Hepatitis E virus infection in chimpanzees: a retrospective analysis.

Different patterns of disease were observed among 11 chimpanzees who were inoculated intravenously with hepatitis E virus (HEV) positive fecal specimens from four different outbreaks (Nepal 1981, Uzbekistan 1981, Pakistan 1985, and Mexico 1986). Five chimpanzees had marginal or no liver enzyme elevations within 70 days of inoculation. Two of the chimpanzees had limited viremia, but did not produce detectable antibody. The four remaining chimpanzees had liver enzyme elevations, viral shedding, viremia, seroconversion to anti-HEV, and detectable HEV antigen in liver biopsy specimens. These results may reflect the range of infection patterns that develop in humans after natural exposure to the HEV.

Hepatitis E seroconversion in United States travelers abroad.

Sporadic cases of symptomatic hepatitis E virus (HEV) infection have been reported in United States travelers to developing countries, including Mexico and Pakistan. To evaluate the risk of exposure in United States travelers, 356 patients seen in our Travel Clinics were tested for antibodies to HEV before and 6 weeks after traveling. Samples obtained 6 months after traveling were available for 211 travelers. IgG and IgM antibodies to HEV were assayed with HEV ELISA diagnostic kits containing 3 recombinant antigens expressed in Escherichia coli representing immunodominant epitopes within open reading frames 2 and 3 of HEV. Nine patients were IgG seropositive in specimens obtained before travel. Four individuals seroconverted. In all 4 patients, IgG seroconversion was demonstrated in samples obtained at least 6 months after return. Samples obtained 6 weeks after return were seronegative for HEV in all 3 patients for whom such samples were available. Travel destinations were diverse: Thailand, China, Russia, and Peru. These data are consistent with an infection acquired while traveling. None of the seropositive subjects reported any symptoms of hepatitis before or after travel. In the absence of overt disease, these results imply that exposure to HEV resulted in subclinical infections.

Hepatitis E virus. Advances in HEV biology and HEV vaccine approaches.

Hepatitis E, previously known as enterically transmitted, enteric, or epidemic hepatitis, is a worldwide public health problem. The causative agent, the hepatitis E virus, is involved in epidemic, sporadic, and fulminant hepatitis cases worldwide. This review describes the advances in the biology of the hepatitis E virus and the progress made to develop simple and robust serologic assays for the diagnosis of HEV infection. Genomic sequence comparisons with a recently identified US isolate now suggests three genetic groups of HEV viruses. A highly conserved animal isolate found in pigs suggest the coexistence of animal and human isolates of HEV. The use of recombinant technology to develop an effective subunit vaccine capable of providing cross-protection for the most divergent HEV strains has been established and is reviewed.

Hepatitis E virus infection in eastern India.

Most cases of enterically transmitted non-A, non-B hepatitis in India have so far been attributed to hepatitis E virus (HEV) infection. Most of the documented studies of hepatitis have focused on the incidence of this disease in northern, western, and south central India. A small seroprevalence study was conducted in the eastern Indian city of Patna to assess the degree of HEV infection among acute sporadic hepatitis cases. Forty-two percent (24 of 57) of the cases of acute sporadic hepatitis were positive for anti-HEV antibodies. Absence of any serologic markers of hepatitis A, B, or E in 58% (33 of 57) of the cases with symptoms of acute hepatitis suggest that there may be as yet unidentified enterically transmitted viruses in this area.

Comparison of tests for antibody to hepatitis E virus.

The results of serologic tests for hepatitis E virus have varied widely from laboratory to laboratory, making interpretation of seroepidemiologic studies difficult. The present study compares serologic results with different antigens and tests developed in two laboratories for their ability to diagnose hepatitis E and measure antibody prevalence in a high risk population in Saudi Arabia. The results confirm that tests based upon open reading frame (ORF) 3 of HEV are of limited value for seroepidemiologic studies, whereas ORF2-based antigens have broad utility and yield data that are reproducible in more than one laboratory.

In vitro infection and replication of hepatitis E virus in primary cynomolgus macaque hepatocytes.

