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Hepatitis E in developed countries: current status and future perspectives
Harry R Dalton*,1, Nassim Kamar2,3,4 & Jacques Izopet3,4,5
ABSTRACT Hepatitis E virus (HEV) was for many years thought to be found almost exclusively in developing countries, where it is a major health issue. Recent studies have shown that HEV causes acute and chronic infection in developed countries. In these geographical settings, HEV is primarily a porcine zoonosis caused by genotypes 3 (HEV3) and 4 (HEV4). The clinical phenotype of hepatitis E continues to emerge, and recent data show that HEV is associated with a range of neurological syndromes including Guillain–Barré syndrome and neuralgic amytrophy.
Basic virology Hepatitis E virus (HEV), the causative agent of hepatitis E, is a small nonenveloped virus (27–34 nm in diameter) with an icosahedral capsid and a positive-sense ssRNA genome. The HEV genome is approximately 7.2 kb long and capped with 7-methyl-guanosine and polyadenylated at the 5′ and 3′ termini, respectively. It contains three open reading frames (ORF) OFR1, ORF2 and ORF3, flanked by noncoding regions [1] . ORF1 encodes a nonstructural protein of 1693 amino acids containing several functional domains as methyltransferase, papaïne-like cysteine protease, macrodomain, helicase and RNA-dependent RNA polymerase [2] . Between the protease and the macrodomain, there is a more variable region containing a proline-rich hinge, the polyproline region (PPR), which may be involved in virus adaptation [3–9] . The PPR is an intrinsically disordered region where segments of human gene have been recently identified in vitro and in vivo [4,7] . In addition, immunocompromised patients who develop chronic infection have greater heterogeneity of PPR quasispecies at the acute phase of the infection compared with individuals with resolving hepatitis [3] . ORF2 encodes the viral capsid protein of 660 amino acids containing a shell domain (S) (amino acids 129–319), a middle domain (M) (amino acids 320–455) and a protruding domain (P) (amino acids 456–606), harboring the neutralizing epitope(s) [10,11] . During assembly, capsid monomers self-assemble into dimers and subsequently in decamers that encapsidate the viral RNA. The virion-sized capsid demonstrates a T = 3 icosahedral lattice [11] . The viral capsid protein is responsible for virion assembly, interaction with target cells and immunogenicity. There is a membrane-binding region at the start of ORF2. Immunological and structural studies of the
KEYWORDS
• chronic infection • cirrhosis • hepatitis • hepatitis E • pigs • zoonosis
Cornwall Gastrointestinal Unit, Royal Cornwall Hospital & European Centre for the Environment & Human Health, University of Exeter Medical School, Truro TR1 3LJ, UK 2 Department of Nephrology & Organ Transplantation, CHU Rangueil, Toulouse, France 3 INSERM U1043, IFR–BMT, CHU Purpan, Toulouse, France 4 Université Paul Sabatier, Toulouse, France 5 Department of Virology, CHU Purpan, Toulouse, France *Author for correspondence: Tel.: + 44 (0)1872 253 858; [email protected] 1
10.2217/FMB.14.89 © 2014 Future Medicine Ltd
Future Microbiol. (2014) 9(12), 1361–1372
ISSN 1746-0913
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Review Dalton, Kamar & Izopet capsid protein have provided a basis for hepatitis E vaccine development. ORF3 encodes a small protein of 113 or 114 amino acids that is a regulatory protein involved in virion morphogenesis and release [12,13] . The ORF3 protein is present on the virion surface in association with lipids. It has been demonstrated that an intact proline-serinealanine-proline (PSAP) motif within the ORF3 protein is important for the formation of membrane-associated HEV particles [14] . Although HEV is a nonenveloped virus, the association of HEV with lipids in the blood of infected individuals might provide protection from neutralizing antibodies [15] . The recent availability of cell culture systems such as PLC/PRF/5 and HepG2/C3A cells, derived from human hepatocellular carcinoma, and A549 cells, derived from human lung cancer permitted the propagation of HEV from fecal and serum samples [7,16] . The propagation of HEV has been also obtained by using cell culture systems that have morphological and functional properties similar to those of primary hepatocytes [17] . Based on sucrose-density fractionation experiments, it has been demonstrated that HEV particles in culture supernatant have similar density to HEV particles circulating in blood of infected individuals (1.15–1.16 g/ml) [16] . HEV particles in feces have higher density (1.27–1.28 g/ml) because lipids and ORF3 protein are removed by bile acids and digestive proteases, respectively [16] . These cell systems are essential for a better understanding of the replication cycle, for the development of inhibitors of HEV replication and for validation studies of viral safety of biopharmaceutical materials, such as plasma-derived medicinal products. Little is known on the physico-chemical properties of HEV with regard to inactivation/removal during industrial processes. Current data indicate that the sensitivity of HEV to heat vary depending on the heating conditions and the composition of the sample [18] . HEV particles are completely removed using 20 nm nanofilters [18] . Several animal models of HEV infection have been developed [19] . Nonhuman primates (chimpanzees, rhesus and cynomolgus macaques) have been the most widely used models for the study of HEV infection pathogenesis and vaccine trials. These models are adequate for all HEV strains whatever the HEV genotype. It has been shown that experimental HEV infection in cynomolgus macaques without an elevation in
