Hepatitis E Virus and Related Viruses in Animals.

Hepatitis E is an acute human liver disease in healthy individuals which may eventually become chronic. It is caused by the hepatitis E virus (HEV) and can have a zoonotic origin. Nearly 57,000 people die yearly from hepatitis E-related conditions. The disease is endemic in both developing and developed countries with distinct epidemiologic profiles. In developing countries, the disease is associated with inadequate water treatment, while in developed countries, transmission is associated with animal contact and the ingestion of raw or uncooked meat, especially liver. All human HEV are grouped into at least four genotypes, while HEV or HEV-related viruses have been identified in an increasing number of domestic and wild animal species. Despite a high genetic diversity, only one single HEV serotype has been described to date for HEV genotypes 1-4. The discovery of new HEV or HEV-related viruses leads to a continuing increase in the number of genotypes. In addition, the genome organization of all these viruses is variable with overlapping open reading frames (ORF) and differences in the location of ORF3. In spite of the role of some domestic and wild animals as reservoir, the origin of HEV and HEV-related viruses in humans and animals is still unclear. This review discusses aspects of the detection, molecular virology, zoonotic transmission and origin of HEV and HEV-related viruses in the context of 'One Health' and establishes a link between the previous and the new taxonomy of this growing virus family.

Primary structure of open reading frame 2 and 3 of the hepatitis E virus isolated from Morocco.

The nucleotide sequence from position 5,014 to 7,186 of the hepatitis E virus (HEV) genome was determined using a set of 10 polymerase chain reaction (PCR) fragments amplified directly from a pool of fecal specimens obtained from patients with well-documented epidemic HEV infection in Morocco. This sequence contains the 3'-terminal region of open reading frame 1 (ORF1), full length ORF2 and ORF3, and a portion of the 3'-noncoding region. The HEV Morocco nucleotide sequence was compared with the corresponding sequences of 13 HEV strains. A region of ORF2 that overlaps with ORF3 was found to be the most conserved region of ORF2, whereas a protein segment encoded by this region was found to be the most variable. Theoretical RNA secondary structure analysis predicted that this region may be folded into a strong secondary structure that may constrain nucleotide sequence variability. In addition, the nucleotide sequence comparison revealed that the HEV Morocco sequence is most homologous to the sequences of the HEV Asian strains compared with the HEV Mexico, swine, and US strains. Phylogenetic analysis performed on the entire ORF2 and ORF3 sequences and on a small fragment of ORF2 allowed classification of the HEV Morocco strain together with a few other known African strains as a separate subtype within the Asian-African genotype.

Hepatitis E: an overview.

Hepatitis E is an enterically transmitted, acute, self-limited, icteric viral disease that occurs in large numbers in countries of the Indian subcontinent, Asia, and Africa. The frequency of epidemics and the high mortality rate among infected pregnant women are strong indicators that hepatitis E is an important cause of morbidity and mortality in humans. Several isolates of hepatitis E virus (HEV) derived from infected humans and experimental animals have recently been cloned and sequenced, allowing investigators to determine the molecular structure of the HEV genome. Laboratory diagnosis of HEV infection is done by detection of HEV antibodies, HEV RNA in stool and serum samples, HEV particles in stool specimens, and HEV antigen in hepatocytes and stool specimens. The detection of anti-HEV by enzyme immunoassay, with the use of several recombinant HEV proteins or synthetic peptides, is the most frequently applied method for the diagnosis of the infection and characterization of its epidemiologic features. Laboratory determination of HEV replication, immune response, and liver pathologic features in patients with hepatitis E and in infected primates has facilitated studies of the disease. Preventive measures against HEV infection include the passive transfer of protective antibodies or active immunization. In efforts to develop HEV vaccines, various recombinant proteins have been used. Although a range of protective immune responses have been induced in primates, further modifications of immunogen, adjuvant, and immunization schedules are necessary to prevent HEV infection. Much remains to be learned about epidemiology of HEV infection, reservoir(s) of the virus, and protective immunity in order to develop effective strategies to prevent hepatitis E.

