Scholarly article on topic 'Hepatitis E: a complex and global disease'

Hepatitis E: a complex and global disease Academic research paper on "Veterinary science"

0
0
Share paper
Academic journal
Emerging Health Threats Journal
OECD Field of science
Keywords
{""}

Academic research paper on topic "Hepatitis E: a complex and global disease"

Emerging Health Threats Journal 2008, 1:e8. doi: 10.3134/ehtj.08.008 © 2008 E Pelosi and I Clarke; licensee Emerging Health Threats Journal.

www.eht-journal.org

REVIEW

Hepatitis E: a complex and global disease

E Pelosi1 and I Clarke2

1 Department of Microbiology and Virology, Health Protection Agency, Southeast Regional Laboratory, Southampton General Hospital, Southampton, UK; and 2Department of Molecular Microbiology, Southampton Medical School, Southampton General Hospital, Southampton, UK.

Thirty years after its discovery, the hepatitis E virus (HEV) continues to represent a major public health problem in developing countries. In developed countries, it has emerged as a significant cause of non-travel-associated acute hepatitis. HEV infects a wide range of mammalian species and a key reservoir worldwide appears to be swine. Genomic sequence similarity between some human HEV genotypes and swine HEV strains has been identified and we know that humans can acquire HEV infection from animals. Although for the most part the clinical course of HEV infection is asymptomatic or mild, significant risk of serious disease exists in pregnant women and those with chronic liver disease. In addition, there are data on the threat of chronic infections in immunocompromised patients. Beyond management of exposure by public health measures, recent data support that active immunisation can prevent hepatitis E, highlighting the need for vaccination programmes. Here we review the current knowledge on HEV, its epidemiology, and the management and prevention of human disease.

Correspondence

Dr E Pelosi, Department of Microbiology and Virology, Southampton University Hospitals NHS Trust, Tremona Road, Southampton SOI 6YD, UK. E-mail: Emanuela. Pelosi@suht.swest.nhs.uk

Received 12 July 2007 Revised 12 March 2008 Accepted 10 April 2008

Introduction

First recognised in Asia almost 30 years ago as the main cause of non-A, non-B enterically transmitted hepatitis,1,2 hepatitis E virus (HEV) is now acknowledged to have worldwide distribution.

In countries with poor sanitation, HEV is endemic and typically causes explosive outbreaks of acute hepatitis, usually associated with faecal contamination of the water supply. The disease is generally mild, yet pregnant women suffer significant morbidity and mortality.3-5

In contrast, in countries with high standards of sanitation, hepatitis E occurs sporadically, initially identified as an imported disease in travellers from highly endemic regions, but subsequently diagnosed in patients with no travel history as well;this latter form has been named 'hepatitis E indigenous to developed countries'.6-26

Phylogenetic analysis of HEV genome from different isolates has led to the identification of four main genotypes, with genotypes 1 and 2 circulating in Africa and Asia, genotype 3 showing a broad distribution worldwide, and genotype 4 being restricted to Asia.

Genotypes 3 and 4 are enzootic in a variety of wild and domestic animals, particularly pigs,8,27-36 which gave rise to the question of whether human HEV infection is a zoonosis? Evidence from Japan37-40 and China28 now confirms that humans can acquire HEV infection from animals.

Hepatitis E represents a significant public health and economic burden particularly in countries where the absence of sanitation infrastructures, or their breakdown as a consequence of wars or natural disasters, brings the hygienic conditions below a safe level.4,41,42 The development of an effective vaccine is expected to dramatically reduce the incidence of the disease, particularly in the most susceptible individuals such as pregnant women.

Virology

Taxonomy and virus structure

In the early 1980s, the observation that individuals involved in epidemics of jaundice were seronegative for markers of acute hepatitis A and B suggested the existence of an unrecognised aetiological agent of enterically transmitted hepatitis.1,2 The confirmation came in 1983, when small, virus-like particles were identified by immune electron microscopy in stool specimens from a volunteer experimentally infected with pooled faecal extracts from human cases of epidemic non-A, non-B hepatitis.43 The pathogen, HEV, is a non-enveloped virus, 27-30 nm in diameter, with an icosahedral capsid. In the early 1990s, the virus genome was cloned44,45 and diagnostic antibody assays were devel-oped.46 After being provisionally assigned to the Caliciviridae family,47 HEV was re-classified as the sole member of the genus Hepevirus, family Hepeviridae, in 2004.48

3This is an Open Access article distributed under the terms of the Creative Commons Attribution licence which permits unrestricted use, distribution, and access reproduction in any medium, provided the original work is properly cited.

Genomic organisation and viral proteins

The HEV genome consists of a single-stranded RNA molecule with positive polarity approximately 7300 nucleotides in length.45,49,50 It comprises a short 5' noncoding region (28 nucleotides), followed by three open reading frames (ORF), a 3' noncoding region (65-74 nucleotides), and a poly(A) tail (Figure 1).45,53 The genome is capped at its 5' terminus54 and capping is required for virus viability.55

ORF1 (approximately 5 kb) encodes a large nonstructural polyprotein with key functions for viral genome replication and viral protein processing. ORF2 (approximately 2 kb) occupies the 3' end of the coding region and encodes the capsid protein.45,51,56-58 The N-terminal region of ORF2 protein binds the 5' noncoding region of the HEV genome59 and is possibly involved in viral encapsidation.41 Only the ORF2 recombinant protein truncated at its N-termini can efficiently self-assemble in vitro into empty, virus-like particles. These share antigenic properties with the native HEV capsid protein, although they are smaller than the

native virions.57,58

ORF3 is a small reading frame (372 bases) with the 5' end overlapping ORF1 by four nucleotides and the 3' end overlapping ORF2 by 331 nucleotides;45,53,51 it encodes a small phosphoprotein that associates with the cytoskeleton and the capsid protein.60,61 The product of ORF3 is possibly involved in modulation of cell signalling62,63 and in the assembly of the HEV nucleocapsid;60 recently, it has been shown that this protein is essential for infectivity in vivo64 but not in vitro.65 The poly(A) tail is necessary for binding of

RNA-dependent RNA polymerase (RdRp) to the 3' noncoding region.41,66

Compared with genotypes 1-3, the genome of genotype 4 contains a nucleotide insertion (U) just after the second AUG codon of ORF3, which changes the downstream reading frames so that, for genotype 4, different AUG codons were initially expected to initiate translation in both ORF2 and ORF3.52,53 However, a recent study by Graff et al.,51 using a replicon, demonstrated that ORF2 and ORF3 proteins are produced from a single subgenomic RNA of approximately

2.2 kb, which initiates downstream of the first two AUG codons of ORF3 in genotypes 1-3 (downstream of the insertion of U in genotype 4), using two closely spaced start codons. It is therefore expected that both ORF2 and ORF3 proteins are similar in size in all four genotypes.51

Resistance to physical and chemical agents

Boiling and chlorination of water represent the main measures to control and prevent infection. However, Emerson et a/.67 reported that HEV is moderately resistant to heat inactivation. This finding was further corroborated in a more recent study that showed how HEV was still infectious on incubation at 56°C for 30min.68

HEV genetic diversity

Comparative nucleotide sequence analysis of whole genomes of HEV isolates has revealed extensive genomic diversity leading to the identification of four major genotypes and several subtypes within each genotype (Figure 2). However, although the separation of HEV into four major genogroups is widely accepted, so far there is no agreement about the number of subtypes within each genotype.69 Genomic regions that have been used for phylogenetic purposes include a 301-nucleotide-long sequence at the 5' end of the ORF2 region (Figure 2)69 and a 306-nucleotide-long sequence in the RdRp of ORF1.70

Replication in cell culture

The lack of efficient cell culture systems has hampered detailed studies on HEV biology, critical for helping to develop diagnostic assays and vaccine research.68 Replication and propagation of HEV was attempted with limited success by using continuous cell lines71,72 and primary hepatocytes from nonhuman primates.73 Recently, more efficient cell-culture systems were developed in cell lines,67,68,74 allowing studies on HEV thermal stability and improving neutralisation tests. It was shown that anti-HEV antibodies are broadly crossreactive because HEV genotype 3 was neutralised by convalescent serum samples from patients

Genomic RNA (approximately 7.3kb)

Poly(A) tail

Met Y Pr P X Hel RdRp

Cap — ORF3 I I

Subgenomic RNA (approximately 2.2 kb)

Poly(A) tail

Figure 1 Schematic organisation of genomic and subgenomic HEV RNAs. ORF1 encodes a nonstructural polyprotein; ORF2 encodes the capsid protein; ORF3 encodes a phosphoprotein. Met, methyltransferase; Y, no function assigned at present; Pr, putative papain-like cysteine protease; P, proline 'hinge'; X, no function assigned at present; Hel, RNA helicase; RdRp, RNA-dependent RNA polymerase.51,52

Figure 2 Phylogeny of HEV genotypes (Gt.) based on the 301-nucleotide-long 5' end of the ORF2 region, the most conserved in all HEV isolates. Sequences determined from this region accounted for the majority of HEV sequences.69

infected with HEV genotypes 1, 3, and 4.68 Similarly, genotype 1 was neutralised by convalescent serum samples obtained from rhesus monkey infected with any of the four mammalian genotypes.74 In addition, serum specimens obtained 24 years after the onset of HEV infection could prevent the propagation of HEV in cell culture, suggesting that long-lasting HEV antibodies with neutralisation activity are induced.68

Epidemiology

Geographical distribution of HEV according to genotype

The geographical distribution of HEV genotypes is complex and continuously evolving (Figure 3). Genotype 1 extensively circulates in Asia (including India,75-78 Pakistan,79 Nepal,80'81 Bangladesh,82 China,49'83-85 Kyrgyzstan,86 and Uzbekistan87) and Africa (including Egypt,88 Algeria,89 Morocco,87 Namibia,90 Sudan,91 and Chad91,92), whereas genotype 2 has been isolated only in Mexico93 and in some African countries (Nigeria,94 Namibia,95 Chad,91 and Sudan91). Genotype 3 has been detected worldwide (Amer-

ica,13,26,32,33,96-99 Europe,9,10,12,15,16,18,20,21,27,34,35,100-102

Asia,25'33'37'39'40'103-111 Australia,69 and New Zealand11) with the exception of Africa, whereas genotype 4 is restricted to India112,113 and East Asia.39,84,103,114-118

Although genotypes 1 and 2 are considered human viruses, genotypes 3 and 4 have been isolated from both hu-mans10'11'16'17'20'25'97'119-122 and animals.32,33,123-126 Of significant interest is the unique distribution of HEV isolates in India, as human and swine HEVs belong to different genotypes (genotype 1 and genotype 4, respectively);113 genotype 4 appears to be specifically restricted to swine and it has never been isolated from humans, in spite of extensive investigations.127,128 Phylo-genetic analysis shows that the Indian HEV genotype 4 represents a distinct variant among HEV genotype 4 isolates with 26 unique amino-acid substitutions (16 in ORF1, 8 in ORF2, and 2 in ORF3).129 Whether the difference in sequence, particularly in ORF2, determines tropism and explains the lack of infectivity in humans remains to be determined.

Mode of transmission

Waterborne

In developing countries, HEV is transmitted through the faecal-oral route, mainly by the consumption of water contaminated with sewage disposal.130 In developed countries, HEV RNA has been detected in human sewage only occasionally.19,20 Interestingly, HEV rescued from sewage in Spain was infectious for rhesus monkey,131 raising the possibility that HEV might occasionally

Gt 1 Gt 2 Gt 3 Gt 4

'HI Gt 1 and 4 □ Gt 1 and 2 Gt 3 and 4 Gt 1 and 3 Gt 2 and 3

Figure 3 Geographical distribution of HEV isolates according to genotypes (Gt). HEV Gt 1 and 2: epidemic strains causing human infection. HEV Gt 3 and 4: zoonotic strains isolated from humans and a variety of animals, particularly pigs. In some countries, different genotypes co-circulate in distinct ecological niches: Gt 1 and 4 in China, India, and Vietnam; Gt 1 and 2 in several African countries, including Namibia, Chad, and Sudan; Gt 3 and 4 in Japan; Gt 1 and 3 in Cambodia; Gt 2 and 3 in Mexico.

contaminate the environment and shellfish even in non-epidemic regions.16,131,132

Foodborne

Evidence that food can transmit HEV came from Japan, where acute hepatitis E was diagnosed in patients who consumed raw or undercooked pig liver and intestine,23 wild boar meat and liver,38,110,133 and deer meat40 contaminated with the virus. HEV with identical nucleotide sequence was detected both in the blood of affected patients and in batches of meat and liver not consumed.40,109,133 A higher HEV IgG seroprevalence in Japanese individuals with frequent dietary consumption of raw deer meat, in comparison with a control group, indirectly supports the foodborne route.134 Food as a vehicle of infection has not yet been proven in other developed countries.