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alanine aminotransferase (ALT) occurred more frequently among animals that received a smaller dose of virus inoculum compared with those given a larger virus inoculum [20] . In these experiments, the highest dilution of the inoculum that caused either the presence of HEV RNA in stool or serum or the development of anti-HEV antibodies (1–10 cynos infections doses) contained 102 –103 viral genome equivalents/milliliter. However, cross-species experiments have shown that similar doses produce considerable variation in pathological outcome. Thus, it seems most likely that a combination of viral factors (including genotype and quantum of viral exposure), host factors and route of transmission are important in clinical disease expression. Several other animal models including pigs, rabbits, rodents and chickens that are naturally infected with particular HEV genotypes have recently been established for the study of various aspects of HEV infection [19] . For example, pigs and rabbits were used to demonstrate the existence of extrahepatic sites of replication by immunohistochemistry and detection of negative strand of HEV RNA [21,22] . The presence of residual infectious virus particles in food products after heat treatments was assessed using an in vivo experimental model in pigs [23] . Heating the food to an internal temperature of 71°C for 20 min was necessary to completely inactivate HEV. Our knowledge on the replication cycle of HEV has improved thanks to the availability of replicons and in vitro infection systems [1] . The virus particle first binds to heparan sulfate proteoglycans on the host cell membrane [24] but the high affinity cellular membrane receptor(s) is still unknown. The following step is internalization through clathrin-mediated endocytosis [25] . The capped viral genome is released from the virion during the uncoating process and directly translated by the host cell ribosomal machinery. The nonstructural proteins generated allow the replication of the viral genome in the cytoplasm and the production of subgenomic RNA that is translated into the structural proteins from ORF2 and ORF3. Full-length RNA progeny is then assembled with the capsid protein into viral particles that are released from the cell in a nonlytic fashion. HEV is classified in the genus Hepevirus in the Hepeviridae family (International Committee on Taxonomy of Viruses). This family contains viruses that infect mammals, birds and fish. A new nomenclature with four
future science group
Hepatitis E in developed countries: current status & future perspectives tentative genera has been recently proposed [26] : Orthohepevirus, including most mammalian strains; Chiropteranhepevirus, including bat strains; Avihepevirus, including avian strains; and Piscihepevirus, including cutthroat trout virus. Avihepevirus, Piscihepevirus and Chiropteranhepevirus whose genome share approximately 50% of the nucleotide sequence of Orthohepevirus have not been associated with human cases. Recent studies with complete genome sequences from ORF1 and ORF2 both in humans and animals suggest the existence of two groups of Orthohepeviruses [27] . One of these corresponds to the four recognized HEV genotypes (HEV1 to HEV4). The second corresponds to new genotypes infecting a range of mammals including humans, pigs, wild boar, rabbit, moose, mongoose, fox, mink, camels and musk shrew. There is a 31 amino acid insertion in the macrodomain of rabbit HEV strains [28] . HEV1 and HEV2 solely infect humans while HEV belonging to the other genotypes are zoonotic agents with their main reservoir in domestic pigs. The second group corresponds to viruses infecting rats and ferrets. Although no consistent criteria can be defined for the assignment of subgenotypes within each genotype, molecular epidemiological data have been obtained with a classification system dividing the four major HEV genotypes into 24 sub genotypes (five for HEV1, two for HEV2, ten for HEV3 and seven for HEV4) [29] . Epidemiology HEV1 and HEV2 are prevalent in many developing countries in Africa and Asia and are transmitted via contaminated water. Sporadic cases occur throughout the year while outbreaks involving thousands of cases are often seasonal. Fecal shedding of HEV by humans with clinical or subclinical infection maintains a circulating pool of infectious individuals who contaminate water supplies [30] . In many developing countries, anti-HEV IgG seroprevalence is low under the age of 10 years and increases dramatically between the ages of 15 and 30 years. However, the seroprevalence rates in children in Egypt are generally higher. HEV transmission from mother to infant occurs mainly during the third trimester of pregnancy. HEV transmissions are associated with a high neonatal mortality, including premature birth, increased fetal loss and neonatal acute hepatitis [30] . A high frequency of HEV transmission from mother to
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Review
infant may be associated with high viral loads in the mother. In developed countries, most cases of human HEV infection are autochthonous (locally acquired). They are not imported from developing countries, as previously believed, but are caused by HEV3 and HEV4 which are transmitted zoonotically from animal reservoirs to humans. HEV3 is found worldwide. By contrast, HEV4 is found largely in China and Japan, however it has recently been detected in Europe, in both pigs and humans [31] . Subgenotypes 3f, 3c and 3e strains have been reported in human and pig populations in France in similar proportions [32,33] . Although the major animal reservoir is domestic pigs, the complete range of mammalian species that might act as reservoirs for HEV is still unclear. HEV3, HEV4 and new HEV genotypes are found in wild boar and deer, but the prevalence is low compared with that in domestic pigs. Rabbit HEV strains have been documented in farmed and wild rabbits in China, the USA and Europe [28] . A human strain closely related to rabbit HEV containing a molecular signature in the macrodomain of ORF1 has been identified, supporting zoonotic transmission from rabbit to humans [28] . Zoonotic transmission of HEV is thought to occur mostly via the consumption of uncooked or undercooked infected pork. Consumption of game meat (wild boar, deer, rabbit) may also be a route of infection, but probably of less importance. HEV has been detected in numerous food products in Europe, Japan and the USA [34–36] . HEV seroprevalence in Germany is associated with consumption of offal and wild boar [37] . In France, HEV infection is associated with consumption of pork meat, game meat and mussels [38] . Direct contact with HEV-infected animals can lead to HEV transmission, as seroprevalence studies show that veterinarians, swine handlers and forestry workers are more likely to be anti-HEV IgG positive compared with the general population [39] . Last, HEV3 and HEV4 can be transmitted via contaminated water as HEV1 and HEV2. HEV3 has been detected in untreated water and in river water in Europe. HEV3 and HEV4 have also been detected in mussels and oysters [40] . HEV has been transmitted by blood transfusion in a number of countries, including the UK [41] . Surprisingly high numbers of blood donors are viremic at the time of donation: Germany 1:1200, The Netherlands 1:2671, England
