. Typically, both IgG and IgM antibodies are detectable at the onset of disease, which allows serologic diagnosis of infection at the time of presentation of the patient; IgM declines to undetectable levels over a period of 2 to 6 months, and an approximately 10-fold decline in IgG levels is seen over this period. Levels then stabilize, but the duration of protective immunity is unknown. In contrast to the anti-hev responses shown in Figure 177-8, as few as one in three macaques challenged with a common source inoculum have a detectable IgG response to homologous HEV PORF3 and to many of the linear epitopes in PORF2, and this response is transient. 133 As a result, assays based on these proteins are not suitable for seroepidemiologic studies. Immunoglobulin A (IgA) responses to HEV (as a correlate of mucosal immunity) have been detected in around 50% of patients 149 ; these antibodies rapidly declined to undetectable levels, although IgA may persist somewhat longer than IgM. 150,151 The role of IgA in immunity to HEV infection is unknown, but because passive immunization

7 177 Hepatitis E Virus 2417 Antibody ALT Viral particles in feces Viral replication Viremia Jaundice Symptoms Days Months Time after exposure Figure Diagram of clinical and serologic events in typical case of acute hepatitis E. Antibody patterns are based on enzymelinked immunosorbent assay (ELISA) results; viremia and fecal shedding are based on polymerase chain reaction data. After reaching high levels during the acute phase, HEV PORF2-specific IgG declines rapidly over 6 to 12 months and might not persist at protective levels for life, and HEV-specific IgM disappears over 3 to 6 months and is diagnostic for acute HEV infection. (Modified from Purcell RH, Hoofnagle JH, Ticehurst J, et al. Hepatitis viruses. In: Schmidt NJ, Emmons RW, eds. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. 6th ed. Washington, DC: American Public Health Association; 1989: ) with IgG appears to be sufficient for protection, 75 IgA is likely not essential. Detection of IgA (in addition to IgM) may have some value in diagnosis of HEV, but further studies are needed. 150,151 Cellular immune responses during HEV infection have not been extensively studied. In patients with HEV, T-cell responses (proliferation) to peptide libraries derived from the PORF2 and PORF3 proteins are observed with peptide pools derived from PORF2 but not PORF Clinical Manifestations Histopathology IgM SYMPTOMS Hepatitis E is clinically indistinguishable from other forms of acute viral hepatitis. The clinical presentation of acute viral hepatitis commonly begins with nonspecific, flu-like prodromal symptoms that last from 1 to 10 days and consist of fatigue, malaise, anorexia, nausea, vomiting, and some alteration in taste and smell. A low-grade fever between 38 C and 39 C is common. The first distinctive signs of hepatitis are often dark urine and pale clay-colored stools followed by onset of clinical jaundice. With the appearance of clinical jaundice, the prodromal symptoms usually subside; however, some patients may not show visible signs of jaundice despite severe symptoms. Abdominal examination may reveal enlarged and tender liver associated with pain and discomfort in the right upper quadrant. Spleen may be enlarged in 10% to 15% of patients. Occasionally, patients may present with cholestasis, which is more common in pregnant women. Resolution of HEV is accompanied by normalization of biochemical markers (serum alanine aminotransferase [ALT] levels, bilirubin) over a period of 6 weeks in most patients, whereas histologic changes may persist for up to 6 months without overt disease. Small numbers of patients appear to have a protracted course of disease, with resolution taking many months. IgG Hepatitis E virus infection during pregnancy is associated with a high mortality rate from fulminant hepatitis that peaks at around 25% in the third trimester. 141,144,145 The basis of the high mortality during pregnancy is not understood; studies in pregnant macaques have not shown any increase in disease severity. 127 Although hormonal factors may contribute to pathogenesis during pregnancy, other factors may also be important, such as the underlying general health status or chronic infection with hepatitis B or C in patients at the time of HEV infection. 153,154 No specific pathogenic factors have yet been identified. Vertical transmission of HEV with severe hepatitis in the infant has been seen. 153,154 HEV has not been associated with congenital abnormalities. Liver function tests are an important adjunct to diagnosis of acute HEV. The serum aminotransferases ALT and aspartate aminotransferase (AST) show variable increases during the prodromal phase. The ALT level peaks at the onset of symptoms before the serum bilirubin begins to increase. Peak levels of ALT vary from 1000 U/L to 2000 U/L at the onset. ALT levels progressively diminish during the recovery phase. Some patients with anicteric acute HEV infection have only raised ALT levels. Jaundice is visible in sclera or skin when the serum total bilirubin level exceeds 