An in vitro model was developed to replicate hepatitis E virus (HEV) in normal primary cynomolgus macaque hepatocytes using a hormonally defined, serum-free medium formulation. Primary hepatocytes were infected in tissue culture following isolation by collagenase treatment of liver wedge biopsy material. Viral replication was monitored by a highly strand-specific reverse transcription-polymerase chain reaction (RT-PCR) assay, which could detect the positive- and negative-strands of HEV RNA independently in a sensitive and specific manner. Several infectious HEV (Burma strain) inocula were titered by this RT-PCR assay, and a minimum effective infectious dose was determined. Appearance of newly replicated virus was demonstrated by detection of both strands of HEV RNA in experimentally infected hepatocytes as well as the genomic positive-strand viral RNA in the culture medium. Infectivity of the virus particles present in the media was confirmed by serial passage and replication of the virus in culture. Using this in vitro infection system, a neutralization assay was developed to assess the ability of anti-HEV antibodies to block virus infection of liver cells. Results presented in this report represent the first in vitro demonstration of a neutralizing anti-HEV antibody directed against the ORF2-encoded putative capsid protein.

Expression, characterization, and immunoreactivities of a soluble hepatitis E virus putative capsid protein species expressed in insect cells.

The hepatitis E virus (HEV) open reading frame-2 (ORF-2) is predicted to encode a 71-kDa putative capsid protein involved in virus particle formation. When insect Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus containing the entire ORF-2 sequence, two types of recombinant proteins were produced; an insoluble protein of 73 kDa and a soluble protein of 62 kDa. The 62-kDa species was shown to be a proteolytic cleavage product of the 73-kDa protein. N-terminal sequence analysis of the 62-kDa protein indicated that it lacked the first 111 amino acids that are present in the full-length 73-kDa protein. A soluble 62-kDa protein was produced without the proteolytic processing by inserting the coding sequence of amino acids 112 to 660 of ORF-2 in a baculovirus expression vector and using the corresponding virus to infect Sf9 cells. The two recombinant 62-kDa proteins made by different mechanisms displayed immunoreactivities very compatible to each other. The 62-kDa proteins obtained by both proteolytic processing and reengineering demonstrated much higher sensitivities in detecting anti-HEV antibodies in human sera than the antigens made from bacteria, as measured by enzyme-linked immunosorbent assay. The data suggest that the soluble 62-kDa protein made from insect cells contains additional epitopes not present in recombinant proteins made from bacteria. Therefore, the 62-kDa protein may be useful for HEV diagnostic improvement and vaccine development. The reengineered construct allows for the consistent large-scale production of the soluble 62-kDa protein without proteolytic processing.

Seroreactivity to hepatitis E virus in areas where the disease is not endemic.

If the occurrence of hepatitis E virus antibody (anti-HEV) in regions where the disease is not endemic represents infection, rates may be greater in high-risk populations and behavioral correlates may reflect recognized transmission modes. Serum samples from 300 homosexual males, 300 injection drug users (IDUs), and 300 blood donors from Baltimore, Md., were tested for anti-HEV by enzyme immunoassay. Anti-HEV was found in an unexpectedly high percentage of homosexual men (15.9%) and IDUs (23.0%). However, anti-HEV was present in a similar proportion of blood donors (21.3%) (P > 0.05), while hepatitis A, B, and C virus antibodies were more prevalent in the high-risk groups (P < 0.001). Among homosexual men, anti-HEV was not significantly correlated with a history of hepatitis, high-risk sexual practices, or sexually transmitted infections, in contrast to hepatitis A and B antibodies. Among IDUs, anti-HEV was not significantly associated with a history of hepatitis or high-risk drug-using practices, as was found with hepatitis C antibodies. In a setting without endemic hepatitis E disease, there was no evidence that anti-HEV reflected subclinical infection. Until the basis for HEV seroreactivity in such areas is elucidated, anti-HEV results should be interpreted with caution.

The role of hepatitis E virus infection among patients with acute viral hepatitis in southern Saudi Arabia.