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Review Dalton, Kamar & Izopet 1:7000 and Sweden 1:7986 [42] . However, in a study of 1939 blood donors in the USA, none were found to be viremic at the time of donation [43] . Donors who have HEV viremia at the time of donation have no symptoms, the liver-function tests are frequently normal and the antiHEV IgM may be negative. Blood donations are not currently screened for HEV, and urgent risk assessments are underway to determine if they should be. When such risk assessments are done, it is important that careful attention is played to the emerging clinical phenotypes associated with HEV, including neurological syndromes. Thanks to validated serological assays with good sensitivity and specificity [44,45] , direct comparisons of seroprevalence between populations in distinct areas can now be made. The question of concordance between different assays has been recently addressed [46] . Using the same assay, IgG seroprevalence in blood donors is 52% in southwestern France [36] , 29% in Germany, 27% in The Netherlands, 16% in southwestern England, 12% in the rest of England and 4.6% in Scotland [30] . In most of these studies, a gradual increase of IgG seroprevalence with the age of individuals has been observed suggesting a cumulative exposure to HEV. Estimates of incidence of HEV infections based on IgM seroprevalence, IgG seroconversion or HEV RNA detection during prospective studies are 0.2% in the UK [47] , 0.7% in the USA [48] , 2–3% in the south of France [49] and 4.3% in China [50] . Clinical aspects of HEV: acute infection ●●Developing countries
Hepatitis E is hyperendemic in many developing countries, particularly in southeast Asia and sub-Saharan Africa. In such locations, hepatitis E is caused by HEV1 and 2, which are obligate human pathogens. Hepatitis E mainly affects young adults, who generally have a self-limiting hepatitis illness lasting a few weeks. However, in pregnant females the mortality is approximately 20%, with an associated excess perinatal mortality [51] . In patients with underlying chronic liver disease who develop hepatitis E, the mortality is also high, and approaches 70% [52] . Hepatitis E occurs as sporadic cases and, more dramatically, in large outbreaks which may involve thousands or tens of thousands of cases. The most recent such outbreak was the one in the refugee camps of South Sudan with 5080 cases and 101 deaths, mainly in pregnant women. A recent estimate in 9 of 21 Global Burden of Disease regions
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in developing countries suggests that there are 3.4 million symptomatic cases of hepatitis E each year, with 70,000 deaths, and 3000 stillbirths [53] . This is probably an underestimate, as there are 1000 HEV-related maternal deaths per annum, in Bangladesh alone [54] . ●●Developed countries
Death in pregnant females appears to be a unique ‘signature’ of waterborne HEV1 and 2. Descriptions of outbreaks of jaundice and deaths in pregnant women suggest that 100 to 200 years ago HEV was present in a number of countries including the USA, Germany, France, Italy, the Balkan nations [55] . The first description of such an outbreak of jaundice and deaths in pregnant women was in Germany in 1794. There were 70 cases, including three pregnant women, two of whom died. Over the last 50 years, there have been no further outbreaks of jaundice associated with deaths in pregnant women in Europe, but they started to appear in Asia and Africa. One explanation for these observations is that improvement in sanitary infrastructure in Europe asserted negative ecological pressure on HEV1 and 2, leaving HEV3 ‘hidden’ in its primary host, the pig [55] . For over 20 years following its discovery, HEV was thought to cause human infection only in hyperendemic developing countries. Thus, in developed countries, HEV was only considered a diagnostic possibility in travelers returning from hyperendemic countries. It is now clear that this notion was mistaken, as locally acquired zoonotic acute hepatitis E has been found in every single developed county in which it has been sought [51] . A clear, consistent and striking observation about such cases is the demography of patients affected, who are more likely to be older (median age: 63 years) males (male:female ratio 3:1) [56] . The reason why acute hepatitis E has a predilection for older men is uncertain. It is possible that older males might have increased exposure to HEV at higher viral loads. Another explanation relates to host factors, as clinically apparent hepatitis E appears to be more common in patients who drink excessive alcohol or who are diabetic. Such patients are more likely to have subclinical hepatitis fibrosis/steatosis, and maybe such patients are more likely to have a clinically apparent hepatitis when exposed to HEV [57] . Most patients with locally acquired acute hepatitis E in developed countries have no symptoms [51] . After an incubation period of
future science group
Hepatitis E in developed countries: current status & future perspectives 2–6 weeks, the minority of patients who become symptomatic usually develop symptoms indistinguishable from other causes of viral hepatitis (Box 1) . Approximately 60% are jaundiced and the ALT is usually in the 1000–2000 IUL range, but the range is wide, and symptomatic patients may have a much more modest transaminitis. Most patients have an illness that lasts 4–6 weeks, followed by complete recovery [58] . Occasionally, the hepatitis is more severe and of longer duration. Patients with underlying chronic liver disease may develop acute or subacute liver failure. Untreated, such patients have a high mortality [59] . The differential diagnosis of acute hepatitis E infection is shown in Table 1. One common difficulty in diagnosis of hepatitis E is mistaking it for drug-induced liver injury. The latter is common in the elderly and is defined by a temporal relationship between starting a drug and developing hepatitis; a temporal relationship between stopping the offending drug and resolution of hepatitis; and, most importantly, excluding all other causes of hepatocellular injury. In a study from southwest England, 13% of patients with ‘criterion-referenced’ drug-induced liver injury were misdiagnosed, as on retrospective testing they had locally acquired hepatitis E. The bottom line of this study is that the diagnosis of drug-induced liver injury is not secure without first excluding hepatitis E [61] . As can been seen from Table 1, locally acquired acute hepatitis E is the commonest cause of viral hepatitis in the UK [62] , and probably many other developed countries too. Commonly used diagnostic testing algorithms for acute hepatitis currently used in Europe suggest that patients with hepatitis should first be tested for hepatitis A virus, hepatitis B virus and hepatitis C virus, and if these tests are negative (particularly if the patient has travelled to an area traditionally thought of as endemic) then the patient