Hepatitis E.

Hepatitis E has a world-wide distribution and causes substantial morbidity and mortality in some developing countries, particularly among pregnant women. Hepatitis E virus (HEV) has recently been cloned and sequenced, and new diagnostic tests have been developed. These tests have been used to begin to characterize the natural history and epidemiological features of HEV infection. Experimental vaccines have also been developed that offer the potential to prevent hepatitis E. However, much remains to be learned about HEV, including the mechanisms of transmission, the reservoir(s) of the virus, and the natural history of protective immunity in order to develop effective strategies to prevent this disease.

Viral specificity of hepatitis E virus antigens identified by fluorescent antibody assay using recombinant HEV proteins.

A fluorescent antibody (FA) assay for hepatitis E virus antigen (HEVAg) in infected liver tissue was used to confirm the presence of virus-specific antigens in hepatocytes during the course of infection. With the cloning of the HEV genome it is now possible to determine which viral antigens are recognized by this FA assay. Recombinant HEV proteins covering the carboxyl half of HEV open reading frame 2 (ORF2) were used in this study to demonstrate that some of the most immunoreactive virus-specific antigens detected by FA are contained within this region of ORF2 (nucleotides 6169-7126).

Hepatitis E.

With the introduction of new, albeit noncommercial, diagnostic tests for the detection of HEV, our understanding of this virus has been greatly enhanced. With these tests, it has been possible to begin a delineation of the process of pathogenesis in infected humans. These tests have also shown us that, at least in some individuals, an IgG response to HEV is long lasting and may confer immunity, and it may be possible to develop a recombinant vaccine against HEV. Contrary to past results, HEV appears to be the most common cause of acute sporadic hepatitis in pediatric populations in endemic regions. HEV has been shown to be a positive-sense single-stranded RNA virus that has been tentatively classified as a calicivirus, although its nonstructural proteins exhibit sequence similarity to rubella virus and furoviruses. Despite these advances in our understanding of HEV, it is still necessary to depend on exclusion of hepatitis caused by other viruses to diagnose a hepatitis E infection; however, this situation should change with the anticipated release of commercially available diagnostic tests.

Preliminary evidence that a trpE-HEV fusion protein protects cynomolgus macaques against challenge with wild-type hepatitis E virus (HEV).

Immunization of two cynomolgus macaques (cynos) with trpE-C2 protein, a trpE-HEV fusion protein that represents the carboxyl two thirds of the putative capsid protein, prevented development of biochemical evidence of viral hepatitis in these primates after challenge by wild-type HEV from either a Burmese or Mexican stool isolate. Neither of the immunized animals showed any elevation of alanine aminotransferase activity after challenge with wild-type HEV in marked contrast with the unimmunized (control) cynos. In the case of the Burmese HEV challenged cyno, the protective effect was complete with the animal failing to demonstrate any evidence of HEV infection. The immunized cyno challenged with Burmese HEV did not exhibit any HEV RNA in its stools or HEV antigen in its liver. The immunized cyno (#8902) challenged with Mexican virus exhibited HEV RNA in its stools and HEV antigen in its liver; however, microscopic examination of liver biopsy specimens from this cyno failed to detect histopathologic evidence of viral hepatitis. All of the animals (naive and immunized) developed anti-HEV IgM and IgG responses after HEV challenge. Our preliminary studies indicate that the trpE-C2 protein is a promising candidate HEV vaccine.

The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China.

In this study an IgM antibody-mediated antigen-capture procedure for direct extraction of hepatitis E virus (HEV) RNA from clinical specimens was developed and used with an efficient method for generating viral cDNA that was subsequently sequenced using the dideoxy chain termination method. This is the first time the complete HEV genome has been isolated directly from a single human clinical specimen obtained during an outbreak of enterically transmitted non-A, non-B hepatitis. When the Chinese-derived sequence was compared with the original isolate of Burmese HEV from an experimentally infected cynomolgus macaque, the homology between the two sequences was 94% and 98.5% at the nucleotide and amino acid levels, respectively. The methods we developed for generating and sequencing genomic HEV cDNA dramatically improved the efficiency of cloning the viral genome and should be helpful for continued analysis of this virus as well as other RNA viruses that have proven to be difficult to clone and sequence directly.