Person-to-person transmission

In contrast to hepatitis A virus (HAV) infection, secondary transmission among household members of patients with acute hepatitis E is an uncommon event,135,136 both in the context of outbreaks137 and sporadic infections.138,139

Parenteral transmission

HEV-infected individuals can transmit the infection by donating blood during the viraemic period. Viraemia can be detected even in asymptomatic infections and during the incubation period,140,141 even in the absence of aminotransferase elevation.140 Transmission of HEV via blood transfusion has been documented in several countries, including Saudi Arabia,142 Japan,105,143,144 and the UK,145 where matching RNA sequences were found in blood donors and their recipients.

Mother-to-child transmission

Mother-to-child transmission of HEV has been scarcely documented; however, the available data suggest a significant rate of HEV vertical transmission among the HEV RNA-positive mothers with worsening liver disease.146-149

Epidemic versus sporadic forms and seasonality

In the developing world, HEV infection represents the most common aetiological agent of periodic outbreaks of acute hepatitis.42,130,136,150 Epidemics are most frequent during the monsoon season when flooding causes faecal contamination

of drinking water.41 Between epidemics, HEV is transmitted in a discrete manner leading to the onset of sporadic forms of acute hepatitis. In India, 30-70% of all cases of acute sporadic hepatitis are caused by HEV infection.41,151

In economically developed regions, indigenous hepatitis E is largely a sporadic disease. However, in Japan, small outbreaks have been described as a consequence of consumption of the same contaminated food.152,153 A small outbreak involving a family with two children has also been reported in France.139 No clear seasonality has been observed

in developed countries.24,154

Age- and sex-specific clinical attack rates and case fatality rates

Clinical infection concentrates among adolescents and young adults in countries of high endemicity. During outbreaks, the clinical attack rate (3_30%)136,150,155-157 is highest among pregnant women.4,158,159 The mortality rate,

which is usually low (0.07-0.6%),136,150,160,161 can reach

values as high as 25-31% in pregnant women, particularly during the third trimester of pregnancy.4,136,150,155,162-164

A distinct characteristic of hepatitis E indigenous to developed countries is its high attack rate among older male adults.13,26,100 This has been observed in both

Europe10,16,18,20,100,101 and Japan,24 which experienced the

greatest number of detected cases. No documented case of hepatitis E has been identified in pregnant women in this different epidemiological setting.

A high mortality, ranging between 25 and 70%, has been recently documented among patients already suffering from chronic liver disease of different aetiologies. These observations were made both in highly endemic countries, such as India,165-168 Pakistan,169 and Nepal,170 where hepatitis E is caused by genotype 1, and in Europe,171 where indigenous hepatitis E is linked to genotype 3.

Seroprevalence of HEV infection

HEV seroprevalence studies have been conducted using several antibody assays based on different recombinant antigens, which do not always include the most relevant B-cell epitopes, leading to lack of sensitivity and poor reciprocal concordance.172 In particular, a greater sensitivity of currently available antibody assays has been reported in detecting overt disease in comparison with subclinical infections,173 which may hamper seroepidemiological investigations. Antibody assays based on the ORF2 protein, which exposes at least one major crossreactive epitope shared by all HEV genotypes,56,62,174 should be

used for investigating seroepidemiology46,56,175,176 (see

'Diagnosis').

Seroprevalence studies in HEV epidemic countries, such as India, have revealed that HEV infection is rare in children, reaching peak prevalence (33-40%) only in early adult-hood.177 These data are in striking contrast with HAV serosurveys, showing anti-HAV antibodies in the majority of children by the age of three years.177 The reason for the

difference in age-specific seroprevalence between HAV and HEV, both transmitted by the faecal-oral route, remains unanswered.

In contrast, HEV seroepidemiology closely mirrors that of HAV in Egypt, a country highly endemic for HEV infection (similar to India), where anti-HEV antibodies are detectable in 65% of children younger than 10 years.178 Although the reason for the earlier exposure in life in Egypt remains unknown, the widespread immunity of the population at an early age might account for the absence of large outbreaks of hepatitis E in the general population179 and among pregnant women.180

In rural southern China, where the majority of HEV infections are zoonotic (genotype 4), anti-HEV IgG is rarely detected in children, rapidly increases in young adults, and peaks (60-80%) at the age of 60 years.181 Interestingly, after 30 years of age, but not in younger age groups, the seroprevalence is two times higher for men than for women, suggesting a link with different social roles adopted by men and women once families are established.181

Seroprevalence investigations in developed regions have revealed the ubiquitous presence of anti-HEV antibodies in the analysed populations, although with significant differences between and within countries.11,172,182-186 A significant concern is the high seroprevalence among US blood donors (18-21%)184,187 compared with individuals professionally exposed to swine HEV, such as veterinarians (23-26%).184

Some studies have documented an increasing seropreva-lence with age in both sexes, suggesting a continuous ongoing exposure to HEV.172,182,187,188 In Japan, age-specific profiles of anti-HEV and anti-HAV antibodies suggest silent HEV infection in the last few decades, during which HAV infection rates declined.188

Zoonosis and host range

The possibility that HEV infection can be a zoonosis was raised when a virus, closely related to the HEV human strains, was isolated from pigs, initially in the USA189 and,

subsequently, worldwide.28,32-36,96,112,183,190-193

Experimental cross-species infections between swine and primates have shown that swine HEV could infect primates (surrogates for human infection), and the US-2 strain of human HEV (genotype 3) could infect specific pathogen-free pigs.194 In contrast, US pigs could not support replication of human epidemic strains (genotypes 1 and 2).195 Similarly, Indian pigs could be infected with swine HEV (genotype 4) but not with human HEV (genotype 1).112

These findings are supported by phylogenetic analysis data showing a high degree of nucleotide and amino-acid sequence homology between swine and human HEV isolates of genotypes 3 and 4 from the same geographical regions, suggesting that pigs may act as a reservoir for human

infection.69,35,109,115,116,125 On the contrary, swine HEV

strains are highly divergent from the human strains of HEV classified within genotypes 1 and 2.69

Pigs are infected via the faecal-oral route196 and develop a self-limiting subclinical infection189,197,198 with transient viraemia (one to two weeks) but prolonged viral shedding (three to four weeks) in faeces. Current pig-raising practices perpetuate exposure of pigs to their waste, promoting viral

transmission.196

Seroprevalence studies in pigs have shown the presence of HEV IgG in an unexpectedly high proportion of animals,36,199-201 with peaks up to 85% in the UK,34 95-98% in India,127 and 70-100% in Japan.109,202,203 Although pigs are infected primarily at the early stage of production (1-3 months), HEV can still be detected by PCR at slaughter age, meaning that swine HEV can enter the food chain.28,36,98 This has been shown in Japan, where infectious HEV was found in 2% of the pig liver packages ready for sale,39 and more recently in the USA30 and in The Netherlands.204 HEV RNA was also detected in 3.1% of bile samples from swine in abattoirs in eastern China.28

HEV replicates in different visceral organs.192,205,206 This explains the common foodborne transmission in Japan owing to the gastronomic habit of eating rarely, or poorly cooked, pig liver and intestines. Despite absence of evidence for HEV replication in muscles, HEV infection has been transmitted by consumption of meat from boar and deer as well.38,40,133

The finding of infectious HEV in pig-farm manure slurry samples193 suggests that human exposure to swine waste may represent an alternative mode of transmission of zoonotic strains, particularly in regions where the water supply comes from wells, rivers, and streams, and where sewage treatment is not generally available.181 In rural eastern China, where 9.6% of pig herds were found to be HEV RNA-positive by stool samples, a 74% higher risk of infection among people professionally engaged in swine farming was observed. Seroprevalence increased with the duration of occupational exposure to swine.28 There was also a 29% higher risk of infection in people without occupational exposure to swine and residing in communities downstream of the Chinese swine farms, compared with those living in communities upstream.28 Compared with control individuals, increased HEV IgG seroprevalence was also detected in people with occupational exposure to pigs in other countries, including Sweden and The Nether-lands.183,207

Seroprevalence studies have shown that HEV natural infection is widespread in many species of wild and domestic

mammals, including rats,208-212 cattle,115,209 goats,127 wild mongooses,213 monkeys,214 dogs,215 and pet cats,216 with

antibody positivity increasing with age in Japanese macaques and Japanese domestic pet cats.214,216 Although these data show the prevalence of HEV circulation among animals, they have not established the possible role of these animals in transmitting HEV infection to humans. Of particular interest is the high HEV seroprevalence among wild rats,208-212 as these rodents, ubiquitous worldwide, have the potential to be infected with swine and human HEV

strains. Rats may therefore be an important intermediate host between pigs and humans or, alternatively, a reservoir for both human and swine infection.

The majority of studies on animals other than swine have been more successful in detecting HEV antibodies than viral RNA. Although HEV RNA from genotypes 3 and 4 has been systematically found in pigs, boars, and deer, it has not yet been defined with certainty which genotypes circulate within other species. Two studies document HEV genotype 1 in horses217 and pigs.218 The finding of HEV genotype 1 in rats219 has subsequently been found to be a laboratory error.220 More studies are necessary to assess if HEV genotype 1 and 2 strains can induce sustainable infection in some animal species. In view of the large number of animal species that are potentially involved, further exploration of zoonotic transmission of HEV is warranted.23

HEV strains were identified in poultry as well as mammals; phylogenetic analysis indicates that avian HEV is genetically related to, but distinct from, mammalian HEV strains221,222 and does not represent a risk for cross-infection to humans.

Pathogenesis, immune response, and time course of infection

It is thought that HEV infection initiates via cells lining the alimentary tract (primary site of virus replication) (Figure 4).223 The virus then reaches the liver through the portal vein41 and replicates in the cytoplasm of hepatocytes without causing direct cytolytic damage.223 Several observations suggest that, in analogy with other hepatitis viruses, liver injury is largely immune-mediated:150,223,224 first, viraemia precedes the onset of alanine transaminase elevation and liver histopathological changes;225-227 secondly, experimental infection of nonhuman primates has shown how the liver damage coincides with the detection of serum anti-HEV antibodies and with a decreasing level of HEV antigens in the hepatocytes;150,225 and finally, the lymphocytes infiltrating the liver have a cytotoxic/suppressor

immunophenotype.150,225

HEV RNA is detectable in blood from as early as two weeks before173 and for two to four weeks after the onset of symptoms.228-230 HEV faecal excretion shows a similar temporal pattern.229 Once liver function has normalised, HEV RNA is usually undetectable in blood and stool.229 Viraemia and faecal shedding beyond the duration of biochemical hepatitis are uncommon,229-231 suggesting that prolonged faecal shedding is not important in maintaining the environmental reservoir of HEV.229

The antibody responses are directed primarily against epitopes in the ORF2 and ORF3 proteins and are typically detectable at the onset of the disease, with IgM antibodies persisting for two to six months.223,228,232 Anti-HEV IgG appears soon after IgM, and persists for a longer period of time.41,223,228,232,233 However, the possibility of repeated infections being the cause of IgG persistence cannot be

excluded.41

Symptoms

Convalescent period

ill HEV RNA in stool

HEV RNA in blood

Anti-HEV IgG

Exposure to HEV

Months post-infection

Figure 4 Time course of HEV infection and specific humoral immune response. This figure shows the correlation of HEV RNA in stool and blood, sign of liver damage and development of anti-HEV antibody response with time. ALT, alanine aminotransferase; IgG, immunoglobulin G; IgM, immunoglobulin m.150,153,173

Overt disease in young adults is commonly the result of primary infection.234 The importance of antibodies in protecting from clinical hepatitis E has been proven experimentally in primates, in which passive immunisation with anti-HEV antibodies was able to protect them against overt disease after challenge with virulent HEV.235

Limited data are available on anti-HEV cellular immune response. Evidence for anti-HEV T-cell response was provided by a study on patients with acute hepatitis E whose T-lymphocytes showed sensitisation to HEV peptides.236 The same group was recently able to map CD4 T-cell epitopes in the ORF2 and ORF3 proteins of HEV using lymphocyte proliferation assays in patients with acute hepatitis E,237 providing the basis for future studies on the immunopatho-genesis of hepatitis E. No data are currently available regarding anti-HEV-specific CD8 T-cell responses or the role of cellular responses in the protection against viral infection.