Review
should be considered for HEV testing. Given that locally acquired acute hepatitis E is the commonest cause of hepatitis, it would make much more sense to flip existing algorithms on their head and test all patients with hepatitis for HEV, and if this is negative then test for the other causes of viral hepatitis [63] . The travel history is irrelevant, and should be excluded from future testing algorithms. In Europe zoonotic locally acquired acute hepatitis E is caused by HEV3, in Japan by HEV3 and 4 and in China by HEV4 [51] . HEV4 has recently been found in pigs in Europe [64] and there have been clusters of human cases of hepatitis E caused by HEV4 in France, Italy and Denmark [65,66] . HEV4 infections are also zoonotic and produce a similar illness to HEV3 in older men. However, it is unknown if HEV4 can produce neurological injury or chronic infection (see below). Clinical aspects of HEV: chronic infection During the few last years, it has been clearly demonstrated that HEV3 infection can cause chronic hepatitis leading to cirrhosis [30,67–68] . Chronic HEV infection was described initially in solid-organ-transplant (SOT) patients, and thereafter in pediatric SOT patients, in patients infected by the human immunodeficiency virus (HIV), in patients having a hematological disease and receiving chemotherapy and in stem-celltransplant patients [30,51] . No chronic cases have been reported in patients infected by HEV1, 2 or 4. Chronic hepatitis has previously been defined as HEV replication in the serum and/or in the stools persisting for at least 6 months. However, in SOT patients, it has recently been shown that no spontaneous HEV clearance occurs between months 3 and 6 after infection, suggesting that chronic HEV infection may be more accurately defined as persisting HEV replication for at least 3 months [69] .
Box 1. Symptoms of acute hepatitis E. Common
Less common
No symptoms Jaundice Anorexia Lethargy Abdominal pain Vomiting
Myalgia Pruritis Weight loss Fever Arthralgia Neurological†
A total of 5–8% of patients present with a neurological illness. In such patients, the neurological symptoms and signs dominate the clinical picture and the liver function tests are often only modestly abnormal (see section ‘Extrahepatic manifestations of HEV’). Data taken from [58,60]. †
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Review Dalton, Kamar & Izopet Table 1. The differential diagnosis of a raised alanine aminotransferase. Diagnosis
Notes
Drug-induced hepatitis
Occurs mainly in the elderly. Other causes of hepatitis need to be excluded, including HEV Most common in females >60 years This may be caused by an, as yet, unidentified hepatotropic virus Middle aged/elderly males Median ALT: 1400 IUL, wide range Occurs at any age. Splenomegaly and lymphocytosis are found in 95% which suggests the diagnosis ALT >3000 IUL, usually associated with a period of hypotension ALT usually >3000 IUL Becoming increasingly rare in most developed countries
Autoimmune hepatitis Seronegative hepatitis† Acute HEV EBV hepatitis Ischemic hepatitis Acute HBV HAV
Defined as ALT >500 UL with exclusion of all the other causes of hepatitis. The diagnoses are placed in the order of frequency in which they occur in >2000 consecutive patients presenting with jaundice to a rapid access jaundice clinic in southwest England. ALT: Alanine aminotransferase; EBV: Epstein–Barr virus; HAV: Hepatitis A virus; HBV: Hepatitis B virus; HEV: Hepatitis E virus. Data taken from [61,62]. †
The natural history of HEV infection has been well documented in SOT patients. In a large retrospective multicenter study, nearly 60% of SOT patients infected by HEV develop chronic infection, and nearly 10% develop cirrhosis [70] . In contrast to immunocompetent patients, liver enzyme levels are modestly elevated (≈350 IU/l). However, the progression of liver fibrosis is very rapid leading to cirrhosis within 2–3 years after infection in some patients [71] . There appears to be no correlation between HEV viral load and liver fibrosis progression [38] . In untreated patients, there is no significant difference in HEV viral load in the acute and chronic phases of infection [38] . In SOT patients, chronic hepatitis occurs mainly in the context of more profound immunosuppression. Chronic HEV infection is more common in patients with low CD3 positive lymphocyte counts and lower specific HEV-T-cell responses. SOT patients receiving tacrolimus compared with cyclosporine A, and those with a lower platelet count during the acute phase of infection are also at higher risk of developing chronic infection [67,70,72] . Patients who develop chronic HEV infection have lower serum concentrations of IL-1 receptor antagonist and IL-2 receptor during the acute phase of infection, compared with those with resolving hepatitis [73] . In addition, increased quasispecies heterogeneity in the M and P domains and the polyproline region is seen in patients with chronic infection, compared with those with resolving hepatitis [73,74] . Finally, patients with higher ALT levels during
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the acute phase of infection are more likely to have a spontaneous HEV clearance [70] . HEV reinfection has been observed in anti-HEV IgGpositive transplant recipients with an anti-HEV IgG concentration
Hepatitis E in developed countries: current status and future perspectives
Harry R Dalton*,1, Nassim Kamar2,3,4 & Jacques Izopet3,4,5
ABSTRACT Hepatitis E virus (HEV) was for many years thought to be found almost exclusively in developing countries, where it is a major health issue. Recent studies have shown that HEV causes acute and chronic infection in developed countries. In these geographical settings, HEV is primarily a porcine zoonosis caused by genotypes 3 (HEV3) and 4 (HEV4). The clinical phenotype of hepatitis E continues to emerge, and recent data show that HEV is associated with a range of neurological syndromes including Guillain–Barré syndrome and neuralgic amytrophy.