Acute sporadic hepatitis E in an Egyptian pediatric population.

A study was conducted to determine the etiology of acute hepatitis among 261 children (age range 1-11 years) living in Cairo, Egypt. A blood sample was obtained from each subject when initially evaluated and a questionnaire was used to collect demographic and risk factor data. Sera were tested by enzyme immunoassay for acute hepatitis A (anti-hepatitis A virus IgM), hepatitis B (anti-hepatitis B core antigen IgM and hepatitis B surface antigen [HBsAg]), hepatitis C (total anti-HCV), delta hepatitis (total anti-delta), and cytomegalovirus infection (anti-CMV IgM). In addition, hepatitis E virus (HEV) infection was diagnosed using a new Western blot technique to test patients with non-A, non-B hepatitis for anti-HEV IgM. Among 261 children, acute hepatitis A was diagnosed in 85 (32.6%) patients, acute hepatitis B in 19 (7.3%), delta hepatitis in 3 (1.1%), mixed hepatitis A and B infection in 2 (0.8%), CMV infection in 1 (0.4%), hepatitis E in 58 (22.2%), and non-A, non-B hepatitis of unknown type in 51 (19.5%). Forty-two (16.1%) subjects had HBsAg without other markers of acute infection. Risk factor analysis indicated that patients living in homes not connected to a municipal source of water were at increased risk of hepatitis E infection. These data provide additional evidence that hepatitis E virus is a common cause of acute sporadic hepatitis in children living in Egypt.

Molecular characterization of hepatitis C and E viruses.

The molecular features of each of the major viruses of non-A, non-B hepatitis, namely hepatitis C virus (HCV) and hepatitis E virus (HEV) are briefly described. The organization of the genome of each of these viruses is discussed and compared to those of other related or distantly related viruses that contain single-stranded, positive-sense RNA genomes. HCV has been tentatively classified as a separate genus within the Flaviviridae, whereas HEV has been loosely associated with caliciviruses and subsequently assigned to the Caliciviridae, although it does possess unique genetic features not found in other caliciviruses.

Molecular organization and replication of hepatitis E virus (HEV).

The recently characterized fecal-orally transmitted agent of hepatitis E (formerly known as enterically transmitted non-A, non-B hepatitis) has been determined to be a new type of positive strand RNA virus. The complete sequencing of four different geographic isolates of the hepatitis E virus (HEV) has confirmed a similar genetic organization not previously recognized in nonenveloped positive strand RNA viruses. The approximately 7.5 kb RNA genome (including polyA tail) has nonstructural genes located at the 5' end and structural genes at the 3' end. Expression of these viral genes occurs in at least 3 different forward open reading frames. The largest open reading frame begins 27 nucleotides (nt) downstream of the apparent noncoding 5' end and extends 5,079 nt. Multiple nonstructural gene motifs/domains have been recognized in this 5' ORF1 including a methyltransferase, a papain-like protease, a helicase and the RNA-dependent, RNA polymerase. The second major ORF2 begins 37nt downstream of ORF1 and extends 1980 nt before terminating 65 nt upstream of the polyadenylation site. A third ORF of only 369 nt was identified by immunoscreening experiments as encoding an immunogenic epitope of the virus. Expression of the downstream ORF2 may occur through internal subgenomic RNA initiation at a sequence element found to have homology to internal RNA initiation sequences in Sindbis virus. This element in the HEV genome maps near the apparent 5' end of one of two identified subgenomic messages. The genomic organization and expression of HEV will be discussed and a hypothesis presented regarding the viral replication strategy.

Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses.