Of great interest are the mechanisms determining the severity of disease during pregnancy, in which fulminant hepatitis is a common complication. HEV infection studied in pregnant and nonpregnant healthy women has shown that infection in pregnancy is associated with a shift in the Th cell type 1/Th cell type 2 balance toward Th cell type 2 response.238 However, at this time it is difficult to link the clinical severity of the illness to this observation because the mechanism of liver injury in HEV infection has not yet been clarified.239 A recent Indian study suggested that a subset of CD4-positive interferon-g-secreting cells, which do not belong to either the

helper Th cell type 1 or type 2 phenotype, might be involved in liver damage during acute HEV infection.240

Clinical features

Acute infection

The incubation period ranges from two to 10 weeks with an average of 40 days.43,163,228 Hepatitis E is indistinguishable from other forms of viral hepatitis. Typical clinical features are one- to 10-day prodrome of malaise, fever, gastrointestinal symptoms (abdominal pain, anorexia, nausea, vomiting), followed by the onset of jaundice.41,150,155,161,241 Once jaundice appears, prodromal symptoms subside. Clinical jaundice is not a constant feature and anicteric forms of hepatitis are well recognised.242 Serum investigations reveal raised levels of bilirubin (predominantly conjugated) and alanine aminotransferase. The magnitude of the alanine aminotransferase elevation does not correlate with the severity of the liver injury, better expressed by the liver synthetic function, as determined by coagulation function estimation. Acute infection resolves in one to four weeks; however, some patients develop a more prolonged clinical illness with cholestasis (cholestatic hepatitis).150 HEV infection is not known to progress to chronicity or cirrhosis2,243 in immunocompetent patients.

Complications

A small proportion of patients develop fulminant or subacute hepatic failure with high mortality as a result of

massive liver necrosis.150,244,245 Fulminant hepatitis E has been described worldwide but it is particularly common in developing countries among pregnant women, mainly

during the third trimester.164,239,246-249 In this setting, HEV

adversely affects both pregnant women and foetal outcome, with high mortality rate, increased frequency of abortions, preterm delivery, stillbirth, and neonatal

death.23,99,148,150,239,250

In Japan, HEV genotype 4 appears to cause severe hepatitis more frequently than genotype 3,23,121,251 possibly as a consequence of specific genomic mutations.119 In Argentina, a country not endemic for hepatitis E, fulminant hepatitis has been recently diagnosed in three children infected with HEV genotype 3.99

Severe forms of hepatitis E have also been increasingly documented among patients, mainly men, with stable chronic liver disease of different aetiologies, including chronic hepatitis B and C, autoimmune hepatitis, alcoholic

liver disease, cryptogenetic hepatitis, and Wilson's

disease.165-171,252,253

Prolonged mild hepatitis with viral shedding has been described in immunocompromised patients during chemotherapy for T-cell lymphoma.143,254 More recently, the evolution of HEV infection into chronic hepatitis E has been reported in solid organ transplant patients in France:255,256 they not only persistently shed the virus in the presence of deranged alanine aminotransferase values, but also showed histopathological changes similar to those observed in chronic hepatitis C.

Uncommon HEV infection complications, described in anecdotal reports, include the Guillain-Barre syndrome,257

258 259

acute transverse myelitis,258 acute pancreatitis,259 nonimmune haemolytic anaemia,260 lymphocytic destructive cholangitis,261 and prolonged polyarthritis.262

Asymptomatic infections

The number of asymptomatic infections far exceeds that of icteric hepatitis, as a large proportion of individuals who test positive for anti-HEV antibodies in highly endemic countries, such as India,150 China,28,181 and Egypt,179 do not recall having suffered from jaundice. Similar data, based on seroprevalence, have been obtained from a variety of population profiles in developed countries: blood donors in Japan and the USA,140,184 prisoners and drug users in Denmark,172 and individuals living in the community in Spain.182

Direct evidence of ongoing subclinical HEV infection in the general population comes from a study conducted in Honshu (Japan) on 6700 asymptomatic blood donors with elevated aminotranferase levels during a three-year period: about 3% of the individuals with an aminotransferase level of X 201 IU/l (normal value <60IU/l) were HEV RNA-positive.140 Based on the number of asymptomatic viraemic individuals and the incidence of clinical hepatitis E in Honshu, the authors estimate that less than 0.1% of HEV-infected cases exhibit clinical manifestation of the infec-

tion.140 Asymptomatic viraemia has been detected in about 0.3% of individuals in rural eastern China.28

Diagnosis Antibody detection

HEV antibody assays represent the routine diagnostic tool for acute hepatitis E cases. Test formats mostly consist of indirect enzyme-linked immunosorbent assays (EIA), with recombinant HEV proteins or peptides as detecting antigens. Currently available commercial assays are based mainly on HEV epidemic strains but EIA tests specific for genotypes 3 and 4 have also been developed.25,128,135 The specificity and sensitivity of these tests have not been established with precision, limiting the reliability of laboratory results.263

One of the most widely available commercial antibody assays, the Genelabs-EIA, uses short recombinant proteins derived from the 3' termini of ORF2 (42 amino acids) and ORF3 (33 amino acids) from the Burmese (genotype 1) and Mexican (genotype 2) prototype sequences.263 However, according to Zhou et al.,175 a truncated form of the ORF2 protein, encompassing amino acids 112-607, contains the neutralisation epitopes (with amino acids 458-607 representing the major neutralisation site) and elicits the greatest and most long-lasting anti-HEV antibody response, being therefore suitable both for diagnostic and seroprevalence estimation purposes. In contrast, the amino acids 1-111 (N-terminus) and 607-660 (C-terminus) of the ORF2 protein and the ORF3 recombinant antigens elicit a weaker and transient antibody response, and are consequently of limited value in diagnosing acute HEV infection, and are not useful for seroprevalence studies.46,176 Importantly, all genotypes share at least one major serologically crossreactive epitope, despite substantial genomic variability.62,174

Diagnosis of acute hepatitis E is made by detecting HEV-specific IgM in acute-phase sera or by detecting a rise in anti-HEV IgG titre between acute and convalescent serum samples. Cases of aberrant IgM and IgG serological profiles, including those with immunologically silent acute hepatitis E, have been documented.84,173 Although atypical serologi-cal profiles from patients with proven HEV RNA viraemia may be the expression of a modified immune response, the insensitivity of diagnostic assays should be taken into account when interpreting these data.101,264,265

Molecular detection

Reverse transcription-polymerase chain reaction (RT-PCR) assays represent the most commonly used molecular investigation for HEV genome detection. The usage of RT-PCR as a diagnostic tool has become feasible since the development of the real-time PCR platforms, closed systems that minimise the risk of contamination by the amplified target.

The majority of HEV RT-PCR assays used for diagnosis were developed as in-house assays by choosing different conserved HEV genomic regions as the target for amplifica-

tion.230,266-268 Considering the wide genetic heterogeneity of HEV isolates, it is critical to design primers and probes that guarantee the development of highly sensitive and broadly reactive assays.268

RT-PCR is a useful complementary diagnostic tool for the diagnosis of acute HEV infection, as it can confirm cases of hepatitis E with atypical serological profiles. RT-PCR assays are also critically important for public health purposes when used for detecting HEV-contaminated environmental samples.

Prevention and control of the infection

Active immunisation

The observation that passive immune prophylaxis with convalescent serum samples prevented hepatitis E in primates has indicated that vaccination against HEV based on humoral immunity is feasible.55,235,269 This has prompted HEV immunisation studies based mainly on recombinant proteins, because the unavailability of an efficient cell culture system for HEV replication270 has precluded the development of vaccines based on inactivated or attenuated whole-virus particles. However, other approaches, such as DNA-based vaccines,271,272 able to induce both cellular and antibody response, are also under evaluation.

The ORF2 protein has been considered the best candidate for HEV vaccine because it contains the neutralisation epitope located between amino acids 458 and 60756 and is crossreactive with all mammalian HEV.56,273 Animal studies have shown that ORF2 recombinant proteins274-278 elicit

neutralising antibodies and mediate protective immunity in vaccinated primates.277,279

One such vaccine with a 56 kDa protein encompassing amino acids 112-60755,280 was recently evaluated in young adults in Nepal in a phase 2, randomised, doubleblind, placebo-controlled trial.281 The study had encouraging results establishing that three doses of hepatitis E vaccine were 95.5% effective in protecting against clinical hepatitis E after a median of 804 days. The primary endpoint of this study was the prevention of clinically overt HEV infection, but the ability of the vaccine to prevent asymptomatic infection and asymptomatic virus shedding was not investigated.174 Asymptomatic HEV shedding in vaccine recipients, shown previously in primates,280 may be relevant in maintaining the environmental reservoir of HEV for human infection.174 Another aspect that this trial could not clarify is the duration of the induced immunity. Based on currently available data, this vaccine may be useful for travellers to highly endemic areas and for susceptible pregnant women,174 particularly during outbreaks. However, its use in children and adolescents in hepatitis E endemic countries,174 or in individuals with chronic liver disease, requires further study assessing the duration of its protective efficacy.

Protection of the environment and control of the outbreaks

The most important measure to prevent HEV infection is the protection of water supply from faecal contamination.

Table 1 Clinical-epidemiological characteristics of HEV infection

HEV Genotypes Geographical distribution Hosts Mode of transmission Epidemic versus sporadic forms Clinical attack rate Disease severity

1 Asia Africa Humans ? Animals Contamination of water supply Sporadic and epidemic forms Highest in young adults Highest in pregnant women and individuals with chronic liver disease

2 Central America, Africa

3 Asia, America, Europe, Oceania Humans and a variety of animals, particularly swine Food (Japan) Sporadic and small outbreaks Highest in older males Highest in individuals with chronic liver disease

4 Asia Food (Japan), Environmental contamination by swine waste

Abbreviation: HEV, hepatitis E virus. Zoonotic strains (HEV genotype 3 and 4) cause infection both in developed and in developing countries (in italics), such as China.

Chlorination and filtration systems are generally inadequate if the source water is heavily contaminated.223 Travellers to highly endemic regions should strictly consume only bottled or boiled water.

During outbreaks, it is critical to provide clean water to all pregnant women. The isolation of individuals affected by acute hepatitis E is not justified because person-to-person transmission is uncommon. Infected people should refrain from food handling and food preparation.223

Conclusion

HEV infection has complex, and not yet completely clarified, clinical-epidemiological characteristics, which are summarised in Table 1. Two forms of infection have been identified: hepatitis E caused by epidemic strains, affecting mainly young adults and particularly pregnant women, and hepatitis E caused by zoonotic strains, which mostly affect older males.282 These differences in sex- and age-specific attack rates are puzzling because the route of transmission in developed countries, apart from Japan, has not yet been identified.

More extensive epidemiological studies are needed, not only to assess the HEV seroprevalence in humans, but also in several animal species. For this purpose, the development of broadly reactive reliable antibody assays, which include immunodominant neutralisation antigens, is critical. Equally important is to establish which HEV genotypes circulate among the different animal species, and their role in human infections.

A high level of suspicion is needed in developed countries where the increasing number of recognised cases of hepatitis E16,18,100-102 suggests significant underdiagnosis.100 Awareness of HEV infection should exist in immunocompromised patients with signs of liver damage in view of the recent finding that hepatitis E can evolve to a chronic

infection.255,256

The results of a HEV vaccination phase 2 trial have provided encouraging preliminary data for the prevention of hepatitis E.281 Data are now awaited regarding the duration of the immune response induced by the vaccine before routine immunisation of children can be promoted in epidemic countries. It will also be important to establish the vaccine efficacy among older adults and the elderly, who are targets of overt HEV infection and in whom most cases of chronic liver disease, a risk factor for severe hepatitis E, concentrate.