Basic virology Hepatitis E virus (HEV), the causative agent of hepatitis E, is a small nonenveloped virus (27–34 nm in diameter) with an icosahedral capsid and a positive-sense ssRNA genome. The HEV genome is approximately 7.2 kb long and capped with 7-methyl-guanosine and polyadenylated at the 5′ and 3′ termini, respectively. It contains three open reading frames (ORF) OFR1, ORF2 and ORF3, flanked by noncoding regions [1] . ORF1 encodes a nonstructural protein of 1693 amino acids containing several functional domains as methyltransferase, papaïne-like cysteine protease, macrodomain, helicase and RNA-dependent RNA polymerase [2] . Between the protease and the macrodomain, there is a more variable region containing a proline-rich hinge, the polyproline region (PPR), which may be involved in virus adaptation [3–9] . The PPR is an intrinsically disordered region where segments of human gene have been recently identified in vitro and in vivo [4,7] . In addition, immunocompromised patients who develop chronic infection have greater heterogeneity of PPR quasispecies at the acute phase of the infection compared with individuals with resolving hepatitis [3] . ORF2 encodes the viral capsid protein of 660 amino acids containing a shell domain (S) (amino acids 129–319), a middle domain (M) (amino acids 320–455) and a protruding domain (P) (amino acids 456–606), harboring the neutralizing epitope(s) [10,11] . During assembly, capsid monomers self-assemble into dimers and subsequently in decamers that encapsidate the viral RNA. The virion-sized capsid demonstrates a T = 3 icosahedral lattice [11] . The viral capsid protein is responsible for virion assembly, interaction with target cells and immunogenicity. There is a membrane-binding region at the start of ORF2. Immunological and structural studies of the
KEYWORDS
• chronic infection • cirrhosis • hepatitis • hepatitis E • pigs • zoonosis
Cornwall Gastrointestinal Unit, Royal Cornwall Hospital & European Centre for the Environment & Human Health, University of Exeter Medical School, Truro TR1 3LJ, UK 2 Department of Nephrology & Organ Transplantation, CHU Rangueil, Toulouse, France 3 INSERM U1043, IFR–BMT, CHU Purpan, Toulouse, France 4 Université Paul Sabatier, Toulouse, France 5 Department of Virology, CHU Purpan, Toulouse, France *Author for correspondence: Tel.: + 44 (0)1872 253 858; [email protected] 1
10.2217/FMB.14.89 © 2014 Future Medicine Ltd
Future Microbiol. (2014) 9(12), 1361–1372
ISSN 1746-0913
1361
Review Dalton, Kamar & Izopet capsid protein have provided a basis for hepatitis E vaccine development. ORF3 encodes a small protein of 113 or 114 amino acids that is a regulatory protein involved in virion morphogenesis and release [12,13] . The ORF3 protein is present on the virion surface in association with lipids. It has been demonstrated that an intact proline-serinealanine-proline (PSAP) motif within the ORF3 protein is important for the formation of membrane-associated HEV particles [14] . Although HEV is a nonenveloped virus, the association of HEV with lipids in the blood of infected individuals might provide protection from neutralizing antibodies [15] . The recent availability of cell culture systems such as PLC/PRF/5 and HepG2/C3A cells, derived from human hepatocellular carcinoma, and A549 cells, derived from human lung cancer permitted the propagation of HEV from fecal and serum samples [7,16] . The propagation of HEV has been also obtained by using cell culture systems that have morphological and functional properties similar to those of primary hepatocytes [17] . Based on sucrose-density fractionation experiments, it has been demonstrated that HEV particles in culture supernatant have similar density to HEV particles circulating in blood of infected individuals (1.15–1.16 g/ml) [16] . HEV particles in feces have higher density (1.27–1.28 g/ml) because lipids and ORF3 protein are removed by bile acids and digestive proteases, respectively [16] . These cell systems are essential for a better understanding of the replication cycle, for the development of inhibitors of HEV replication and for validation studies of viral safety of biopharmaceutical materials, such as plasma-derived medicinal products. Little is known on the physico-chemical properties of HEV with regard to inactivation/removal during industrial processes. Current data indicate that the sensitivity of HEV to heat vary depending on the heating conditions and the composition of the sample [18] . HEV particles are completely removed using 20 nm nanofilters [18] . Several animal models of HEV infection have been developed [19] . Nonhuman primates (chimpanzees, rhesus and cynomolgus macaques) have been the most widely used models for the study of HEV infection pathogenesis and vaccine trials. These models are adequate for all HEV strains whatever the HEV genotype. It has been shown that experimental HEV infection in cynomolgus macaques without an elevation in
1362
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alanine aminotransferase (ALT) occurred more frequently among animals that received a smaller dose of virus inoculum compared with those given a larger virus inoculum [20] . In these experiments, the highest dilution of the inoculum that caused either the presence of HEV RNA in stool or serum or the development of anti-HEV antibodies (1–10 cynos infections doses) contained 102 –103 viral genome equivalents/milliliter. However, cross-species experiments have shown that similar doses produce considerable variation in pathological outcome. Thus, it seems most likely that a combination of viral factors (including genotype and quantum of viral exposure), host factors and route of transmission are important in clinical disease expression. Several other animal models including pigs, rabbits, rodents and chickens that are naturally infected with particular HEV genotypes have recently been established for the study of various aspects of HEV infection [19] . For example, pigs and rabbits were used to demonstrate the existence of extrahepatic sites of replication by immunohistochemistry and detection of negative strand of HEV RNA [21,22] . The presence of residual infectious virus