Computer-assisted comparison of the nonstructural polyprotein of hepatitis E virus (HEV) with proteins of other positive-strand RNA viruses allowed the identification of the following putative functional domains: (i) RNA-dependent RNA polymerase, (ii) RNA helicase, (iii) methyltransferase, (iv) a domain of unknown function ("X" domain) flanking the papain-like protease domains in the polyproteins of animal positive-strand RNA viruses, and (v) papain-like cysteine protease domain distantly related to the putative papain-like protease of rubella virus (RubV). Comparative analysis of the polymerase and helicase sequences of positive-strand RNA viruses belonging to the so-called "alpha-like" supergroup revealed grouping between HEV, RubV, and beet necrotic yellow vein virus (BNYVV), a plant furovirus. Two additional domains have been identified: one showed significant conservation between HEV, RubV, and BNYVV, and the other showed conservation specifically between HEV and RubV. The large nonstructural proteins of HEV, RubV, and BNYVV retained similar domain organization, with the exceptions of relocation of the putative protease domain in HEV as compared to RubV and the absence of the protease and X domains in BNYVV. These observations show that HEV, RubV, and BNYVV encompass partially conserved arrays of distinctive putative functional domains, suggesting that these viruses constitute a distinct monophyletic group within the alpha-like supergroup of positive-strand RNA viruses.

Acute sporadic hepatitis E in children living in Cairo, Egypt.

Seventy-three pediatric patients with acute hepatitis and 19 control patients without liver disease living in Cairo, Egypt, were evaluated with a newly developed Western blot assay for IgM antibody to hepatitis E virus (IgM anti-HEV). The mean age of acute hepatitis patients was 6.4 years (range, 1-13 years); 56% were male. Among the 73 acute cases, hepatitis A was diagnosed in 30 (41%), possible acute hepatitis B in three (4%), hepatitis E in nine (12%), and by exclusion, non-A, non-B hepatitis in 29 (40%). Two additional acute cases were positive for both IgM anti-HAV and IgM anti-HEV. None of the 19 control subjects had IgM anti-HEV. Parenteral risk factors were associated with cases of non-A, non-B hepatitis but were not associated with acute hepatitis E. Contact with a family member with jaundice was associated with acute hepatitis A. In contrast to prior epidemics of enterically-transmitted non-A, non-B hepatitis, HEV was found to be a common cause of acute hepatitis in a pediatric population. This study provides additional evidence that HEV may be a frequent cause of acute sporadic hepatitis among children living in some developing countries.

Recent developments in the molecular cloning and characterization of hepatitis C and E viruses.

The molecular properties of the genomes of both hepatitis C virus (HCV) and hepatitis E virus (HEV), the major etiologic agents of non-A, non-B hepatitis, are briefly described. The organization of the genome of each of these viruses is discussed and compared to those of other related or distantly related viruses that contain single-stranded, positive-sense RNA genomes. In the case of HEV, the reactivity of expressed proteins and genetic divergence of geographically distinct isolates are also described within the context of sequence variation, type-common epitopes and type-specific epitopes.

Acute sporadic hepatitis E in Sudanese children: analysis based on a new western blot assay.

A newly developed Western blot assay for antibody to hepatitis E virus (anti-HEV) was used to evaluate 39 cases of acute pediatric hepatitis and 39 control patients in Khartoum, Sudan. The mean age of cases was 6.5 years (range, 2-14); 64% were male. Acute hepatitis A (IgM anti-HAV-positive) was diagnosed in 13 cases, acute hepatitis B (IgM anti-HBc-positive) in 1, and acute hepatitis E (positive for IgM anti-HEV) in 23 (59%). None of the cases with IgM anti-HAV or IgM anti-HBc had IgM anti-HEV; 3 controls had IgM anti-HEV. Acute hepatitis E was associated with recent contact with a family member or acquaintance with jaundice and the presence of indoor plumbing. The newly developed hepatitis E assay appeared to be specific for the diagnosis of acute icteric non-A, non-B hepatitis. Hepatitis E was found to be the most common cause of acute sporadic hepatitis in children living in an urban area of Africa.