2.5 mg/dl, usually following the peak levels of ALT. Peak serum total bilirubin levels range from 5 to 25 mg/dl; both conjugated and unconjugated fractions are increased. In cholestatic HEV infection (around 10% of patients), serum bilirubin may remain elevated for prolonged periods. Prothrombin time may be increased in acute viral hepatitis, especially in fulminant hepatitis, indicating extensive hepatocellular necrosis and a worse prognosis. Similarly, a reduction in the serum albumin level may occur. Reduced blood glucose levels leading to hypoglycemia may be observed in patients with prolonged nausea, vomiting, and inadequate carbohydrate intake. Neutropenia, lymphopenia and atypical lymphocytes may occasionally be observed during the acute phase of viral hepatitis. COMPLICATIONS The risk and severity of clinical HEV disease increases with age at exposure, as with hepatitis A. Fever as the presenting sign of hepatitis E may be more common in the young. A major complication of HEV infection is fulminant hepatitis, observed in less than 1% of patients from the general population but in up to 30% during the third trimester of pregnancy, with a death rate as high as 100%. 153,154 Cholestatic hepatitis is seen in around 10% to 25% of patients, 155 with a prolonged period of cholestasis observed in some studies. Evidence shows that HEV infection contributes to fetal death and other complications when the mother has symptomatic hepatitis E, 141,145 but the situation with subclinical infections is unclear. Laboratory Diagnosis Acute hepatitis E has no distinguishing clinical characteristics that allow a differential diagnosis from other forms of acute viral hepatitis, and the detection of rare cases of locally acquired HEV in nonendemic countries argues for HEV to be considered in many cases of acute hepatitis, even without a relevant travel history to regions where the virus is endemic. Because of their identical clinical presentation and predominant modes of transmission, hepatitis E may be suspected in the same circumstances as hepatitis A. However, despite declining rates of hepatitis A infection through widespread use of inactivated hepatitis A vaccines, HAV infection remains more common than HEV infection in most developed countries because of much higher rates of HAV transmission through personal contact. Although more sensitive serologic assays are now available for detection of HEV IgM as a marker of acute infection, a low level of false-positive results (around 0.3% to 2%) is still detected in these assays, and the more common agents (HAV, hepatitis B virus, hepatitis C virus) should first be excluded for patients in nonendemic countries who do not have a relevant travel

8 2418 Part III Infectious Diseases and Their Etiologic Agents history or other exposure risk. Conversely, in endemic countries, HEV is often the most common cause of acute hepatitis and should be a first-line test. In those developing countries in which improved public health has resulted in a high prevalence of young adults who remain susceptible to HAV infection, waterborne epidemics may be caused by HAV or HEV. Unusually severe hepatitis in pregnant women suggests hepatitis E, but tests for both viruses may be prudent, given their potential for cocirculation in some populations. 52 Early detection of epidemic HEV can also allow improved public health responses, in particular the protection of pregnant women from exposure to contaminated water where possible. Specific diagnosis of HEV infection remains problematic. A variety of serologic and molecular assays are used in research laboratories, 62 but routine (commercial) diagnostic assays vary widely in sensitivity and specificity. Virus isolation and detection of viral antigen are not appropriate for diagnosis of HEV because of low sensitivity. SEROLOGIC TESTS A number of research and commercial immunoassays are available in various countries but have shown major differences in sensitivity and specificity. 156 More recent commercial assays appear to have improved on this performance through the use of better recombinant antigens and assay formats (detailed subsequently), but first-generation and second-generation tests are still commercially available in most parts of the world. The appropriate use and interpretation of serologic assays for HEV infection must take into account the quality of different tests and the widely varying prevalence of clinical HEV infection worldwide. 