Acknowledgements

Competing interest

The authors state no competing interests. Provenance and peer review

Commissioned without payment, externally peer-reviewed.

References

1 Wong DC, Purcell RH, Sreenivasan MA, Prasad SR, Pavri KM. Epidemic and endemic hepatitis in India: evidence for a non-A, non-B hepatitis virus aetiology. Lancet 1980;2:876-9.

2 Khuroo MS, Saleem M, Teli MR, Sofi MA. Failure to detect chronic liver disease after epidemic non-A, non-B hepatitis. Lancet 1980;2:97-8.

3 Jary C. Hepatitis E and meat carcasses. Br J Gen Pract 2005;55: 557-558.

4 Boccia D, Guthmann JP, Klovstad H, Hamid N, Tatay M, Ciglenecki I, et al. High mortality associated with an outbreak of hepatitis E among displaced persons in Darfur, Sudan. Clin Infect Dis 2006;42:1679-84.

5 Banait VS, Sandur V, Parikh F, Murugesh M, Ranka P, Ramesh VS, et al. Outcome of acute liver failure due to acute hepatitis E in pregnant women. Indian J Gastroenterol 2007;26:6-10.

6 Reuter G, Fodor D, Szucs G. Molecular detection of hepatitis E virus in non-imported hepatitis E cases: identification of a potential new human hepatitis E virus lineage in Hungary. Orv 'Hetil 2005;146:2389-94. [article in Hungarian].

7 Preiss JC, Plentsz A, Engelman E, Scneider T, Jilq W, Zeitz M, et al. Autochthonous hepatitis E virus infection in Germany with sequence similarities to other European isolates. Infection 2006;24:173-5.

8 Widdowson MA, Jaspers WJ, van der Poel WH, Verschoor F, de Roda Husman AM, Winter HL, et al. Cluster of cases of acute hepatitis associated with hepatitis E virus infection acquired in The Netherlands. Clin Infect Dis 2003;36:29-33.

9 Worm HC, Wurzer H, Frosner G. Sporadic hepatitis E in Austria. N Engl J Med 1998;339:1554-5.

10 Mansuy JM, Peron JM, Abravanel F, Poirson H, Dubois M, Miedouge M, et al. Hepatitis E in the south west of France in individuals who have never visited an endemic area. J Med Virol 2004;74:419-24.

11 Dalton HR, Fellows HJ, Gane EJ, Wong P, Gerred S, Schroeder B, et al. Hepatitis E in New Zealand. J Gastroenterol Hepatol 2007;22:1236-40.

12 Schlauder GG, Desai SM, Zanetti AR, Tassopoulos NC, Mush-ahwar IK. Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV. J Med Virol 1999;57:243-51.

13 Schlauder GG, Dawson GJ, Erker JC, Kwo PY, Knigge MF, Smalley DL, et al. The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States. J Gen Virol 1998;79 (Pt 3): 447-56.

14 Teo CG. Hepatitis E indigenous to economically developed countries: to what extent a zoonosis? Curr Opin Infect Dis 2006;19:460-6.

15 Zanetti AR, Schlauder GG, Romano L, Tanzi E, Fabris P, Dawson GJ, et al. Identification of a novel variant of hepatitis E virus in Italy. J Med Virol 1999;57:356-60.

16 Ijaz S, Arnold E, Banks M, Bendall RP, Cramp ME, Cunningham R, et al. Non-travel-associated hepatitis E in England and Wales: demographic, clinical, and molecular epidemiological characteristics. J Infect Dis 2005;192:1166-72.

17 Amon JJ, Drobeniuc J, Bower WA, Magana JC, Escobedo MA, Williams IT, et al. Locally acquired hepatitis E virus infection, El Paso, Texas. J Med Virol 2006;78:741-6.

18 Dalton HR, Thurairajah PH, Fellows HJ, Hussaini HS, Mitchell J, Bendall R, et al. Autochthonous hepatitis E in southwest England. J Viral Hepat 2007;14:304-9.

19 Clemente-Casares P, Pina S, Buti M, Jardi R, MartIn M, Bofill-Mas S, et al. Hepatitis E virus epidemiology in industrialized countries. Emerg Infect Dis 2003;9:448-54.

20 Buti M, Clemente-Casares P, Jardi R, Formiga-Cruz M, Schaper M, Valdes A, et al. Sporadic cases of acute autochthonous hepatitis E in Spain. J Hepatol 2004;41:126-31.

21 McCrudden R, O'Connell S, Farrant T, Beaton S, Iredale JP, Fine D. Sporadic acute hepatitis E in the United Kingdom: an underdiagnosed phenomenon? Gut 2000;46:732-3.

22 Levine DF, Bendall RP, Teo CG. Hepatitis E acquired in the UK. Gut 2000;47:740.

23 Mizuo H, Yazaki Y, Sugawara K, Tsuda F, Takahashi M, Nishizawa T, et al. Possible risk factors for the transmission of hepatitis E virus and for the severe form of hepatitis E acquired locally in Hokkaido, Japan. J Med Virol 2005;76:341-9.

24 Okamoto H, Takahashi M, Nishizawa T. Features of hepatitis E virus infection in Japan. Intern Med 2003;42:1065-71.

25 Mizuo H, Suzuki K, Takikawa Y, Sugai Y, Tokita H, Akahane Y, et al. Polyphyletic strains of hepatitis E virus are responsible for sporadic cases of acute hepatitis in Japan. J Clin Microbiol 2002;40:3209-18.

26 Kwo PY, Schlauder GG, Carpenter HA, Murphy PJ, Rosenblatt JE, Dawson GJ, et al. Acute hepatitis E by a new isolate acquired in the United States. Mayo Clin Proc 1997;72:1133-6.

27 Herremans M, Vennema H, Bakker J, van der Veer B, Duizer E, Benne CA, et al. Swine-like hepatitis E viruses are a cause of unexplained hepatitis in The Netherlands. J Viral Hepat 2007;14:140-6.

28 Zheng Y, Ge S, Zhang J, Guo Q Ng MH, Wang F, et al. Swine as a principal reservoir of hepatitis E virus that infects humans in eastern China. J Infect Dis 2006;193:1643-9.

29 Tanaka Y, Takahashi K, Orito E, Karino Y, Kang JH, Suzuki K, et al. Molecular tracing of Japan-indigenous hepatitis E viruses. J Gen Virol 2006;87 (Pt 4): 949-54.

30 Feagins AR, Opriessnig T, Guenette DK, Halbur PG, Meng XJ. Detection and characterization of infectious hepatitis E virus from commercial pig livers sold in local grocery stores in the USA. J Gen Virol 2007;88 (Pt 3): 912-17.

31 Jung K, Kang B, Song DS, Chae C. Prevalence and genotyping of hepatitis E virus in swine population in Korea between 1995 and 2004: a retrospective study. Vet J 2007;173:683-7.

32 Munne MS, Vladimirsky S, Otegui L, Castro R, Brajterman L, Soto S, et al. Identification of the first strain of swine hepatitis E virus in South America and prevalence of anti-HEV antibodies in swine in Argentina. J Med Virol 2006;78:1579-83.

33 Cooper K, Huang FF, Batista L, Rayo CD, Bezanilla JC, Toth TE, et al. Identification of genotype 3 hepatitis E virus (HEV) in serum and fecal samples from pigs in Thailand and Mexico, where genotype 1 and 2 HEV strains are prevalent in the respective human populations. J Clin Microbiol 2005; 43:1684-8.

34 Banks M, Heath GS, Grierson SS, King DP, Gresham A, Girones R, et al. Evidence for the presence of hepatitis E virus in pigs in the United Kingdom. Vet Rec 2004;154:223-7.

35 van der Poel WH, Verschoor F, van der Heide R, Herrera MI, Vivo A, Kooreman M, et al. Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerg Infect Dis 2001;7:970-6.

36 Seminati C, Mateu E, Peralta B, de Deus N, Martin M. Distribution of hepatitis E virus infection and its prevalence in pigs on commercial farms in Spain. Vet J 2008;175:130-2.

37 Takahashi K, Kitajima N, Abe N, Mishiro S. Complete or near-complete nucleotide sequences of hepatitis E virus genome recovered from a wild boar, a deer, and four patients who ate the deer. Virology 2004;330:501-5.

38 Masuda J, Yano K, Tamada Y, Takii Y, Ito M, Omagari K, et al. Acute hepatitis E of a man who consumed wild boar meat prior to the onset of illness in Nagasaki, Japan. Hepatol Res 2005;31:178-83.

39 Yazaki Y, Mizuo H, Takahashi M, Nishizawa T, Sasaki N, Gotanda Y, et al. Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food. J Gen Virol 2003;84 (Pt 9): 2351-7.

40 Tei S, Kitajima N, Takahashi K, Mishiro S. Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 2003;362:371-3.

41 Panda SK, Thakral D, Rehman S. Hepatitis E virus. Rev Med Virol 2007;17:151-80.

42 Corwin AL, Tien NT, Bounlu K, Winarno J, Putri MP, Laras K, et al. The unique riverine ecology of hepatitis E virus transmission in South-East Asia. Trans R Soc Trop Med Hyg 1999;93:255-60.

43 Balayan MS, Andjaparidze AG, Savinskaya SS, Ketiladze ES, Braginsky DM, Savinov AP, et al. Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology 1983;20:23-31.

44 Reyes GR, Purdy MA, Kim JP, Luk KC, Young LM, Fry KE, et al. Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis. Science 1990;247:1335-9.

45 Tam AW, Smith MM, Guerra ME, Huang CC, Bradley DW, Fry KE, et al. Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 1991;185:120-31.

46 Dawson GJ, Chau KH, Cabal CM, Yarbough PO, Reyes GR, Mushahwar IK. Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides. J Virol Methods 1992;38:175-86.

47 Berke T, Golding B, Jiang X, Cubitt DW, Wolfaardt M, Smith AW, et al. Phylogenetic analysis of the Caliciviruses. J Med Virol 1997;52:419-24.

48 Emerson SU, Anderson D, Arankalle A, Meng XJ, Purdy M, Schlauder GG, et al. Hepevirus. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (eds) Virus Taxonomy: VIIIth Report of the ICTV. Elsevier/Academic Press: London, 2004, pp 851-5.

49 Aye TT, Uchida T, Ma XZ, Iida F, Shikata T, Zhuang H, et al. Complete nucleotide sequence of a hepatitis E virus isolated from the Xinjiang epidemic (1986-1988) of China. Nucleic Acids Res 1992;20:3512.

50 Reyes GR, Huang CC, Tam AW, Purdy MA. Molecular organization and replication of hepatitis E virus (HEV). Arch Virol Suppl 1993;7:15-25.

51 Graff J, Torian U, Nguyen H, Emerson SU. A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus. J Virol 2006;80:5919-26.

52 Okamoto H. Genetic variability and evolution of hepatitis E virus. Virus Res 2007;127:216-28.

53 Wang Y, Zhang H, Ling R, Li H, Harrison TJ. The complete sequence of hepatitis E virus genotype 4 reveals an alternative strategy for translation of open reading frames 2 and 3. J Gen Virol 2000;81 (Pt 7): 1675-86.

54 Kabrane-Lazizi Y, Meng XJ, Purcell RH, Emerson SU. Evidence that the genomic RNA of hepatitis E virus is capped. J Virol 1999;73:8848-50.

55 Emerson SU, Purcell RH. Recombinant vaccines for hepatitis E. Trends Mol Med 2001;7:462-6.

56 Zhou YH, Purcell RH, Emerson SU. A truncated ORF2 protein contains the most immunogenic site on ORF2: antibody responses to non-vaccine sequences following challenge of vaccinated and non-vaccinated macaques with hepatitis E virus. Vaccine 2005;23:3157-65.

57 Xing L, Kato K, Li T, Takeda N, Miyamura T, Hammar L, et al. Recombinant hepatitis E capsid protein self-assembles into a dual-domain T = 1 particle presenting native virus epitopes. Virology 1999;265:35-45.

58 Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, et al. Expression and self-assembly of empty virus-like particles of hepatitis E virus. J Virol 1997;71:7207-13.

59 Surjit M, Jameel S, Lal SK. The ORF2 protein of hepatitis E virus binds the 5' region of viral RNA. J Virol 2004;78:320-8.