Human linear B-cell epitopes encoded by the hepatitis E virus include determinants in the RNA-dependent RNA polymerase.

Hepatitis E virus is responsible for both sporadic and epidemic hepatitis in developing countries. The nonenveloped virus is 27-34 nm in diameter and has been shown to contain a single-strand, positive-sense, polyadenylylated RNA genome of approximately 7.5 kilobases. The nucleotide sequence of the Burma strain of hepatitis E virus has been reported and three open reading frames (ORFs) have been identified. The deduced amino acid sequence from each of these ORFs was used to synthesize overlapping peptides (decamers overlapping at every fourth amino acid) on a solid phase. These peptides were then tested in an ELISA with pooled acute-phase sera from known cases of enterically transmitted non-A, non-B hepatitis collected in the Sudan. Linear B-cell epitopes were identified in all three ORFs. Epitopes were identified throughout the polyprotein encoded by ORF1, but they appeared to be particularly concentrated in the region of the RNA-dependent RNA polymerase. Distinct epitopes were identified in the presumed structural protein encoded by ORF2, and one epitope was identified close to the carboxyl terminus of the protein encoded by ORF3. These data precisely pinpoint linear B-cell epitopes recognized by antibodies from patients with acute hepatitis E and identify an antibody response directed against the RNA-dependent RNA polymerase.

Hepatitis E virus (HEV): strain variation in the nonstructural gene region encoding consensus motifs for an RNA-dependent RNA polymerase and an ATP/GTP binding site.

Hepatitis is transmitted by a number of infectious agents. The epidemiological characterization of waterborne or enterically transmitted non-A, non-B hepatitis (ET-NANBH) is unique when compared with other known hepatitides. We have reported on the molecular cloning of a cDNA clone derived from the etiologic agent associated with ET-NANBH, the hepatitis E virus (HEV). The complete sequence of these first molecular clones, isolated from an HEV-infected human after passage in Macaca fascicularis (cynomolgus macaques), illustrates a distant relationship to other known positive-strand RNA viruses of plants and animals. The translated major open reading frame (ORF-1) from these clones indicates that this portion of the genome encodes a polyprotein with consensus sequences found in RNA-dependent RNA polymerase and ATP/GTP binding domains. The latter activity has been associated with putative helicases of positive-strand RNA viruses. These viral-encoded enzymatic activities identify this region and ORF-1 as containing at least two different nonstructural genes involved in HEV replication. Molecular clones obtained from two other geographically distinct HEV isolates demonstrated sequence heterogeneity in this nonstructural gene region. Further study will be required to elucidate the pathogenic significance (if any) of this observed divergence in the nonstructural region.

Serologic identification of hepatitis E virus infections in epidemic and endemic settings.

Recombinant chimeric protein C2 containing the N-terminal region of trpE (37 kilodaltons [kDa]) and the C-terminal half (46.8 kDa) of the polypeptide encoded by ORF2 of the hepatitis E virus (HEV) genome was used for the construction of a Western blot diagnostic test for IgG and IgM antibodies to the virus (anti-HEV). (The C2 protein and the trpE protein devoid of C2 activity and used as a control for non-specific reactions were purified by recovery from sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] and used for preparation of strips). Specificity of the test was proven with sera obtained from patients with acute hepatitis non-A, non-B, non-C (NANBNC) from outbreaks in different geographic regions of the world. IgG antibodies reactive to the recombinant C2 protein were detected in 93% of patients with acute hepatitis NANBNC and remained detectable in 89-100% of these patients 1-24 months after onset of jaundice. IgM antibodies were detected in 73% of patients within 26 days after onset of jaundice, in 50% 1-4 months after onset, in 6% 6-7 months after onset, and in no patients by 8 months after onset. When this test was used to identify sporadic hepatitis E cases in different regions of the world, such cases were found almost exclusively in areas where outbreaks of the disease had occurred and rarely in any other regions.