88 Diagnosis of Hepatitis E Virus Infection in Areas of Low or Intermediate Prevalence In areas with a low incidence of clinical HEV infection (such as the United States, Australia, and western Europe) or intermediate prevalence (such as Japan, Korea, and Taiwan), assay specificity has a large impact on the predictive value of HEV serologic tests. Diagnostic assays for the detection of HEV-specific IgG (manufactured by Genelabs Diagnostics/MP Biomedicals Asia Pacific, Singapore, and Abbott Diagnostics, Chicago) have considerable value for the diagnosis of acute hepatitis in travelers returned from endemic areas, 157 among whom the incidence rate may be higher than the background rate of reactivity, around 2% in the healthy population. However, with the recognition that HEV should be considered in the diagnosis of sporadic acute hepatitis without a travel history to recognized endemic regions, 58,158 the need for more specific tests becomes evident. The detection of HEV-specific IgM should therefore become the method of choice for serologic diagnosis of acute HEV infection in areas of low prevalence. Until recently, a single HEV IgM assay was commercially available in most countries (Genelabs Diagnostics/MP Biomedicals Asia Pacific), based on the same recombinant HEV antigens as the widely available IgG tests. This assay had a reported falsepositive rate in U.S. blood donors of 26/856 (3%) (Genelabs, 1998 technical bulletin), which made it no more suitable than HEV IgG for diagnosis in nonendemic areas. Published studies have also shown that the recombinant antigens used in this assay may fail to detect antibody in around 40% of patients with acute HEV infection. 36 Enzyme-linked immunosorbent assays with improved reactivity are in use in research laboratories, based on recombinant antigens derived from baculovirus-based 29,30,36,78,159 or E. coli based expression systems, 84,103 but these have not been widely adopted for commercially available assays. However, the ORF2.1 antigen, representing amino acids 394 to 660 derived from a Chinese strain of HEV expressed in E. coli, 103,133,160 has been shown to represent immunodominant and highly conserved epitopes of HEV 79 and forms the basis of the recently developed commercial HEV IgM ELISA 3.0 and the ASSURE rapid point of care (RPOC) tests produced by MP Biomedicals Asia Pacific (formerly Genelabs Diagnostics). In the first published study of these new tests, the IgM ELISA 3.0 showed sensitivity of 99.3% (150/151 Figure Representative test results obtained with ASSURE HEV IgM rapid point of care test. Sample with no detectable HEVspecific IgM (S982) shows single line (control, C), and samples with HEV-specific IgM (J89, J60, J70) show both control and test (T) lines (data from the authors laboratories). (Modified from Anderson DA, Shrestha IL. Hepatitis E virus. In: Richman DD, Whitley RJ, Hayden FG. Clinical Virology. Washington, DC: ASM Press; 2008: ) patients with HEV infection) and specificity of 97.6% (203/208 control subjects), and the RPOC test showed slightly reduced sensitivity (96.7%; 146/151) and slightly higher specificity (98.6%; 205/208) on the same samples. 80 In a separate study of the HEV IgM RPOC test, in which the comparator assay was an in-house quantitative IgM ELISA based on baculovirus-expressed HEV antigen, 159 the RPOC assay was also found to have high sensitivity (93%; 186/200) and specificity (99.7%; 320/321). 161 The RPOC test takes around 5 minutes in total and requires minimal training and no specialized equipment; representative assay results with the ASSURE HEV IgM test are shown in Figure The sensitivity of both the HEV IgM ELISA 3.0 and the ASSURE HEV IgM tests is clearly adequate for diagnosis of acute HEV infection, but the likely false-positive rate of between 0.3% and 2.4% urges some caution in the interpretation of positive results from patients without a history of travel outside areas of lowest endemicity. Where available, RT-PCR can provide useful confirmation. Compared with the United States and most of western Europe, larger numbers of HEV cases have been reported from Taiwan, 70,71 Korea, 72 and Japan. 73,74 Serologic diagnosis with HEV IgM tests is likely to have a higher positive predictive value in these populations than in the United States and other countries of lowest endemicity. Serologic detection of HEV infection in animals also poses a challenge; the quality of species-specific antibody conjugates and variable background reactivity can be problematic. Hu and colleagues 82 have recently described a double-antigen sandwich-based ELISA for total anti-hev antibodies, in which ET2.1 antigen (equivalent to ORF2.1) and its peroxidase-labeled counterpart are able to detect antibody from patients and from pigs; this assay should prove useful in the search for potential zoonotic reservoirs of HEV or HEV-like viruses. Diagnosis of Hepatitis E Virus Infection in Areas of High Prevalence Although the titer of HEV-specific IgG tends to decline markedly in the first year after infection, this relationship is not reliable enough to form the basis of differential diagnosis. 102,103 The detection of HEVspecific IgG is therefore of little use for diagnosis of acute infection in developing countries where HEV is endemic and where large numbers of patients have antibody from past infections. Detection of HEVspecific IgM must therefore be the method of choice in endemic areas. Because HEV accounts for as much as 70% of the acute sporadic hepatitis in endemic countries, the specificity of assays is less critical than assay sensitivity in these settings. In addition, HEV often is seen in areas where standard equipment, such as ELISA washers and readers, is not available. In this context, the ASSURE HEV IgM rapid