60 Tyagi S, Korkaya H, Zafrullah M, Jameel S, Lal SK. The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2. J Biol Chem 2002;277:22759-67.

61 Zafrullah M, Ozdener MH, Panda SK, Jameel S. The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton. J Virol 1997;71:9045-53.

62 Emerson SU, Purcell RH. Hepatitis E virus. Rev Med Virol 2003;13:145-54.

63 Korkaya H, Jameel S, Gupta D, Tyagi S, Kumar R, Zafrullah M, et al. The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK. J Biol Chem 2001;276:42389-400.

64 Huang YW, Opriessnig T, Halbur PG, Meng XJ. Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo. J Virol 2007;81:3018-26.

65 Emerson SU, Nguyen H, Torian U, Purcell RH. ORF3 protein of hepatitis E virus is not required for replication, virion assembly, or infection of hepatoma cells in vitro. J Virol 2006;80: 10457-64.

66 Agrawal S, Gupta D, Panda SK. The 3' end of hepatitis E virus (HEV) genome binds specifically to the viral RNA-dependent RNA polymerase (RdRp). Virology 2001;282:87-101.

67 Emerson SU, Arankalle VA, Purcell RH. Thermal stability of hepatitis E virus. J Infect Dis 2005;192:930-3.

68 Tanaka T, Takahashi M, Kusano E, Okamoto H. Development and evaluation of an efficient cell-culture system for hepatitis E virus. J Gen Virol 2007;88 (Pt 3): 903-11.

69 Lu L, Li C, Hagedorn CH. Phylogenetic analysis of global hepatitis E virus sequences: genetic diversity, subtypes and zoonosis. Rev Med Virol 2006;16:5-36.

70 Zhai L, Dai X, Meng J. Hepatitis E virus genotyping based on full-length genome and partial genomic regions. Virus Res 2006;120:57-69.

71 Huang R, Li D, Wei S, Li Q Yuan X, Geng L, et al. Cell culture of sporadic hepatitis E virus in China. Clin Diagn Lab Immunol 1999;6:729-33.

72 Wei S, Walsh P, Huang R, To SS. 93G, a novel sporadic strain of hepatitis E virus in South China isolated by cell culture. J Med Virol 2000;61:311-18.

73 Tam AW, White R, Reed E, Short M, Zhang Y, Fuerst TR, et al. In vitro propagation and production of hepatitis E virus from in vivo-infected primary macaque hepatocytes. Virology 1996;215:1-9.

74 Emerson SU, Clemente-Casares P, Moiduddin N, Arankalle VA, Torian U, Purcell RH. Putative neutralization epitopes and broad cross-genotype neutralization of hepatitis E virus confirmed by a quantitative cell-culture assay. J Gen Virol 2006;87 (Pt 3): 697-704.

75 Jameel S, Zafrullah M, Chawla YK, Dilawari JB. Reevaluation of a North India isolate of hepatitis E virus based on the full-length genomic sequence obtained following long RT-PCR. Virus Res 2002;86:53-8.

76 Aggarwal R, McCaustland KA, Dilawari JB, Sinha SD, Robertson BH. Genetic variability of hepatitis E virus within and between three epidemics in India. Virus Res 1999;59:35-48.

77 Arankalle VA, Paranjape S, Emerson SU, Purcell RH, Walimbe AM. Phylogenetic analysis of hepatitis E virus isolates from India (1976-1993). J Gen Virol 1999;80 (Pt 7): 1691-700.

78 Vaidya SR, Chitambar SD, Arankalle VA. Polymerase chain reaction-based prevalence of hepatitis A, hepatitis E and TT viruses in sewage from an endemic area. J Hepatol 2002;37: 131-136.

79 Tsarev SA, Emerson SU, Reyes GR, Tsareva TS, Legters LJ, Malik IA, et al. Characterization of a prototype strain of hepatitis E virus. Proc Natl Acad Sci USA 1992;89:559-63.

80 Shrestha SM, Shrestha S, Tsuda F, Nishizawa T, Gotanda Y, Takeda N, et al. Molecular investigation of hepatitis E virus

infection in patients with acute hepatitis in Kathmandu, Nepal. J Med Virol 2003;69:207-14.

81 Shrestha SM, Shrestha S, Tsuda F, Nishizawa T, Takahashi M, Gotanda Y, et al. Genetic changes in hepatitis E virus of subtype 1a in patients with sporadic acute hepatitis E in Kathmandu, Nepal, from 1997 to 2002. J Gen Virol 2004;85 (Pt 1): 97-104.

82 Drabick JJ, Gambel JM, Gouvea VS, Caudill JD, Sun W, Hoke Jr CH, et al. A cluster of acute hepatitis E infection in United Nations Bangladeshi peacekeepers in Haiti. Am J Trop Med Hyg 1997;57:449-54.

83 Bi SL, Purdy MA, McCaustland KA, Margolis HS, Bradley DW. The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China. Virus Res 1993;28:233-47.

84 Wang Y, Zhang H, Li Z, Gu W, Lan H, Hao W, et al. Detection of sporadic cases of hepatitis E virus (HEV) infection in China using immunoassays based on recombinant open reading frame 2 and 3 polypeptides from HEV genotype 4. J Clin Microbiol 2001;39: 4370-4379.

85 Yin S, Purcell RH, Emerson SU. A new Chinese isolate of hepatitis E virus: comparison with strains recovered from different geographical regions. Virus Genes 1994;9:23-32.

86 Lu L, Drobeniuc J, Kobylnikov N, Usmanov RK, Robertson BH, Favorov MO, et al. Complete sequence of a Kyrgyzstan swine hepatitis E virus (HEV) isolated from a piglet thought to be experimentally infected with human HEV. J Med Virol 2004;74: 556-562.

87 Chatterjee R, Tsarev S, Pillot J, Coursaget P, Emerson SU, Purcell RH. African strains of hepatitis E virus that are distinct from Asian strains. J Med Virol 1997;53:139-44.

88 Tsarev SA, Binn LN, Gomatos PJ, Arthur RR, Monier MK, van Cuyck-Gandre H, et al. Phylogenetic analysis of hepatitis E virus isolates from Egypt. J Med Virol 1999;57:68-74.

89 Grandadam M, Tebbal S, Caron M, Siriwardana M, Larouze B, Koeck JL, et al. Evidence for hepatitis E virus quasispecies. J Gen Virol 2004;85 (Pt 11): 3189-94.

90 He J, Binn LN, Tsarev SA, Hayes CG, Frean JA, Isaacson M, et al. Molecular characterization of a hepatitis E virus isolate from Namibia. J Biomed Sci 2000;7:334-8.

91 Nicand E, Armstrong GL, Enouf V, Guthmann JP, Guerin JP, Caron M, et al. Genetic heterogeneity of hepatitis E virus in Darfur, Sudan, and neighboring Chad. J Med Virol 2005;77: 519-21.

92 van Cuyck H, Juge F, Roques P. Phylogenetic analysis of the first complete hepatitis E virus (HEV) genome from Africa. FEMS Immunol Med Microbiol 2003;39:133-9.

93 Huang CC, Nguyen D, Fernandez J, Yun KY, Fry KE, Bradley DW, et al. Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV). Virology 1992;191:550-8.

94 Buisson Y, Grandadam M, Nicand E, Cheval P, van Cuyck-Gandre H, Innis B, et al. Identification of a novel hepatitis E virus in Nigeria. J Gen Virol 2000;81 (Pt 4): 903-9.

95 Maila HT, Bowyer SM, Swanepoel R. Identification of a new strain of hepatitis E virus from an outbreak in Namibia in 1995. J Gen Virol 2004;85 (Pt 1): 89-95.

96 Huang FF, Haqshenas G, Guenette DK, Halbur PG, Schommer SK, Pierson FW, et al. Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. J Clin Microbiol 2002;40:1326-32.

97 Kabrane-Lazizi Y, Zhang M, Purcell RH, Miller KD, Davey RT, Emerson SU. Acute hepatitis caused by a novel strain of hepatitis E virus most closely related to United States strains. J Gen Virol 2001;82 (Pt 7): 1687-93.

98 Leblanc D, Ward P, Gagne MJ, Poitras E, Muller P, Trottier YL, et al. Presence of hepatitis E virus in a naturally infected swine herd from nursery to slaughter. Int J Food Microbiol 2007;177: 160-166.

99 Munne MS, Vladimirsky S, Otegui L, Brajterman L, Castro R, Soto S, et al. Molecular characterization of hepatitis E virus in

three acute liver failure cases in children in Argentina. Acta Gastroenterol Latinoam 2006;36:125-30.

100 Lewis H, Morgan D, Ijaz S, Boxall E. Indigenous hepatitis E virus infection in England and Wales. BMJ 2006;332:1509-10.

101 De Silva AN, Muddu AK, Iredale JP, Sheron N, Khakoo SI, Pelosi

E. Unexpectedly high incidence of indigenous acute hepatitis E within South Hampshire: time for routine testing? J Med Virol 2008;80:283-8.

102 Peron JM, Mansuy JM, Poirson H, Bureau C, Dupuis E, Alric L, et al. Hepatitis E is an autochthonous disease in industrialized countries. Analysis of 23 patients in South-West France over a 13-month period and comparison with hepatitis A. Gastroenterol Clin Biol 2006;30:757-62.

103 Takahashi K, Kang JH, Ohnishi S, Hino K, Mishiro S. Genetic heterogeneity of hepatitis E virus recovered from Japanese patients with acute sporadic hepatitis. J Infect Dis 2002;185: 1342-5.

104 Takahashi K, Iwata K, Watanabe N, Hatahara T, Ohta Y, Baba K, et al. Full-genome nucleotide sequence of a hepatitis E virus strain that may be indigenous to Japan. Virology 2001;287:9-12.

105 Matsubayashi K, Nagaoka Y, Sakata H, Sato S, Fukai K, Kato T, et al. Transfusion-transmitted hepatitis E caused by apparently indigenous hepatitis E virus strain in Hokkaido, Japan. Transfusion 2004;44:934-40.

106 Takahashi M, Nishizawa T, Okamoto H. Identification of a genotype III swine hepatitis E virus that was isolated from a Japanese pig born in 1990 and that is most closely related to Japanese isolates of human hepatitis E virus. J Clin Microbiol 2003;41:1342-3.

107 Okamoto H, Takahashi M, Nishizawa T, Fukai K, Muramatsu U, Yoshikawa A. Analysis of the complete genome of indigenous swine hepatitis E virus isolated in Japan. Biochem Biophys Res Commun 2001;289:929-36.

108 Tokita H, Harada H, Gotanda Y, Takahashi M, Nishizawa T, Okamoto H. Molecular and serological characterization of sporadic acute hepatitis E in a Japanese patient infected with a genotype III hepatitis E virus in 1993. J Gen Virol 2003;84 (Pt 2): 421-7.

109 Takahashi M, Nishizawa T, Miyajima H, Gotanda Y, Iita T, Tsuda

F, et al. Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus. J Gen Virol 2003;84 (Pt 4): 851-62.

110 Tamada Y, Yano K, Yatsuhashi H, Inoue O, Mawatari F, Ishibashi H. Consumption of wild boar linked to cases of hepatitis E. J Hepatol 2004;40:869-70.

111 Ning H, Yu S, Zhu Y, Dong S, Yu R, Shen S, et al. Genotype 3 hepatitis E has been widespread in pig farms of Shanghai suburbs. Vet Microbiol 2008;126:257-63.

112 Arankalle VA, Chobe LP, Joshi MV, Chadha MS, Kundu B, Walimbe AM. Human and swine hepatitis E viruses from Western India belong to different genotypes. J Hepatol 2002;36: 417-25.

113 Arankalle VA, Chobe LP, Walimbe AM, Yergolkar PN, Jacob GP. Swine HEV infection in south India and phylogenetic analysis (1985-1999). J Med Virol 2003;69:391-6.

114 Li K, Zhuang H, Zhu W. Partial nucleotide sequencing of hepatitis E viruses detected in sera of patients with hepatitis E from 14 cities in China. Chin Med J (Engl) 2002;115:1058-63.

115 Wang YC, Zhang HY, Xia NS, Peng G, Lan HY, Zhuang H, et al. Prevalence, isolation, and partial sequence analysis of hepatitis E virus from domestic animals in China. J Med Virol 2002;67:516-21.