We investigated the etiology of acute sporadic viral hepatitis in southern Saudi Arabia in a series of 132 patients admitted with acute viral hepatitis. Of these cases, 108 (81.8%) were due to acute hepatitis A virus infection, of which 11 (8.3%) patients had been previously exposed to hepatitis E virus, and another 10 (7.6%) were chronic carriers of hepatitis B virus. Three cases (2.3%) were acute hepatitis B virus infection. The overall prevalence of hepatitis E IgG antibodies was found to be 9.1%. The remaining 21 (15.9%) patients were tested for hepatitis E IgM, EBV-VCA IgM and hepatitis C IgG antibodies by sensitive enzyme immunoassays. In none of them could hepatitis E IgM, EBV-VCA IgM or hepatitis C IgG antibodies be demonstrated, and these patients were thus considered as acute non-A, non-B hepatitis. Acute hepatitis C virus infection, however, could not be ruled out from this group. We therefore concluded that the majority of clinically apparent viral hepatitis cases were due to HAV, while HBV accounted for a small proportion of the cases. Clinically apparent HEV infection does not appear to be common in the population studied, since even those with serologic evidence of previous exposure to HEV did not recall a history suggestive of acute viral hepatitis.

Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry.

A protein with a molecular mass of approximately 62.10(3), derived from open reading frame 2 (ORF-2) of the hepatitis E virus (HEV: Burma strain), was expressed in a baculovirus expression vector and purified to homogeneity. The recombinant 62 kDa protein appeared to be a doublet, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tryptic digestion in conjunction with laser desorption mass spectrometry (LD-MS) and sequence analysis of the tryptic peptides indicated that the amino terminus was blocked, although no proteolytic degradation occurred. The determined internal sequences of peptides were in agreement with the predicted ORF-2 protein. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS) resolved the doublet proteins into two major components with molecular masses of 56548.5 and 58161.4. Confirmation of the amino terminus of the molecule by LD-MS post-ion decay enabled us to tentatively assign the carboxyl terminus of each species at residues 540 and 525. Sequencing of the intact protein by automated carboxyl terminal sequencing confirmed that the carboxyl terminus was truncated and that the sequence assignment predicted by LC-MS was correct.

Purification of a soluble hepatitis E open reading frame 2-derived protein with unique antigenic properties.

The second open reading frame (ORF2) of hepatitis E virus (HEV) is predicted to encode a 73-kDa capsid protein (1). When full-length ORF2 was expressed in insect cells (Spodoptera frugiperda (Sf9)) using a recombinant baculovirus, two distinct HEV polypeptides were observed: a full-length insoluble 73-kDa protein, and a soluble 56.5-kDa protein. Following purification and sequence analysis, it was determined that the 56.5-kDa protein was derived from endoproteolytic cleavage site that was between the Thr and Ala residues located at amino acids 111 and 112 in the ORF2 sequence with the carboxy terminus corresponding to residue 636 of the ORF2 sequence. Comparative ELISA data using human acute-phase antisera demonstrated that the 56.5-kDa protein served as a highly reactive antigen in detecting anti-HEV antibodies. These data suggest that the 56.5-kDa protein may serve as a particularly useful antigen for both diagnostic and vaccine purposes.

Purification of a baculovirus-expressed hepatitis E virus structural protein and utility in an enzyme-linked immunosorbent assay.

We report on the purification of the full-length structural protein encoded by open reading frame 2 (ORF-2) of hepatitis E virus. The ORF-2 protein, expressed in Sf9 cells by using a recombinant baculovirus vector system, was successfully purified to homogeneity. Gel electrophoresis of the purified ORF-2 protein showed a single polypeptide of 75 kDa by Coomassie blue staining and by Western blot (immunoblot) analysis. We demonstrated that the partially purified ORF-2 protein could be used successfully in a sensitive and specific enzyme-linked immunosorbent assay for the detection of antibodies to hepatitis E virus.

Acute viral hepatitis in Saudi Arabia: seroepidemiological analysis, risk factors, clinical manifestations, and evidence for a sixth hepatitis agent.