particles in food products after heat treatments was assessed using an in vivo experimental model in pigs [23] . Heating the food to an internal temperature of 71°C for 20 min was necessary to completely inactivate HEV. Our knowledge on the replication cycle of HEV has improved thanks to the availability of replicons and in vitro infection systems [1] . The virus particle first binds to heparan sulfate proteoglycans on the host cell membrane [24] but the high affinity cellular membrane receptor(s) is still unknown. The following step is internalization through clathrin-mediated endocytosis [25] . The capped viral genome is released from the virion during the uncoating process and directly translated by the host cell ribosomal machinery. The nonstructural proteins generated allow the replication of the viral genome in the cytoplasm and the production of subgenomic RNA that is translated into the structural proteins from ORF2 and ORF3. Full-length RNA progeny is then assembled with the capsid protein into viral particles that are released from the cell in a nonlytic fashion. HEV is classified in the genus Hepevirus in the Hepeviridae family (International Committee on Taxonomy of Viruses). This family contains viruses that infect mammals, birds and fish. A new nomenclature with four
future science group
Hepatitis E in developed countries: current status & future perspectives tentative genera has been recently proposed [26] : Orthohepevirus, including most mammalian strains; Chiropteranhepevirus, including bat strains; Avihepevirus, including avian strains; and Piscihepevirus, including cutthroat trout virus. Avihepevirus, Piscihepevirus and Chiropteranhepevirus whose genome share approximately 50% of the nucleotide sequence of Orthohepevirus have not been associated with human cases. Recent studies with complete genome sequences from ORF1 and ORF2 both in humans and animals suggest the existence of two groups of Orthohepeviruses [27] . One of these corresponds to the four recognized HEV genotypes (HEV1 to HEV4). The second corresponds to new genotypes infecting a range of mammals including humans, pigs, wild boar, rabbit, moose, mongoose, fox, mink, camels and musk shrew. There is a 31 amino acid insertion in the macrodomain of rabbit HEV strains [28] . HEV1 and HEV2 solely infect humans while HEV belonging to the other genotypes are zoonotic agents with their main reservoir in domestic pigs. The second group corresponds to viruses infecting rats and ferrets. Although no consistent criteria can be defined for the assignment of subgenotypes within each genotype, molecular epidemiological data have been obtained with a classification system dividing the four major HEV genotypes into 24 sub genotypes (five for HEV1, two for HEV2, ten for HEV3 and seven for HEV4) [29] . Epidemiology HEV1 and HEV2 are prevalent in many developing countries in Africa and Asia and are transmitted via contaminated water. Sporadic cases occur throughout the year while outbreaks involving thousands of cases are often seasonal. Fecal shedding of HEV by humans with clinical or subclinical infection maintains a circulating pool of infectious individuals who contaminate water supplies [30] . In many developing countries, anti-HEV IgG seroprevalence is low under the age of 10 years and increases dramatically between the ages of 15 and 30 years. However, the seroprevalence rates in children in Egypt are generally higher. HEV transmission from mother to infant occurs mainly during the third trimester of pregnancy. HEV transmissions are associated with a high neonatal mortality, including premature birth, increased fetal loss and neonatal acute hepatitis [30] . A high frequency of HEV transmission from mother to
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Review
infant may be associated with high viral loads in the mother. In developed countries, most cases of human HEV infection are autochthonous (locally acquired). They are not imported from developing countries, as previously believed, but are caused by HEV3 and HEV4 which are transmitted zoonotically from animal reservoirs to humans. HEV3 is found worldwide. By contrast, HEV4 is found largely in China and Japan, however it has recently been detected in Europe, in both pigs and humans [31] . Subgenotypes 3f, 3c and 3e strains have been reported in human and pig populations in France in similar proportions [32,33] . Although the major animal reservoir is domestic pigs, the complete range of mammalian species that might act as reservoirs for HEV is still unclear. HEV3, HEV4 and new HEV genotypes are found in wild boar and deer, but the prevalence is low compared with that in domestic pigs. Rabbit HEV strains have been documented in farmed and wild rabbits in China, the USA and Europe [28] . A human strain closely related to rabbit HEV containing a molecular signature in the macrodomain of ORF1 has been identified, supporting zoonotic transmission from rabbit to humans [28] . Zoonotic transmission of HEV is thought to occur mostly via the consumption of uncooked or undercooked infected pork. Consumption of game meat (wild boar, deer, rabbit) may also be a route of infection, but probably of less importance. HEV has been detected in numerous food products in Europe, Japan and the USA [34–36] . HEV seroprevalence in Germany is associated with consumption of offal and wild boar [37] . In France, HEV infection is associated with consumption of pork meat, game meat and mussels [38] . Direct contact with HEV-infected animals can lead to HEV transmission, as seroprevalence studies show that veterinarians, swine handlers and forestry workers are more likely to be anti-HEV IgG positive compared with the general population [39] . Last, HEV3 and HEV4 can be transmitted via contaminated water as HEV1 and HEV2. HEV3 has been detected in untreated water and in river water in Europe. HEV3 and HEV4 have also been detected in mussels and oysters [40] . HEV has been transmitted by blood transfusion in a number of countries, including the UK [41] . Surprisingly high numbers of blood donors are viremic at the time of donation: Germany 1:1200, The Netherlands 1:2671, England
