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Hepatitis E: a complex and global disease

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.


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][4][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'.  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.
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 cloned 44,45 and diagnostic antibody assays were developed. 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 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 0 noncoding region (28 nucleotides), followed by three open reading frames (ORF), a 3 0 noncoding region (65-74 nucleotides), and a poly(A) tail ( Figure 1). 45,53 The genome is capped at its 5 0 terminus 54 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 0 end of the coding region and encodes the capsid protein. 45,51,[56][57][58] The N-terminal region of ORF2 protein binds the 5 0 noncoding region of the HEV genome 59 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 0 end overlapping ORF1 by four nucleotides and the 3 0 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 signalling 62,63 and in the assembly of the HEV nucleocapsid; 60 recently, it has been shown that this protein is essential for infectivity in vivo 64 but not in vitro. 65 The poly(A) tail is necessary for binding of RNA-dependent RNA polymerase (RdRp) to the 3 0 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 al. 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 561C for 30 min. 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 0 end of the ORF2 region ( Figure 2) 69 and a 306-nucleotidelong sequence in the RdRp of ORF1. 70

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 contaminate the environment and shellfish even in nonepidemic 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 meat 40 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 outbreaks 137 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 RNApositive mothers with worsening liver disease. [146][147][148][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][156][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][163][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 Europe 10,16,18,20,100,101 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][166][167][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 Bcell 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 seroepidemiology 46,56,175,176 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 adulthood. 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 population 179 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 seroprevalence 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 USA 189 and, subsequently, worldwide. 28,[32][33][34][35][36]96,112,183,[190][191][192][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 route 196 and develop a self-limiting subclinical infection 189,197,198 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][200][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 USA 30 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 samples 193 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 Netherlands. 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 horses 217 and pigs. 218 The finding of HEV genotype 1 in rats 219 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 strains 221,222 and does not represent a risk for cross-infection to humans. (Figure 4). 223 The virus then reaches the liver through the portal vein 41 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][226][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 before 173 and for two to four weeks after the onset of symptoms. [228][229][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][230][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 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 Tlymphocytes 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 immunopathogenesis 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.

It is thought that HEV infection initiates via cells lining the alimentary tract (primary site of virus replication)
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

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 cirrhosis 2,243 in immunocompetent patients. Hepatitis E Emerging Health Threats Journal E Pelosi and I Clarke 2008, 1:e8 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][247][248][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][166][167][168][169][170][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.

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 X201 IU/l (normal value o60 IU/l) were HEV RNApositive. 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 HEVinfected 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 0 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 (Nterminus) 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 HEVspecific 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 serological 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][267][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.

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 replication 270 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 607 56 and is crossreactive with all mammalian HEV. 56,273 Animal studies have shown that ORF2 recombinant proteins 274-278 elicit neutralising antibodies and mediate protective immunity in vaccinated primates. 277,279 One such vaccine with a 56 kDa protein encompassing amino acids 112-607 55,280 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. 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 E 16,18,100-102 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.