116 Nishizawa T, Takahashi M, Mizuo H, Miyajima H, Gotanda Y, Okamoto H. Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99% identity over the entire genome. J Gen Virol 2003;84 (Pt 5): 1245-51.

117 Wibawa ID, Muljono DH, Mulyanto, Suryadarma IG, Tsuda F, Takahashi M, et al. Prevalence of antibodies to hepatitis E virus among apparently healthy humans and pigs in Bali, Indonesia:

identification of a pig infected with a genotype 4 hepatitis E virus. J Med Virol 2004;73:38-44.

118 Hijikata M, Hayashi S, Trinh NT, Ha le D, Ohara H, Shimizu YK, et al. Genotyping of hepatitis E virus from Vietnam. Intervirology 2002;45:101-4.

119 Inoue J, Nishizawa T, Takahashi M, Aikawa T, Mizuo H, Suzuki K, et al. Analysis of the full-length genome of genotype 4 hepatitis E virus isolates from patients with fulminant or acute self-limited hepatitis E. J Med Virol 2006;78:476-84.

120 Ahn JM, Kang SG, Lee DY, Shin SJ, Yoo HS. Identification of novel human hepatitis E virus (HEV) isolates and determination of the seroprevalence of HEV in Korea. J Clin Microbiol 2005;43:3042-8.

121 Suzuki K, Aikawa T, Okamoto H. Fulminant hepatitis E in Japan. N Engl J Med 2002;347:1456.

122 Takahashi M, Nishizawa T, Yoshikawa A, Sato S, Isoda N, Ido K, et al. Identification of two distinct genotypes of hepatitis E virus in a Japanese patient with acute hepatitis who had not travelled abroad. J Gen Virol 2002;83 (Pt 8): 1931-40.

123 Michitaka K, Takahashi K, Furukawa S, Inoue G, Hiasa Y, Horiike N, et al. Prevalence of hepatitis E virus among wild boar in the Ehime area of western Japan. Hepatol Res 2007;37:214-20.

124 Martelli F, Caprioli A, Zengarini M, Marata A, Fiegna C, Di Bartolo I, et al. Detection of hepatitis E virus (HEV) in a demographic managed wild boar (Sus scrofa scrofa) population in Italy. Vet Microbiol 2008;126:74-81.

125 Banks M, Bendall R, Grierson S, Heath G, Mitchell J, Dalton H. Human and porcine hepatitis E virus strains, United Kingdom. Emerg Infect Dis 2004;10:953-5.

126 Pei Y, Yoo D. Genetic characterization and sequence heterogeneity of a Canadian isolate of swine hepatitis E virus. J Clin Microbiol 2002;40:4021-9.

127 Shukla P, Chauhan UK, Naik S, Anderson D, Aggarwal R. Hepatitis E virus infection among animals in northern India: an unlikely source of human disease. J Viral Hepat 2007;14: 310-17.

128 Arankalle VA, Lole KS, Deshmukh TM, Chobe LP, Gandhe SS. Evaluation of human (genotype 1) and swine (genotype 4)-ORF2-based ELISAs for anti-HEV IgM and IgG detection in an endemic country and search for type 4 human HEV infections. J Viral Hepat 2007;14:435-45.

129 Chobe LP, Lole KS, Arankalle VA. Full genome sequence and analysis of Indian swine hepatitis E virus isolate of genotype 4. Vet Microbiol 2006;114:240-51.

130 Jothikumar N, Aparna K, Kamatchiammal S, Paulmurugan R, Saravanadevi S, Khanna P. Detection of hepatitis E virus in raw and treated wastewater with the polymerase chain reaction. Appl Environ Microbiol 1993;59:2558-62.

131 Pina S, Jofre J, Emerson SU, Purcell RH, Girones R. Characterization of a strain of infectious hepatitis E virus isolated from sewage in an area where hepatitis E is not endemic. Appl Environ Microbiol 1998;64:4485-8.

132 Li TC, Miyamura T, Takeda N. Detection of hepatitis E virus RNA from the bivalve Yamato-Shijimi (Corbicula japonica) in Japan. Am J Trop Med Hyg 2007;76:170-2.

133 Li TC, Chijiwa K, Sera N, Ishibashi T, Etoh Y, Shinohara Y, et al. Hepatitis E virus transmission from wild boar meat. Emerg Infect Dis 2005;11:1958-60.

134 Tei S, Kitajima N, Ohara S, Inoue Y, Miki M, Yamatani T, et al. Consumption of uncooked deer meat as a risk factor for hepatitis E virus infection: an age- and sex-matched case-control study. J Med Virol 2004;74:67-70.

135 Arankalle VA, Chadha MS, Mehendale SM, Tungatkar SP. Epidemic hepatitis E: serological evidence for lack of intrafami-lial spread. Indian J Gastroenterol 2000;19:24-8.

136 Naik SR, Aggarwal R, Salunke PN, Mehrotra NN. A large waterborne viral hepatitis E epidemic in Kanpur, India. Bull World Health Organ 1992;70:597-604.

137 Aggarwal R, Naik SR. Hepatitis E: intrafamilial transmission versus waterborne spread. J Hepatol 1994;21:718-23.

138 Somani SK, Aggarwal R, Naik SR, Srivastava S, Naik S. A serological study of intrafamilial spread from patients with sporadic hepatitis E virus infection. J Viral Hepat 2003;10:446-9.

139 Ducancelle A, Payan C, Nicand E, Le Guillou H, Cales P, Lunel-Fabiani F. Intrafamilial hepatitis E in France. J Clin Virol 2007;39:51-3.

140 Gotanda Y, Iwata A, Ohnuma H, Yoshikawa A, Mizoguchi H, Endo K, et al. Ongoing subclinical infection of hepatitis E virus among blood donors with an elevated alanine aminotransferase level in Japan. J Med Virol 2007;79:734-42.

141 Mitsui T, Tsukamoto Y, Suzuki S, Yamazaki C, Masuko K, Tsuda F, et al. Serological and molecular studies on subclinical hepatitis E virus infection using periodic serum samples obtained from healthy individuals. J Med Virol 2005;76:526-33.

142 Khuroo MS, Kamili S, Yattoo GN. Hepatitis E virus infection may be transmitted through blood transfusions in an endemic area. J Gastroenterol Hepatol 2004;19:778-84.

143 Tamura A, Shimizu YK, Tanaka T, Kuroda K, Arakawa Y, Takahashi K, et al. Persistent infection of hepatitis E virus transmitted by blood transfusion in a patient with T-cell lymphoma. Hepatol Res 2007;37:113-20.

144 Mitsui T, Tsukamoto Y, Yamazaki C, Masuko K, Tsuda F, Takahashi M, et al. Prevalence of hepatitis E virus infection among hemodialysis patients in Japan: evidence for infection with a genotype 3 HEV by blood transfusion. J Med Virol 2004; 74:563-72.

145 Boxall E, Herborn A, Kochethu G, Pratt G, Adams D, Ijaz S, et al. Transfusion-transmitted hepatitis E in a 'nonhyperendemic' country. Transfus Med 2006;16:79-83.

146 Kumar A, Beniwal M, Kar P, Sharma JB, Murthy NS. Hepatitis E in pregnancy. Int J Gynaecol Obstet 2004;85:240-4.

147 Kumar RM, Uduman S, Rana S, Kochiyil JK, Usmani A, Thomas L. Sero-prevalence and mother-to-infant transmission of hepatitis E virus among pregnant women in the United Arab Emirates. Eur J Obstet Gynecol Reprod Biol 2001;100:9-15.

148 Khuroo MS, Kamili S, Jameel S. Vertical transmission of hepatitis E virus. Lancet 1995;345:1025-6.

149 Singh S, Mohanty A, Joshi YK, Deka D, Mohanty S, Panda SK. Mother-to-child transmission of hepatitis E virus infection. Indian J Pediatr 2003;70:37-9.

150 Aggarwal R, Krawczynski K. Hepatitis E: an overview and recent advances in clinical and laboratory research. J Gastroenterol Hepatol 2000;15:9-20.

151 Arankalle VA, Chobe LP, Jha J, Chadha MS, Banerjee K, Favorov MO, et al. Aetiology of acute sporadic non-A, non-B viral hepatitis in India. J Med Virol 1993;40:121-5.

152 Ishida S, Yoshizumi S, Miyoshi M, Okui T, Ishida A, Abe S, et al. A cluster of hepatitis E virus infection in Hokkaido, Japan. Jpn J Infect Dis 2006;59:135-6.

153 Nakano Y, Yamauchi A, Yano T, Nakayama O, Sakai H, Nagasaka Y, et al. A diffuse outbreak of hepatitis E in Mie Prefecture, 2005. Jpn J Infect Dis 2006;59:136-8.

154 Sainokami S, Abe K, Kumagai I, Miyasaka A, Endo R, Takikawa Y, et al. Epidemiological and clinical study of sporadic acute hepatitis E caused by indigenous strains of hepatitis E virus in Japan compared with acute hepatitis A. J Gastroenterol 2004; 39:640-8.

155 Khuroo MS. Study of an epidemic of non-A, non-B hepatitis. Possibility of another human hepatitis virus distinct from posttransfusion non-A, non-B type. Am J Med 1980;68:818-24.

156 Kane MA, Bradley DW, Shrestha SM, Maynard JE, Cook EH, Mishra RP, et al. Epidemic non-A, non-B hepatitis in Nepal. Recovery of a possible etiologic agent and transmission studies in marmosets. JAMA 1984;252:3140-5.

157 Tsega E, Hansson BG, Krawczynski K, Nordenfelt E. Acute sporadic viral hepatitis in Ethiopia: causes, risk factors, and effects on pregnancy. Clin Infect Dis 1992;14:961-5.

158 Guthmann JP, Klovstad H, Boccia D, Hamid N, Pinoges L, Nizou JY, et al. A large outbreak of hepatitis E among a displaced population in Darfur, Sudan, 2004: the role of water treatment methods. Clin Infect Dis 2006;42:1685-91.

159 Rab MA, Bile MK, Mubarik MM, Asghar H, Sami Z, Siddiqi S, et al. Water-borne hepatitis E virus epidemic in Islamabad, Pakistan: a common source outbreak traced to the malfunction of a modern water treatment plant. Am J Trop Med Hyg 1997; 57:151-7.

160 Sanyal MC. Epidemic of infectious hepatitis amongst personnel of the armed forces, Delhi (1955-56): epidemiology. Indian J Med Res 1957;45 (Suppl): 91-9.

161 Viswanathan R, Sidhu AS. Infectious hepatitis; clinical findings. Indian J Med Res 1957;45 (Suppl): 49-58.

162 Balayan MS. Epidemiology of hepatitis E virus infection. J Viral Hepat 1997;4:155-65.

163 Vishwanathan. Infectious hepatitis in Delhi (1955-1956). A critical study: epidemiology. Indian J Med Res 1957;45:49-58.

164 Jilani N, Das BC, Husain SA, Baweja UK, Chattopadhya D, Gupta RK, et al. Hepatitis E virus infection and fulminant hepatic failure during pregnancy. J Gastroenterol Hepatol 2007;22:676-82.

165 Kumar AS, Kumar SP, Singh R, Kumar MS, Madan K, Kumar JJ, et al. Hepatitis E virus (HEV) infection in patients with cirrhosis is associated with rapid decompensation and death. J Heat 2007;46:387-94.

166 Kumar A, Aggarwal R, Naik SR, Saraswat V, Ghoshal UC, Naik S. Hepatitis E virus is responsible for decompensation of chronic liver disease in an endemic region. Indian J Gastroenterol 2004;23:59-62.

167 Monga R, Garg S, Tyagi P, Kumar N. Superimposed acute hepatitis E infection in patients with chronic liver disease. Indian J Gastroenterol 2004;23:50-2.

168 Ramachandran J, Eapen CE, Kang G, Abraham P, Hubert DD, Kurian G, et al. Hepatitis E superinfection produces severe decompensation in patients with chronic liver disease. J Gastroenterol Hepatol 2004;19:134-8.

169 Hamid SS, Atiq M, Shehzad F, Yasmeen A, Nissa T, Salam A, et al. Hepatitis E virus superinfection in patients with chronic liver disease. Hepatology 2002;36:474-8.