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Review Dalton, Kamar & Izopet 1:7000 and Sweden 1:7986 [42] . However, in a study of 1939 blood donors in the USA, none were found to be viremic at the time of donation [43] . Donors who have HEV viremia at the time of donation have no symptoms, the liver-function tests are frequently normal and the antiHEV IgM may be negative. Blood donations are not currently screened for HEV, and urgent risk assessments are underway to determine if they should be. When such risk assessments are done, it is important that careful attention is played to the emerging clinical phenotypes associated with HEV, including neurological syndromes. Thanks to validated serological assays with good sensitivity and specificity [44,45] , direct comparisons of seroprevalence between populations in distinct areas can now be made. The question of concordance between different assays has been recently addressed [46] . Using the same assay, IgG seroprevalence in blood donors is 52% in southwestern France [36] , 29% in Germany, 27% in The Netherlands, 16% in southwestern England, 12% in the rest of England and 4.6% in Scotland [30] . In most of these studies, a gradual increase of IgG seroprevalence with the age of individuals has been observed suggesting a cumulative exposure to HEV. Estimates of incidence of HEV infections based on IgM seroprevalence, IgG seroconversion or HEV RNA detection during prospective studies are 0.2% in the UK [47] , 0.7% in the USA [48] , 2–3% in the south of France [49] and 4.3% in China [50] . Clinical aspects of HEV: acute infection ●●Developing countries
Hepatitis E is hyperendemic in many developing countries, particularly in southeast Asia and sub-Saharan Africa. In such locations, hepatitis E is caused by HEV1 and 2, which are obligate human pathogens. Hepatitis E mainly affects young adults, who generally have a self-limiting hepatitis illness lasting a few weeks. However, in pregnant females the mortality is approximately 20%, with an associated excess perinatal mortality [51] . In patients with underlying chronic liver disease who develop hepatitis E, the mortality is also high, and approaches 70% [52] . Hepatitis E occurs as sporadic cases and, more dramatically, in large outbreaks which may involve thousands or tens of thousands of cases. The most recent such outbreak was the one in the refugee camps of South Sudan with 5080 cases and 101 deaths, mainly in pregnant women. A recent estimate in 9 of 21 Global Burden of Disease regions
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in developing countries suggests that there are 3.4 million symptomatic cases of hepatitis E each year, with 70,000 deaths, and 3000 stillbirths [53] . This is probably an underestimate, as there are 1000 HEV-related maternal deaths per annum, in Bangladesh alone [54] . ●●Developed countries
Death in pregnant females appears to be a unique ‘signature’ of waterborne HEV1 and 2. Descriptions of outbreaks of jaundice and deaths in pregnant women suggest that 100 to 200 years ago HEV was present in a number of countries including the USA, Germany, France, Italy, the Balkan nations [55] . The first description of such an outbreak of jaundice and deaths in pregnant women was in Germany in 1794. There were 70 cases, including three pregnant women, two of whom died. Over the last 50 years, there have been no further outbreaks of jaundice associated with deaths in pregnant women in Europe, but they started to appear in Asia and Africa. One explanation for these observations is that improvement in sanitary infrastructure in Europe asserted negative ecological pressure on HEV1 and 2, leaving HEV3 ‘hidden’ in its primary host, the pig [55] . For over 20 years following its discovery, HEV was thought to cause human infection only in hyperendemic developing countries. Thus, in developed countries, HEV was only considered a diagnostic possibility in travelers returning from hyperendemic countries. It is now clear that this notion was mistaken, as locally acquired zoonotic acute hepatitis E has been found in every single developed county in which it has been sought [51] . A clear, consistent and striking observation about such cases is the demography of patients affected, who are more likely to be older (median age: 63 years) males (male:female ratio 3:1) [56] . The reason why acute hepatitis E has a predilection for older men is uncertain. It is possible that older males might have increased exposure to HEV at higher viral loads. Another explanation relates to host factors, as clinically apparent hepatitis E appears to be more common in patients who drink excessive alcohol or who are diabetic. Such patients are more likely to have subclinical hepatitis fibrosis/steatosis, and maybe such patients are more likely to have a clinically apparent hepatitis when exposed to HEV [57] . Most patients with locally acquired acute hepatitis E in developed countries have no symptoms [51] . After an incubation period of
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Hepatitis E in developed countries: current status & future perspectives 2–6 weeks, the minority of patients who become symptomatic usually develop symptoms indistinguishable from other causes of viral hepatitis (Box 1) . Approximately 60% are jaundiced and the ALT is usually in the 1000–2000 IUL range, but the range is wide, and symptomatic patients may have a much more modest transaminitis. Most patients have an illness that lasts 4–6 weeks, followed by complete recovery [58] . Occasionally, the hepatitis is more severe and of longer duration. Patients with underlying chronic liver disease may develop acute or subacute liver failure. Untreated, such patients have a high mortality [59] . The differential diagnosis of acute hepatitis E infection is shown in Table 1. One common difficulty in diagnosis of hepatitis E is mistaking it for drug-induced liver injury. The latter is common in the elderly and is defined by a temporal relationship between starting a drug and developing hepatitis; a temporal relationship between stopping the offending drug and resolution of hepatitis; and, most importantly, excluding all other causes of hepatocellular injury. In a study from southwest England, 13% of patients with ‘criterion-referenced’ drug-induced liver injury were misdiagnosed, as on retrospective testing they had locally acquired hepatitis E. The bottom line of this study is that the diagnosis of drug-induced liver injury is not secure without first excluding hepatitis E [61] . As can been seen from Table 1, locally acquired acute hepatitis E is the commonest cause of viral hepatitis in the UK [62] , and probably many other developed countries too. Commonly used diagnostic testing algorithms for acute hepatitis currently used in Europe suggest that patients with hepatitis should first be tested for hepatitis A virus, hepatitis B virus and hepatitis C virus, and if these tests are negative (particularly if the patient has travelled to an area traditionally thought of as endemic) then the patient