We conducted a prospective, descriptive cohort study of all 217 cases of acute viral hepatitis (AVH) seen in adults during 1992 at the sole hospitals with infectious disease departments in the second and third largest cities in the Kingdom of Saudi Arabia. In addition, we undertook a nested case-control study. Our goals were (1) to determine the causes, demographics, risk factors, and clinical characteristics of AVH in the Kingdom; (2) to evaluate the reliability of diagnostic tests for acute hepatitis C and E; and (3) to assess the relative importance, characteristics, and risk factors of a sixth hepatitis agent, non-A-E. All cases and controls completed a questionnaire. Cases provided blood samples for studies of serum bilirubin, alanine and aspartate aminotransferases, and antibody to hepatitis viruses as well as genome detection studies. The results of serological and molecular tests were used to categorize each case as hepatitis A, B, C, D, E, or non-A-E. Historical, clinical, and laboratory determinants were statistically analyzed by comparisons between groups with different types of AVH and controls. Analysis of risk factors suggested that hepatitis C and D were parenterally transmitted, while hepatitis A, E, and non-A-E were not; the route of transmission of hepatitis B was unclear. Hepatitis E was strongly associated with living or traveling on the Indian subcontinent. The clinical disease caused by all six agents was indistinguishable. The putative sixth agent caused 13% of cases. The second-generation tests for antibody to HCV and HEV were relatively reliable for the diagnosis of AVH.

The risk of viral hepatitis A, B, C, and E among North American missionaries.

The seroprevalence and incidence of hepatitis A, B, C, and E virus infection were determined among North American missionaries (n = 328) serving in various geographic locations between 1967 and 1984. The mean age of subjects at entry into the study was 39.7 years (range 5-73 years); 65% were female; 89% had lived outside the United States before the study began. Seventy-eight percent of subjects served in sub-Saharan Africa during the study. At initial evaluation, 50.9% of the subjects had antibodies to hepatitis A virus (total anti-HAV), 8.5% to hepatitis B virus core antigen (total anti-HBc), 0.6% to hepatitis C virus (total anti-HCV by second-generation immunoblot assay), and 0% to hepatitis E virus (IgG anti-HEV). After an average period of service of 7.3 years (2,396 person-years total), 5.8% of the missionaries seroconverted to anti-HAV, 5.5% to anti-HBc, 0.6% to anti-HCV, and 0% to anti-HEV. This study indicates a relatively low risk of hepatitis C and E virus infection among missionaries while confirming the previously reported high risk of hepatitis A and B virus infection. Hepatitis A and B vaccination is recommended for long-term travelers to developing countries.

Detection of hepatitis E virus genome and gene products in two patients with fulminant hepatitis E.

Non-isotopic in situ hybridization (digoxigenin-labeled probe directed towards hepatitis E virus ORF1) and immunohistochemistry (against hepatitis E virus ORF2 and ORF3) were applied to detect hepatitis E virus genome and gene product in the liver tissue of two patients with fulminant hepatitis E seropositive for hepatitis E virus RNA. Both hepatitis E virus RNA and hepatitis E virus antigens were detected exclusively in the cytoplasm of hepatocytes and not detected in other cell types. In both patients, more than 50% of the hepatocytes were positive for both hepatitis E virus RNA and hepatitis E virus antigens, most of which showed degenerative changes. This is consistent with the histological appearance of marked loss of hepatocytes with acinar collapse. Interestingly, denaturation of the RNA before in situ hybridization was found to enhance hepatitis E virus RNA detection. We conclude that: (1) hepatitis E virus RNA and hepatitis E virus antigens can be demonstrated in the liver in hepatitis E virus-related fulminant hepatitic failure, (2) hepatitis E virus is hepatocyte-tropic within the liver, (3) cytoplasmic localization of hepatitis E virus RNA and hepatitis E virus antigens is consistent with cytoplasmic replication, and (4) the presence of degenerative changes in hepatitis E virus positive cells, together with the histological appearance of hepatocyte loss in the absence of significant inflammatory infiltrate, suggests that hepatitis E virus-related fulminant hepatitic failure is mediated by a cytopathic mechanism.

Infectivity titration of a prototype strain of hepatitis E virus in cynomolgus monkeys.

