Hepatitis E Virus Isolates Research Articles

Phylogenetic analysis of hepatitis E virus isolates from India (1976-1993).

Seventeen Indian hepatitis E virus (HEV) isolates, representing epidemic and sporadic hepatitis E cases during 1976-1991, were sequenced in the RNA polymerase (RNAP) region. Five isolates were also sequenced in the non-structural hypervariable region of open reading frame 1. Open reading frames 2 and 3 were sequenced only for the prototype isolate. On the basis of the comparison of all the available sequences of the conserved RNAP region, the HEV isolates were divided into three genotypes, differing from each other by >15%. Genotype I included African and Asian isolates, whereas II and III were represented by Mexican and US isolates, respectively. Genotype I was further divided into four sub-genotypes. The majority of the Indian isolates (15/20), along with the Burmese and Nepali isolates, belonged to genotype IA. Genotype IB included HEV isolates from China, Pakistan and the former USSR and 2/20 Indian isolates, which represented the oldest (1976) HEV sequenced so far. Genotype IC included both the African isolates, whereas 3/20 Indian isolates formed genotype ID. Nucleotide sequence analysis of other regions of the HEV genome also placed isolates in the same genotypes. Both the Indian cities experiencing second HEV epidemics, after intervals of 8 and 10 years, showed shifts in the sub-genotypes found; from IB (Ahm-76) to IA (Ahm-84) and from IA (Kol-81) to ID (Kol-91). However, no major shift in the genotypes was noted. Overall, HEV genotypes appear to be segregated geographically.

Identification of a novel variant of hepatitis E virus in Italy.

Hepatitis E infection is typically associated with areas in which hepatitis E virus (HEV) is endemic. Except for a few cases in Europe and in the United States, acute hepatitis E is usually associated with travel to endemic areas. We set out to determine the etiologic role of HEV in acute non-A-C hepatitis in Italy. The presence of HEV-RNA and antibody was determined in 218 patients diagnosed with acute viral non-A-C hepatitis. Acute hepatitis E infection was defined by the presence of HEV-RNA in sera and positivity for IgM anti-HEV and seroconversion to IgG anti-HEV. Acute hepatitis E was found in 10.1% of the patients with acute non-A-C, with 95.5% exhibiting a benign course. A more severe course was observed in a patient co-infected with HAV and HEV. Most cases were travelers to endemic areas, although 18.2% reported no travel. One patient was from a household with an infected patient. Sequence analyses of the polymerase chain reaction (PCR) product derived from a patient who never visited endemic areas, identified an isolate that is divergent significantly from all reported isolates of HEV (79.5-85.8% nucleotide identity). Evidence from this study suggests that HEV accounts for approximately 10% of acute non-A-C viral hepatitis in Italy, diagnosed generally in travelers returning from endemic areas. However, the identification of a new HEV variant in an individual who never indicated travel or contact with individuals associated with endemic areas, suggests that this virus may be native to Italy.

Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV.

Hepatitis E infection is associated with areas in which hepatitis E virus (HEV) infection is endemic. Acute infections in industrialized nations are usually linked to travel to endemic areas. Recently, an acute hepatitis infection in a patient from the United States (US), with no recent foreign travel history, was linked to a novel strain of HEV. Although a few additional cases have been reported from patients who have not traveled to endemic areas, the source of these infections has not been determined. The objective of this study was to identify additional HEV isolates from patients with acute infection who had no recent history of travel to areas where HEV is considered endemic, and to determine the genetic relationship between these and other HEV isolates. Viral RNA was isolated from serum and polymerase chain reaction (PCR) was performed using consensus primers based on a number of HEV isolates. HEV sequence in open reading frame (ORF) 1 and ORF2 was identified in three patients from nonendemic areas, one from Italy and two from Greece. Comparative and phylogenetic analyses were performed. The Greek and Italian isolates were significantly divergent from two isolates from the US and isolates identified previously from HEV-endemic regions. The Italian isolate was distinct from the two Greek isolates. In addition, the two Greek isolates were significantly divergent from each other. Phylogenetic analysis indicated that the Italian and two Greek isolates represent three new genotypes of HEV, distinct from the Burmese, Mexican, and US genotypes.

Phylogenetic analysis of hepatitis E virus isolates from Egypt.