170 Kc S, Mishra AK, Shrestha R. Hepatitis E virus infection in chronic liver disease causes rapid decompensation. JNMA J Nepal Med Assoc 2006;45:212-15.

171 Dalton HR, Hazeldine S, Banks M, Ijaz S, Bendall R. Locally acquired hepatitis E in chronic liver disease. Lancet 2007;369:1260.

172 Christensen PB, Engle RE, Jacobsen SE, Krarup HB, Georgsen J, Purcell RH. High prevalence of hepatitis E antibodies among Danish prisoners and drug users. J Med Virol 2002;66:49-55.

173 Nicand E, Grandadam M, Teyssou R, Rey JL, Buisson Y. Viraemia and faecal shedding of HEV in symptom-free carriers. Lancet 2001;357:68-9.

174 Krawczynski K. Hepatitis E vaccine—ready for prime time? N Engl J Med 2007;356:949-51.

175 Zhou YH, Purcell RH, Emerson SU. An ELISA for putative neutralizing antibodies to hepatitis E virus detects antibodies to genotypes 1, 2, 3, and 4. Vaccine 2004;22:2578-85.

176 Ghabrah TM, Tsarev S, Yarbough PO, Emerson SU, Strickland GT, Purcell RH. Comparison of tests for antibody to hepatitis E virus. J Med Virol 1998;55:134-7.

177 Arankalle VA, Tsarev SA, Chadha MS, Alling DW, Emerson SU, Banerjee K, et al. Age-specific prevalence of antibodies to hepatitis A and E viruses in Pune, India, 1982 and 1992. J Infect Dis 1995;171:447-50.

178 Fix AD, Abdel-Hamid M, Purcell RH, Shehata MH, Abdel-Aziz F, Mikhail N, et al. Prevalence of antibodies to hepatitis E in two rural Egyptian communities. Am J Trop Med Hyg 2000;62: 519-23.

179 Stoszek SK, Engle RE, Abdel-Hamid M, Mikhail N, Abdel-Aziz F, Medhat A, et al. Hepatitis E antibody seroconversion without

disease in highly endemic rural Egyptian communities. Trans R Soc Trop Med Hyg 2006;100:89-94.

180 Stoszek SK, Abdel-Hamid M, Saleh DA, El Kafrawy S, Narooz S, Hawash Y, et al. High prevalence of hepatitis E antibodies in pregnant Egyptian women. Trans R Soc Trop Med Hyg 2006;100: 95-101.

181 Li RC, Ge SX, Li YP, Zheng YJ, Nong Y, Guo QS, et al. Seroprevalence of hepatitis E virus infection, rural southern People's Republic of China. Emerg Infect Dis 2006;12:1682-8.

182 Buti M, Dominguez A, Plans P, Jardi R, Schaper M, Espunes J, et al. Community-based seroepidemiological survey of hepatitis E virus infection in Catalonia, Spain. Clin Vaccine Immunol 2006;13:1328-32.

183 Olsen B, Axelsson-Olsson D, Thelin A, Weiland O. Unexpected high prevalence of IgG-antibodies to hepatitis E virus in Swedish pig farmers and controls. Scand J Infect Dis 2006;38:55-8.

184 Thomas DL, Yarbough PO, Vlahov D, Tsarev SA, Nelson KE, Saah AJ, et al. Seroreactivity to hepatitis E virus in areas where the disease is not endemic. J Clin Microbiol 1997;35:1244-7.

185 Kikuchi K, Yoshida T, Kimata N, Sato C, Akiba T. Prevalence of hepatitis E virus infection in regular hemodialysis patients. Ther Apher Dial 2006;10:193-7.

186 Cheng PN, Wang RH, Wu IC, Wu JC, Tseng KC, Young KC, et al. Seroprevalence of hepatitis E virus infection among institutionalized psychiatric patients in Taiwan. J Clin Virol 2007;38:44-8.

187 Meng XJ, Wiseman B, Elvinger F, Guenette DK, Toth TE, Engle RE, et al. Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries. J Clin Microbiol 2002;40:117-22.

188 Tanaka E, Matsumoto A, Takeda N, Li TC, Umemura T, Yoshizawa K, et al. Age-specific antibody to hepatitis E virus has remained constant during the past 20 years in Japan. J Viral Hepat 2005;12:439-42.

189 Meng XJ, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, et al. A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci USA 1997;94:9860-5.

190 Karetnyi YV, Gilchrist MJ, Naides SJ. Hepatitis E virus infection prevalence among selected populations in Iowa. J Clin Virol 1999;14:51-5.

191 Nakai I, Kato K, Miyazaki A, Yoshii M, Li TC, Takeda N, et al. Different fecal shedding patterns of two common strains of hepatitis E virus at three Japanese swine farms. Am J Trop Med Hyg 2006;75:1171-7.

192 Choi C, Chae C. Localization of swine hepatitis E virus in liver and extrahepatic tissues from naturally infected pigs by in situ hybridization. J Hepatol 2003;38:827-32.

193 Kasorndorkbua C, Opriessnig T, Huang FF, Guenette DK, Thomas PJ, Meng XJ, et al. Infectious swine hepatitis E virus is present in pig manure storage facilities on United States farms, but evidence of water contamination is lacking. Appl Environ Microbiol 2005;71:7831-7.

194 Meng XJ, Halbur PG, Shapiro MS, Govindarajan S, Bruna JD, Mushahwar IK, et al. Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. J Virol 1998;72:9714-21.

195 Meng XJ, Halbur PG, Haynes JS, Tsareva TS, Bruna JD, Royer RL, et al. Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV. Arch Virol 1998;143:1405-15.

196 Kasorndorkbua C, Guenette DK, Huang FF, Thomas PJ, Meng XJ, Halbur PG. Routes of transmission of swine hepatitis E virus in pigs. J Clin Microbiol 2004;42:5047-52.

197 Halbur PG, Kasorndorkbua C, Gilbert C, Guenette D, Potters MB, Purcell RH, et al. Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human. J Clin Microbiol 2001;39:918-23.

198 Martin M, Segales J, Huang FF, Guenette DK, Mateu E, de Deus N, et al. Association of hepatitis E virus (HEV) and postweaning

multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs. Vet Microbiol 2007;122:16-24.

199 Rutjes SA, Lodder WJ, Bouwknegt M, de Roda Husman AM. Increased hepatitis E virus prevalence on Dutch pig farms from 33 to 55% by using appropriate internal quality controls for RT-PCR. J Virol Methods 2007;143:112-16.

200 Chandler JD, Riddell MA, Li F, Love RJ, Anderson DA. Serological evidence for swine hepatitis E virus infection in Australian pig herds. Vet Microbiol 1999;68:95-105.

201 Meng XJ, Dea S, Engle RE, Friendship R, Lyoo YS, Sirinarumitr T, et al. Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population. J Med Virol 1999;59:297-302.

202 Takahashi M, Nishizawa T, Tanaka T, Tsatsralt-Od B, Inoue J, Okamoto H. Correlation between positivity for immuno-globulin A antibodies and viraemia of swine hepatitis E virus observed among farm pigs in Japan. J Gen Virol 2005;86 (Pt 6): 1807-13.

203 Yoo D, Willson P, Pei Y, Hayes MA, Deckert A, Dewey CE, et al. Prevalence of hepatitis E virus antibodies in Canadian swine herds and identification of a novel variant of swine hepatitis E virus. Clin Diagn Lab Immunol 2001;8:1213-19.

204 Bouwknegt M, Lodder-Verschoor F, van der Poel WH, Rutjes SA, de Roda Husman AM. Hepatitis E virus RNA in commercial porcine livers in The Netherlands. J Food Prot 2007;70:2889-95.

205 Williams TP, Kasorndorkbua C, Halbur PG, Haqshenas G, Guenette DK, Toth TE, et al. Evidence of extrahepatic sites of replication of the hepatitis E virus in a swine model. J Clin Microbiol 2001;39:3040-6.

206 Ha SK, Chae C. Immunohistochemistry for the detection of swine hepatitis E virus in the liver. J Viral Hepat 2004;11:263-7.

207 Bouwknegt M, Engel B, Herremans MM, Widdowson MA, Worm HC, Koopmans MP, et al. Bayesian estimation of hepatitis E virus seroprevalence for populations with different exposure levels to swine in The Netherlands. Epidemiol Infect 2007;20:1-0.

208 Hirano M, Ding X, Li TC, Takeda N, Kawabata H, Koizumi N, et al. Evidence for widespread infection of hepatitis E virus among wild rats in Japan. Hepatol Res 2003;27:1-5.

209 Arankalle VA, Joshi MV, Kulkarni AM, Gandhe SS, Chobe LP, Rautmare SS, et al. Prevalence of anti-hepatitis E virus antibodies in different Indian animal species. J Viral Hepat 2001;8:223-7.

210 Favorov MO, Kosoy MY, Tsarev SA, Childs JE, Margolis HS. Prevalence of antibody to hepatitis E virus among rodents in the United States. J Infect Dis 2000;181:449-55.

211 Kabrane-Lazizi Y, Fine JB, Elm J, Glass GE, Higa H, Diwan A, et al. Evidence for widespread infection of wild rats with hepatitis E virus in the United States. Am J Trop Med Hyg 1999;61:331-5.

212 Easterbrook JD, Kaplan JB, Vanasco NB, Reeves WK, Purcell RH, Kosoy MY, et al. A survey of zoonotic pathogens carried by Norway rats in Baltimore, Maryland, USA. Epidemiol Infect 2007;15:1-8.

213 Nakamura M, Takahashi K, Taira K, Taira M, Ohno A, Sakugawa H, et al. Hepatitis E virus infection in wild mongooses of Okinawa, Japan: demonstration of anti-HEV antibodies and a full-genome nucleotide sequence. Hepatol Res 2006;34: 137-40.

214 Hirano M, Ding X, Tran HT, Li TC, Takeda N, Sata T, et al. Prevalence of antibody against hepatitis E virus in various species of non-human primates: evidence of widespread infection in Japanese monkeys (Macaca fuscata). Jpn J Infect Dis 2003;56:8-11.

215 Vitral CL, Pinto MA, Lewis-Ximenez LL, Khudyakov YE, dos Santos DR, Gaspar AM. Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil. Mem Inst Oswaldo Cruz 2005;100:117-22.

216 Okamoto H, Takahashi M, Nishizawa T, Usui R, Kobayashi E. Presence of antibodies to hepatitis E virus in Japanese pet cats. Infection 2004;32:57-8.

217 Saad MD, Hussein HA, Bashandy MM, Kamel HH, Earhart KC, Fryauff DJ, et al. Hepatitis E virus infection in work horses in Egypt. Infect Genet Evol 2007;7:368-73.

218 Caron M, Enouf V, Than SC, Dellamonica L, Buisson Y, Nicand E. Identification of genotype 1 hepatitis E virus in samples from swine in Cambodia. J Clin Microbiol 2006;44:3440-2.

219 He J, Innis BL, Shrestha MP, Clayson ET, Scott RM, Linthicum KJ, et al. Evidence that rodents are a reservoir of hepatitis E virus for humans in Nepal. J Clin Microbiol 2002;40:4493-8.

220 He J, Innis BL, Shrestha MP, Clayson ET, Scott RM, Linthicum KJ, et al. Evidence that rodents are a reservoir of hepatitis E virus for humans in Nepal. J Clin Microbiol 2006;44:1208.

221 Haqshenas G, Shivaprasad HL, Woolcock PR, Read DH, Meng XJ. 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 (Pt 10): 2449-62.

222 Huang FF, Sun ZF, Emerson SU, Purcell RH, Shivaprasad HL, Pierson FW, et al. Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV. J Gen Virol 2004;85 (Pt 6): 1609-18.

223 Anderson DA, Shrestha IL. Hepatitis E Virus. ASM Press: Washington DC, 2002.

224 Zhang J, Ge SX, Huang GY, Li SW, He ZQ, Wang YB, et al. Evaluation of antibody-based and nucleic acid-based assays for diagnosis of hepatitis E virus infection in a rhesus monkey model. J Med Virol 2003;71:518-26.

225 Soe S, Uchida T, Suzuki K, Komatsu K, Azumi J, Okuda Y, et al. Enterically transmitted non-A, non-B hepatitis in cynomolgus monkeys: morphology and probable mechanism of hepato-cellular necrosis. Liver 1989;9:135-45.