Review
should be considered for HEV testing. Given that locally acquired acute hepatitis E is the commonest cause of hepatitis, it would make much more sense to flip existing algorithms on their head and test all patients with hepatitis for HEV, and if this is negative then test for the other causes of viral hepatitis [63] . The travel history is irrelevant, and should be excluded from future testing algorithms. In Europe zoonotic locally acquired acute hepatitis E is caused by HEV3, in Japan by HEV3 and 4 and in China by HEV4 [51] . HEV4 has recently been found in pigs in Europe [64] and there have been clusters of human cases of hepatitis E caused by HEV4 in France, Italy and Denmark [65,66] . HEV4 infections are also zoonotic and produce a similar illness to HEV3 in older men. However, it is unknown if HEV4 can produce neurological injury or chronic infection (see below). Clinical aspects of HEV: chronic infection During the few last years, it has been clearly demonstrated that HEV3 infection can cause chronic hepatitis leading to cirrhosis [30,67–68] . Chronic HEV infection was described initially in solid-organ-transplant (SOT) patients, and thereafter in pediatric SOT patients, in patients infected by the human immunodeficiency virus (HIV), in patients having a hematological disease and receiving chemotherapy and in stem-celltransplant patients [30,51] . No chronic cases have been reported in patients infected by HEV1, 2 or 4. Chronic hepatitis has previously been defined as HEV replication in the serum and/or in the stools persisting for at least 6 months. However, in SOT patients, it has recently been shown that no spontaneous HEV clearance occurs between months 3 and 6 after infection, suggesting that chronic HEV infection may be more accurately defined as persisting HEV replication for at least 3 months [69] .
Box 1. Symptoms of acute hepatitis E. Common
Less common
No symptoms Jaundice Anorexia Lethargy Abdominal pain Vomiting
Myalgia Pruritis Weight loss Fever Arthralgia Neurological†
A total of 5–8% of patients present with a neurological illness. In such patients, the neurological symptoms and signs dominate the clinical picture and the liver function tests are often only modestly abnormal (see section ‘Extrahepatic manifestations of HEV’). Data taken from [58,60]. †
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Review Dalton, Kamar & Izopet Table 1. The differential diagnosis of a raised alanine aminotransferase. Diagnosis
Notes
Drug-induced hepatitis
Occurs mainly in the elderly. Other causes of hepatitis need to be excluded, including HEV Most common in females >60 years This may be caused by an, as yet, unidentified hepatotropic virus Middle aged/elderly males Median ALT: 1400 IUL, wide range Occurs at any age. Splenomegaly and lymphocytosis are found in 95% which suggests the diagnosis ALT >3000 IUL, usually associated with a period of hypotension ALT usually >3000 IUL Becoming increasingly rare in most developed countries
Autoimmune hepatitis Seronegative hepatitis† Acute HEV EBV hepatitis Ischemic hepatitis Acute HBV HAV
Defined as ALT >500 UL with exclusion of all the other causes of hepatitis. The diagnoses are placed in the order of frequency in which they occur in >2000 consecutive patients presenting with jaundice to a rapid access jaundice clinic in southwest England. ALT: Alanine aminotransferase; EBV: Epstein–Barr virus; HAV: Hepatitis A virus; HBV: Hepatitis B virus; HEV: Hepatitis E virus. Data taken from [61,62]. †
The natural history of HEV infection has been well documented in SOT patients. In a large retrospective multicenter study, nearly 60% of SOT patients infected by HEV develop chronic infection, and nearly 10% develop cirrhosis [70] . In contrast to immunocompetent patients, liver enzyme levels are modestly elevated (≈350 IU/l). However, the progression of liver fibrosis is very rapid leading to cirrhosis within 2–3 years after infection in some patients [71] . There appears to be no correlation between HEV viral load and liver fibrosis progression [38] . In untreated patients, there is no significant difference in HEV viral load in the acute and chronic phases of infection [38] . In SOT patients, chronic hepatitis occurs mainly in the context of more profound immunosuppression. Chronic HEV infection is more common in patients with low CD3 positive lymphocyte counts and lower specific HEV-T-cell responses. SOT patients receiving tacrolimus compared with cyclosporine A, and those with a lower platelet count during the acute phase of infection are also at higher risk of developing chronic infection [67,70,72] . Patients who develop chronic HEV infection have lower serum concentrations of IL-1 receptor antagonist and IL-2 receptor during the acute phase of infection, compared with those with resolving hepatitis [73] . In addition, increased quasispecies heterogeneity in the M and P domains and the polyproline region is seen in patients with chronic infection, compared with those with resolving hepatitis [73,74] . Finally, patients with higher ALT levels during
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the acute phase of infection are more likely to have a spontaneous HEV clearance [70] . HEV reinfection has been observed in anti-HEV IgGpositive transplant recipients with an anti-HEV IgG concentration