Hepatitis E virus (HEV) genome was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in fecal samples of two sporadic cases of hepatitis E in Cairo Egypt. Sequence of the complete putative structural region [open reading frame (ORF)-2] and complete region of unknown function (ORF-3) was determined for the two HEV isolates. Phylogenetic analysis of the nucleotide sequences was performed using neighbor joining or maximum parsimony methods of tree reconstruction. Direct correspondence between the HEV evolutionary trees and geographic origin of the HEV isolates was observed. Three genotypes of HEV were identified: genotype I (Asia-Africa), genotype II (US), and genotype III (Mexico). Genotype I was further divided into two subgenotypes (Asia and Africa). In the Asian subgenotype, three smaller genetic clusters were observed (China-like sequences, Burma-like sequences, and sequence from a fulminant case of HEV). The segregation of all these genetic clusters was supported by the high level of bootstrap probabilities. Four regions of the HEV genome were used for phylogenetic analysis. In all four regions, Egyptian HEV isolates were grouped in a separate African clade.

A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis.

Recent studies have reported and provided nucleotide sequence data from divergent isolates of hepatitis E virus (HEV), including isolates from North America and Africa. Sera were investigated from 29 Chinese patients with a diagnosis of acute hepatitis and who were negative for hepatitis viruses A-E by serology (HEV was excluded by testing for IgG antibody only). To determine whether some patients were infected with HEV but had yet to seroconvert to antibody positivity, RT-PCR was carried out with primers designed within conserved sequences of the HEV open reading frame (ORF) 1 and ORF2 regions. Fifteen patients were found to harbour sequences related to HEV. Analysis of the HEV products revealed that nucleotide sequences from nine of the sera closely matched Burmese-like HEV sequences (more than 92% nucleotide identity across ORF1 and 88% in ORF2). The remaining six HEV isolates were similar to each other but divergent from all other known HEV sequences (74 to 83% nucleotide identity in ORF1 or ORF2). Phylogenetic analysis suggests that the six divergent isolates represent a fourth genotype of HEV, distinct from the previously described Burmese, Mexican and United States variants (genotypes 1, 2 and 3). This novel variant, referred to here as the Chinese genotype (genotype 4), may be responsible for a significant proportion of cases of acute hepatitis in China, as seen by the fact that 40% of the HEV-infected patients in this study were genotype 4 positive.

The impact of traveling to endemic areas on the spread of hepatitis E virus infection: epidemiological and molecular analyses.

Traveling to endemic areas carries a risk of hepatitis E virus (HEV) infection, but no molecular analysis to document sources of infection is available. Eighteen (38%) of 47 patients with acute non-A, non-B, non-C hepatitis were positive for antibody to HEV (anti-HEV), and 9 (50%) of these were also positive for serum HEV RNA by polymerase chain reaction following reverse transcription. Only 1 (5%) of the 21 patients with acute hepatitis A was positive for HEV RNA. Travel to endemic areas (mostly to China; odds ratio, 22.2; 95% confidence interval, 4.7-105.8) and deeper jaundice (odds ratio, 5.2; 95% confidence interval, 1.01-27.2) were the only factors associated with HEV infection in multivariate analysis. The two HEV isolates from two patients who had traveled to China and the HEV isolate from a patient whose travel history was obscure formed a monophyletic group with the isolates from Guangzhou. The HEV isolates from our patients show a homology of 72% to 78% in nucleotide sequence with the Burma, Beijing, India, Pakistan, and Xiangjiang strains; a homology of 81% to 91% with the Guangzhou strains; and a homology of 76% with the Mexico strain. The close relationship between the Taiwan isolates and the Guangzhou strains was further supported by the short Kimura's two-parameter distances among them. In summary, HEV infection does occur in this area. Epidemiological and molecular analyses strongly indicate that most cases of HEV infection originated from travel to HEV-endemic areas.

The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States.

A variant of hepatitis E virus (HEV), designated HEV US-1, was identified in a hepatitis patient in the United States (US); the patient had no history of travel to areas where HEV is endemic. Nucleotide sequences were obtained from the 5' end of open reading frame (ORF) 1 (1418 nt), the 3' end of ORF1 (1359 nt), the entire ORF2 and ORF3 regions, and the 3'-untranslated region (2127 nt). The HEV US-1 strain is significantly divergent from other human HEV isolates with nucleotide identities ranging from 76.8 to 77.5%. Phylogenetic analyses indicate that HEV US-1 and a recently discovered HEV variant from swine may represent separate isolates of a new strain of HEV, significantly divergent from the Mexican and Burmese strains. Synthetic peptides derived from the carboxyl amino acids of ORF2 and ORF3 were shown to be useful for detecting exposure to HEV. In addition, IgM class antibodies directed against HEV US-1 synthetic peptides were detected in the US patient infected with HEV US-1, but were absent using synthetic peptides from the Burmese or Mexican strains of HEV. A preferential reactivity to HEV US-1 specific peptides has lead to the identification of a second isolate of this virus also from a patient with acute hepatitis from the US. The discovery of these HEV variants may be important in understanding the worldwide distribution of HEV infection.