The infectivity titer of a standard stock of the SAR-55 strain of hepatitis E virus (HEV) was determined in cynomolgus macaques (Macaca fascicularis) and the effect of dose on the course of the infection was examined by weekly monitoring of alanine aminotransferase (ALT) and anti-HEV levels. Antibody to HEV (anti-HEV) was measured with ELISAs based on ORF-2 recombinant antigens consisting of either a 55 kDa region expressed in insect cells or shorter regions expressed as fusion proteins in bacteria. The ELISA based on the 55 kDa antigen was generally more sensitive. The infectivity titer of SAR-55 was 10(6) cynomolgus 50% infectious doses per gram of feces. The infectivity titer corresponded to the HEV genome titer of the inoculum as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Anti-HEV IgM was detected in only a portion of the animals that had an anti-HEV IgG response. Biochemical evidence of hepatitis was most prominent in animals that were inoculated with the higher concentrations of virus and the incubation period to seroconversion was prolonged in animals that received the lower doses.

Expression of hepatitis E virus putative structural proteins in recombinant vaccinia viruses.

Hepatitis E virus (HEV) is a polyadenylated, positive-stranded RNA virus which is a major cause of enterically transmitted non-A, non-B hepatitis in many developing countries. The viral genome contains three different open reading frames (ORFs): ORF1, which is believed to encode nonstructural proteins, and ORF2 and ORF3, which are believed to encode structural proteins. The full-length putative structural proteins encoded by ORF2 and ORF3 of HEV have been cloned and expressed in recombinant vaccinia virus. Proteins encoded by ORF2 and ORF3 when expressed in vaccinia virus are recognized by pooled sera obtained from individuals with acute hepatitis E. Vaccinia-expressed viral gene products of HEV will have utility in characterizing the cell-mediated immune response to HEV.

Non-A, non-B fulminant hepatitis is also non-E and non-C.

OBJECTIVES: to define the roles of the hepatitis C and E viruses (HCV and HEV) in non-A, non-B (NANB) fulminant hepatitis. METHODS: we utilized the polymerase chain reaction to amplify HCV and HEV RNA sequences and assays to detect antibodies to HCV and HEV in the acute phase sera of eight presumed viral NANB and seven nonviral NANB fulminant hepatic failure (FHF) patients. RESULTS: none of the 15 patients had detectable HCV or HEV RNA or elevated HCV and IgM-HEV antibody titers in their acute phase sera. Three patients, all with features of autoimmune hepatitis, had raised IgG-HEV antibody titers. Due to the possibility of serologically undetectable hepatitis B virus (HBV) infection in fulminant hepatitis patients, we performed polymerase chain reaction amplification of HBV genomic DNA in acute phase sera of the presumed viral NANB FHF patients and subsequently found no evidence of HBV DNA. CONCLUSIONS: we did not find evidence implicating HCV or HEV in presumed viral NANB FHF or as agents contributing to or causing the liver failure in nonviral NANB FHF patients with autoimmune hepatitis, drug-induced hepatotoxicity, or halothane hepatotoxicity.

Expression and diagnostic utility of hepatitis E virus putative structural proteins expressed in insect cells.

The full-length putative structural proteins encoded by open reading frame 2 (ORF2) and ORF3 of hepatitis E virus have been cloned and expressed in recombinant baculovirus. Sera obtained from 28 Sudanese pediatric patients with acute hepatitis and 19 pediatric control patients were analyzed for reactivity to hepatitis E virus by using the baculovirus-expressed ORF2 and ORF3 proteins in a Western blot (immunoblot) format. Seventeen of the 18 patients classified as having non-A, non-B hepatitis, without acute antibody markers for hepatitis A, B, or C viruses, Epstein-Barr virus, or cytomegalovirus, were shown to have immunoglobulin M (IgM) antibodies to the recombinant ORF2 protein, as did two patients with chronic hepatitis B, three of seven patients with acute hepatitis A, and one patient with acute hepatitis B. None of the 19 control patients had IgM antibodies against the ORF2 or ORF3 proteins. The Western blot assay using the baculovirus-expressed ORF3 protein did not appear to be as sensitive as the assay based on the ORF2 protein. Only 10 of the patients classified as having non-A, non-B hepatitis had IgM antibodies to the baculovirus-expressed ORF3 protein. We conclude that a Western blot assay which uses a baculovirus-expressed ORF2 protein is both sensitive and specific for diagnosing acute hepatitis E.