226 Longer CF, Denny SL, Caudill JD, Miele TA, Asher LV, Myint KS, et al. Experimental hepatitis E: pathogenesis in cynomolgus macaques (Macaca fascicularis). J Infect Dis 1993;168:602-9.

227 Tsarev SA, Emerson SU, Tsareva TS, Yarbough PO, Lewis M, Govindarajan S, et al. Variation in course of hepatitis E in experimentally infected cynomolgus monkeys. J Infect Dis 1993;167:1302-6.

228 Chauhan A, Jameel S, Dilawari JB, Chawla YK, Kaur U, Ganguly NK. Hepatitis E virus transmission to a volunteer. Lancet 1993;341:149-50.

229 Aggarwal R, Kini D, Sofat S, Naik SR, Krawczynski K. Duration of viraemia and faecal viral excretion in acute hepatitis E. Lancet 2000;356:1081-2.

230 Zhao ZY, Ruan B, Shao H, Chen ZJ, Liu SL. Detection of hepatitis E virus RNA in sera of patients with hepatitis E by polymerase chain reaction. Hepatobiliary Pancreat Dis Int 2007;6:38-42.

231 Nanda SK, Ansari IH, Acharya SK, Jameel S, Panda SK. Protracted viremia during acute sporadic hepatitis E virus infection. Gastroenterology 1995;108:225-30.

232 Arankalle VA, Chadha MS, Tsarev SA, Emerson SU, Risbud AR, Banerjee K, et al. Seroepidemiology of water-borne hepatitis in India and evidence for a third enterically transmitted hepatitis agent. Proc Natl Acad Sci USA 1994;91:3428-32.

233 Clayson ET, Myint KS, Snitbhan R, Vaughn DW, Innis BL, Chan L, et al. Viremia, fecal shedding, and IgM and IgG responses in patients with hepatitis E. J Infect Dis 1995;172:927-33.

234 Bryan JP, Tsarev SA, Iqbal M, Ticehurst J, Emerson S, Ahmed A, et al. Epidemic hepatitis E in Pakistan: patterns of serologic response and evidence that antibody to hepatitis E virus protects against disease. J Infect Dis 1994;170:517-21.

235 Tsarev SA, Tsareva TS, Emerson SU, Govindarajan S, Shapiro M, Gerin JL, et al. Successful passive and active immunization of cynomolgus monkeys against hepatitis E. Proc Natl Acad Sci USA 1994;91:10198-202.

236 Naik S, Aggarwal R, Naik SR, Dwivedi S, Talwar S, Tyagi SK, et al. Evidence for activation of cellular immune responses in patients with acute hepatitis E. Indian J Gastroenterol 2002;21:149-52.

237 Aggarwal R, Shukla R, Jameel S, Agrawal S, Puri P, Gupta VK, et al. T-cell epitope mapping of ORF2 and ORF3 proteins of human hepatitis E virus. J Viral Hepat 2007;14:283-92.

238 Pal R, Aggarwal R, Naik SR, Das V, Das S, Naik S. Immunological alterations in pregnant women with acute hepatitis E. J Gastroenterol Hepatol 2005;20:1094-101.

239 Aggarwal R. Hepatitis E and pregnancy. Indian J Gastroenterol 2007;26:3-5.

240 Srivastava R, Aggarwal R, Jameel S, Puri P, Gupta VK, Ramesh VS, et al. Cellular immune responses in acute hepatitis E virus infection to the viral open reading frame 2 protein. Viral Immunol 2007;20:56-65.

241 Tsega E, Krawczynski K, Hansson BG, Nordenfelt E, Negusse Y, Alemu W, et al. Outbreak of acute hepatitis E virus infection among military personnel in northern Ethiopia. J Med Virol 1991;34:232-6.

242 Krawczynski K, Kamili S, Aggarwal R. Global epidemiology and medical aspects of hepatitis E. Forum (Genova) 2001;11:166-79.

243 Purcell RH, Emerson SU. Hepatitis E virus in Fields Virology. Lippincott, William and Wilkins: Philadelphia, 2001.

244 Nanda SK, Yalcinkaya K, Panigrahi AK, Acharya SK, Jameel S, Panda SK. Etiological role of hepatitis E virus in sporadic fulminant hepatitis. J Med Virol 1994;42:133-7.

245 Arora NK, Nanda SK, Gulati S, Ansari IH, Chawla MK, Gupta SD, et al. Acute viral hepatitis types E, A, and B singly and in combination in acute liver failure in children in north India. J Med Virol 1996;48:215-21.

246 Tsega E, Krawczynski K, Hansson BG, Nordenfelt E. Hepatitis E virus infection in pregnancy in Ethiopia. Ethiop Med J 1993;31:173-81.

247 Khuroo MS, Teli MR, Skidmore S, Sofi MA, Khuroo MI. Incidence and severity of viral hepatitis in pregnancy. Am J Med 1981;70:252-5.

248 Mirghani OA, Saeed OK, Basama FM. Viral hepatitis in pregnancy. EastAfr Med J 1992;69:445-9.

249 Hussaini SH, Skidmore SJ, Richardson P, Sherratt LM, Cooper BT, O'Grady JG. Severe hepatitis E infection during pregnancy. J Viral Hepat 1997;4:51-4.

250 Peron JM, Bureau C, Poirson H, Mansuy JM, Alric L, Selves J, et al. Fulminant liver failure from acute autochthonous hepatitis E in France: description of seven patients with acute hepatitis E and encephalopathy. J Viral Hepat 2007;14:298-303.

251 Ohnishi S, Kang JH, Maekubo H, Arakawa T, Karino Y, Toyota J, et al. Comparison of clinical features of acute hepatitis caused by hepatitis E virus (HEV) genotypes 3 and 4 in Sapporo, Japan. Hepatol Res 2006;36:301-7.

252 Coursaget P, Buisson Y, N'Gawara MN, Van Cuyck-Gandre H, Roue R. Role of hepatitis E virus in sporadic cases of acute and fulminant hepatitis in an endemic area (Chad). Am J Trop Med Hyg 1998;58:330-4.

253 Sallie R, Chiyende J, Tan KC, Bradley D, Portmann B, Williams R, et al. Fulminant hepatic failure resulting from coexistent Wilson's disease and hepatitis E. Gut 1994;35:849-53.

254 Peron JM, Mansuy JM, Recher C, Bureau C, Poirson H, Alric L, et al. Prolonged hepatitis E in an immunocompromised patient. J Gastroenterol Hepatol 2006;21:1223-4.

255 Gerolami R, Moal V, Colson P. Chronic hepatitis E with cirrhosis in a kidney-transplant recipient. N Engl J Med 2008;358:859-60.

256 Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Guitard J, et al. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N Engl J Med 2008;358:811-17.

257 Kamani P, Baijal R, Amarapurkar D, Gupte P, Patel N, Kumar P, et al. Guillain-Barre syndrome associated with acute hepatitis E. Indian J Gastroenterol 2005;24:216.

258 Mandal K, Chopra N. Acute transverse myelitis following hepatitis E virus infection. Indian Pediatr 2006;43:365-6.

259 Jaroszewicz J, Flisiak R, Kalinowska A, Wierzbicka I, Prokopowicz D. Acute hepatitis E complicated by acute pancreatitis: a case report and literature review. Pancreas 2005;30:382-4.

260 Zamvar V, McClean P, Odeka E, Richards M, Davison S. Hepatitis E virus infection with nonimmune hemolytic anemia. J Pediatr Gastroenterol Nutr 2005;40:223-5.

261 Wendum D, Nachury M, Yver M, Lemann M, Flejou JF, Janin A, et al. Acute hepatitis E: a cause of lymphocytic destructive cholangitis. Hum Pathol 2005;36:436-8.

262 Serratrice J, Disdier P, Colson P, Ene N, de Roux CS, Weiller PJ. Acute polyarthritis revealing hepatitis E. Clin Rheumatol 2007; 20:1973-5.

263 Worm HC, van der Poel WH, Brandstatter G. Hepatitis E: an overview. Microbes Infect 2002;4:657-66.

264 Myint KS, Endy TP, Gibbons RV, Laras K, Mammen Jr MP, Sedyaningsih ER, et al. Evaluation of diagnostic assays for hepatitis E virus in outbreak settings. J Clin Microbiol 2006;44:1581-3.

265 Mast EE, Alter MJ, Holland PV, Purcell RH. Evaluation of assays for antibody to hepatitis E virus by a serum panel. Hepatitis E Virus Antibody Serum Panel Evaluation Group. Hepatology 1998;27:857-61.

266 Orru G, Masia G, Orru G, Romano L, Piras V, Coppola RC. Detection and quantitation of hepatitis E virus in human faeces by real-time quantitative PCR. J Virol Methods 2004;118:77-82.

267 Inoue J, Takahashi M, Yazaki Y, Tsuda F, Okamoto H. Development and validation of an improved RT-PCR assay with nested universal primers for detection of hepatitis E virus strains with significant sequence divergence. J Virol Methods 2006;137:325-33.

268 Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR. A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J Virol Methods 2006;131: 65-71.

269 Wang L, Zhuang H. Hepatitis E: an overview and recent advances in vaccine research. World J Gastroenterol 2004;10: 2157-2162.

270 Worm HC, Wirnsberger G. Hepatitis E vaccines: progress and prospects. Drugs 2004;64:1517-31.

271 Kamili S, Spelbring J, Carson D, Krawczynski K. Protective efficacy of hepatitis E virus DNA vaccine administered by gene gun in the cynomolgus macaque model of infection. J Infect Dis 2004;189:258-64.

272 He J, Hayes CG, Binn LN, Seriwatana J, Vaughn DW, Kuschner RA, et al. Hepatitis E virus DNA vaccine elicits immunologic memory in mice. J Biomed Sci 2001;8:223-6.

273 Meng J, Dai X, Chang JC, Lopareva E, Pillot J, Fields HA, et al. Identification and characterization of the neutralization epitope(s) of the hepatitis E virus. Virology 2001;288:203-11.

274 Im SW, Zhang JZ, Zhuang H, Che XY, Zhu WF, Xu GM, et al. A bacterially expressed peptide prevents experimental infection of primates by the hepatitis E virus. Vaccine 2001;19:3726-32.

275 Li SW, Zhang J, He ZQ, Ge SX, Gu Y, Lin J, et al. The study of aggregate of the ORF2 peptide of hepatitis E virus expressed in Escherichia coli. Sheng Wu Gong Cheng Xue Bao 2002;18:463-7.

276 Robinson RA, Burgess WH, Emerson SU, Leibowitz RS, Sosnovt-seva SA, Tsarev S, et al. Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells. Protein Expr Purif 1998;12:75-84.

277 Zhang M, Emerson SU, Nguyen H, Engle R, Govindarajan S, Blackwelder WC, et al. Recombinant vaccine against hepatitis E: duration of protective immunity in rhesus macaques. Vaccine 2002;20:3285-91.

278 Zhang M, Emerson SU, Nguyen H, Engle RE, Govindarajan S, Gerin JL, et al. Immunogenicity and protective efficacy of a vaccine prepared from 53 kDa truncated hepatitis E virus capsid protein expressed in insect cells. Vaccine 2001;20:853-7.

279 Purdy MA, McCaustland KA, Krawczynski K, Spelbring J, Reyes GR, Bradley DW. Preliminary evidence that a trpE-HEV fusion protein protects cynomolgus macaques against challenge with wild-type hepatitis E virus (HEV). J Med Virol 1993;41: 90-4.

280 Purcell RH, Nguyen H, Shapiro M, Engle RE, Govindarajan S, Blackwelder WC, et al. Pre-clinical immunogenicity and efficacy trial of a recombinant hepatitis E vaccine. Vaccine 2003;21: 2607-15.

281 Shrestha MP, Scott RM, Joshi DM, Mammen Jr MP, Thapa GB, Thapa N, et al. Safety and efficacy of a recombinant hepatitis E vaccine. N Engl J Med 2007;356:895-903.

282 Teo CG. The two clinico-epidemiological forms of hepatitis E. J Viral Hepat 2007;14:295-7.

Copyright of Emerging Health Threats is the property of Forum for Global Health Protection and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.