Genotyping of hepatitis E virus in clinical specimens by restriction endonuclease analysis

The genomic variability of hepatitis E virus (HEV) was examined by restriction endonuclease analysis (REA) of four genomic cDNA copies comprising a 499 bp segment of the putative polymerase gene, a 264 bp segment of the helicase gene, and two, 680 bp and 448 bp, segments of the capsid gene. Analysis of the deduced restriction sites of all 27 HEV sequences currently available in the GenBank, and digestion of reverse-transcribed and nested PCR amplified segments obtained from six Nepali isolates were used to devise and test a REA genotyping assay. The assay allowed easy discrimination between the Mexico and Asian genotypes, and the classification of the Asian genotypes into three, or perhaps four subgenotypes. In addition, endonucleases identifiers of individual isolate or clusters of isolates were found. This assay permits rapid identification of a large number of HEV isolates directly from clinical specimens for studies on the molecular epidemiology and evolution of HEV.

Hepatitis E virus in Nepal: similarities with the Burmese and Indian variants

Hepatitis E has been the predominant type of acute hepatitis in Nepal both in adults and children, in sporadic and epidemic forms. We examined six hepatitis E virus (HEV) isolates obtained during an 8-year period, from 1987 to 1995, in the Kathmandu valley of Nepal. Analysis of portions of the putative helicase, polymerase and capsid genes demonstrated close genetic relatedness among themselves (>96.4% identity) and with the Burmese (>95.5%) and Indian (>95.3%) isolates, and less so with the African (>94.4%) and the Chinese (>91%) isolates within the Asian genotype. Phylogenetic analysis placed the Nepali isolates in the Burma-India evolutionary branch and showed that the oldest isolate, TK78/87 was more similar to the Burmese isolates whereas the most recent isolates were closer to the Indian ones. Assuming no frameshifts, the Nepali isolates showed high amino acid conservation, but also unique changes when compared to other HEV isolates. Amino acid residue 614 of the capsid protein was identified as a possible marker to distinguish the Burma-Nepal-India from the China-Central Asian Republics subgenotype, and the Mexico genotype.

Cross-challenge studies in rhesus monkeys employing different Indian isolates of hepatitis E virus.

The aim of this study was to determine if rhesus monkeys infected with one isolate of hepatitis E virus (HEV) were immune to subsequent challenge with other isolates of the virus. Three epidemic and one sporadic Indian HEV isolates were employed in the study. The interval between primary inoculation and challenge varied from 1 year and 6 months to 2 years and 9 months. Evidence of HEV infection was ascertained by rise in serum alanine transaminase (ALT) levels and/or seroconversion to antibodies to HEV (anti-HEV), and the presence of HEV-RNA in the bile or faeces of the infected monkeys. No evidence for multiplication of virus in monkeys challenged with different HEV isolates was obtained. These results show that immunity generated by one isolate of HEV protects against different isolates of hepatitis E virus.

Epidemiology and serologic diagnosis of hepatitis E

Hepatitis E occurs in both epidemic and sporadic forms (1). Epidemics have been reported in many Asian and African countries, and in Mexico. Sporadic cases have been identified in developed countries, among travellers from endemic areas. Since there are no simple diagnostic tests, the precise epidemiology of hepatitis E virus (HEV) infection remains unclear. Diagnosis of hepatitis E used to be based on clinical and epidemiological evidence of enterically transmitted non-A, non-B hepatitis (ET-NANBH). In the past few years, several diagnostic tests for hepatitis E have been developed: immune electron microscopy is used to detect aggregated virus-like particles in stool by means of serum taken during convalescence (2); and the fluorescent blocking antibody assay detects antibodies that react against HEV antigen in hepatocytes (3). These tests are, however, laborious, technically difficult, and expensive, so they are unsuitable for general diagnostic purposes. Recent cloning of HEV has allowed the development of serologic assays for the detection of antibodies to recombinant expressed HEV antigens (anti-HEV) (4). The HEV genome consists of a single-stranded, positive-sense RNA of approximately 7.5 kb. There are three open reading frames (ORF) (5). ORF 1 contains the RNA-dependent RNA polymerase and nucleoside triphosphate binding motifs. ORF 2 contains sequences that may code for virus capsid proteins. ORF 3 partially overlaps with both ORF 1 and ORF 2. Enzyme immunoassays (EIAs) using recombinant (E. coli or baculovirus) expressed HEV antigens from ORF 2 and ORF 3 of Burmese, Mexican, and Pakistani isolates to detect anti-HEV (IgM and IgG) have been developed (6-8). Studies using these assays showed that most epidemics of ET-NANBH were due to HEV. These studies also showed that EIAs using HEV antigens from one isolate can detect anti-HEV in sera from patients or primates infected with HEV strains from other geographical regions, confirming the previous finding (based on aggregation of virus-like particles in stools using convalescent sera from different geographical areas, and cross-challenge experiments in primates) that there is only one HEV serotype. Comparison of the sequences of HEV isolates from diverse countries also revealed a high degree of homology (5,9). Recent surveys in Hong Kong and Egypt, countries in which epidemics of ET-NANBH have not been reported, found that IgM anti-HEV could be detected in 6%-22% of patients with acute viral hepatitis (10-12), suggesting that HEV infection is more widespread than previously thought. In these countries, hepatitis A, another enterically transmitted viral hepatitis, is also common; thus, hepatitis E may be endemic and not necessarily imported. This is substantiated by the finding of IgG anti-HEV in 16% of healthy subjects in Hong Kong (10). Interestingly, both Egyptian studies were conducted in children (I 1,12), while previous studies on epidemics of ET-NANBH found that clinical hepatitis was rare among children. This discrepancy may be related to milder clinical illness in children (as with hepatitis A), and underdiagnosis in the absence of a diagnostic test. The incidence of hepatitis E in western countries was examined in two studies reported in this issue of the Journal. Buti et al. (13) studied 341 consecutive patients with acute viral hepatitis who attended their hospital in Barcelona. None of 64 patients who were negative for hepatitis A, B, C, and D markers that were tested for IgM and IgG anti-HEV by EIA at Genelabs was reactive. In the other study, Pham et al. (14) tested 65 French patients with

Molecular cloning and sequencing of the mexico isolate of hepatitis E virus (HEV)

Hepatitis E virus (HEV) is the major causative agent of hepatitis E or what was formerly known as enterically transmitted non-A, non-B hepatitis. The disease has a worldwide distribution but occurs principally in developing countries in any of three forms: large epidemics, smaller outbreaks, or sporadic infections. Genetic variation of different HEV strains was previously noted and it will be important to determine the extent to which this variation may pose problems in the diagnosis and treatment of HEV infection. To analyze differences at the genetic level between HEV(Mexico; M) and the previously characterized HEV(Burma; B) and HEV(Pakistan; P) isolates, overlapping cDNAs were cloned from samples obtained from an infected human and an experimentally inoculated cynomolgus macaque. These cDNA clones, representing the nearly complete (7185-bp) genome of HEV(M), confirmed an expression strategy for the virus that involves the use of 3 forward open reading frames (ORFs). The HEV(M) strain has an overall 76 and 77% nucleic acid identity with the HEV(B) strain and HEV(P) strain, respectively; however, the degree of sequence variation was not uniform throughout the viral genome. A hypervariable region was identified in ORF1 that exhibited a 58 and 54% nucleic acid sequence and 13% amino acid similarity with the Burma strain and the Pakistan strain, respectively. A large number of the nucleotide differences occurred at the third codon position, with the deduced amino acid sequences similarity of 83, 93, and 87% between HEV(M) and HEV(B) isolates in ORF1, ORF2, and ORF3, respectively, and with 84, 93, and 87% amino acid identities between HEV(M) and HEV(P) isolates in ORF1, ORF2, and ORF3, respectively. The nucleotide sequences derived from the highly conserved regions of HEV genome will be useful in developing polymerase chain reaction-based tests to confirm the viral infection. Knowledge of the extent of the sequence variation encountered with HEV will not only aid in the future development of diagnostic and vaccine reagents but also further our understanding of how HEV strain variation might impact the pathological outcome of infection.

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.

Hepatitis E virus genome

The strategy for molecular cloning of hepatitis E virus (HEV), the major etiologic agent of enterically-transmitted non-A, non-B hepatitis, is briefly described. The organization of the HEV genome is discussed and compared to those of two other vertebrate viruses that contain single-stranded, positive-sense, polyadenylated RNA genomes with three overlapping ORFs. Serologic cross-reactivity of expressed proteins and genetic divergence of HEV isolates are also described within the context of sequence variation, type-common epitopes, and type-specific epitopes.