Sergei A. Tsarev

Prevalence of Antibody to Hepatitis E Virus among Rodents in the United States

The recent identification of antibody to hepatitis E virus (HEV) in pigs, sheep, and cattle and characterization of an HEV isolated from domestic pigs suggest animal reservoirs for this virus. To investigate whether rodents might be a natural reservoir of HEV, the prevalence of anti-HEV was determined among a variety of species throughout the United States. Serum samples were obtained from 806 rodents of 26 species in 15 genera. Anti-HEV prevalence was assessed by 2 EIAs (mosaic protein- and 55-kDa protein-based), which gave concordant results. The highest prevalence of antibody was found in the genus Rattus (59.7%; 166/278). Overall, rodents from urban habitats had a significantly higher prevalence of anti-HEV than did animals captured from rural areas. A high prevalence of anti-HEV was found in animals captured on mainland versus barrier islands. The results from this study provide convincing evidence of widespread HEV or HEV-like infection in rodents of the United States.

Recombinant vaccine against hepatitis E: dose response and protection against heterologous challenge

Thirty-two rhesus monkeys were used to evaluate the dose response of a recombinant HEV vaccine, and the efficacy of the vaccine based on the ORF2 protein of the Pakistani strain for pre- and post-exposure vaccination against intravenous challenge with homologous or heterologous virus was examined. Post-exposure vaccination did not protect animals against hepatitis. Although primates vaccinated twice with 50-microgram, 10-microgram, 2-microgram, or 0.4-microgram doses of the recombinant 55 kDa ORF-2 protein were infected, they were protected from hepatitis when they were challenged with very high doses of the homologous strain of HEV. Primates vaccinated twice with a 50 micrograms dose of the recombinant protein were protected from hepatitis after heterologous challenge with the Mexican strain, the strain of HEV most genetically distant from the Pakistani strain.

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. J. Med. Virol. 57:68–74, 1999.

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. J. Med. Virol. 55:134–137, 1998.

Characterization of hepatitis E virus (HEV) from Algeria and Chad by partial genome sequence

The purpose of this study was to analyze partial nucleotide sequences and derived peptide sequences of hepatitis E virus (HEV) from two outbreaks of hepatitis E in Africa (Chad 1983–1984; Algeria 1978–1980). A portion of ORF3 and the major portion of ORF2 were amplified by Reverse Transcriptase‐Polymerase Chain Reaction (RT‐PCR). The PCR products were sequenced directly or after cloning into the pCRII vector. Sequences were then compared to the corresponding regions of reported full length HEV sequences. In the ORF2 and ORF3 regions, the homology between the Algerian and the Chad isolates at the nucleic acid level was 92 and 95%, respectively. At the peptide level the homology was 98% in both regions. In these regions, both strains are more related to Asian strains at the nucleic acid level (89 to 95%) and at the amino acid level (95 to 100%) than to the Mexico strain. At the peptide level the differences are less apparent. Both African isolates have amino acid changes in common with some reference strains although the Chad isolate has three unique changes. These African strains of HEV, based on the ORF2 and ORF3 phylogenetic trees, appear to be a distinct phylogenetic group, separate from the Mexican and Asian strains. J. Med. Virol. 53:340–347, 1997.

African strains of hepatitis E virus that are distinct from Asian strains

Partial genomic sequences of four hepatitis E virus (HEV) strains from Africa (Morocco and Tunisia) and one from Central Asia (Tashkent, Uzbekistan) were obtained. The reverse transcriptase‐polymerase chain reaction was used to amplify 5′ and hypervariable regions of open reading frame 1 (ORF1) and a region overlapping all 3 ORFs. Sequence analysis of these regions revealed the African strains to be quite distinct from all known Asian strains but more similar to them than to the Mexican strain. Sequence analysis of the Tashkent strain revealed almost complete identity with another central Asian strain from Osh, Kirgizia. These results thus further confirm the geographical origin of HEV strain divergence. J. Med. Virol. 53:139–144, 1997.

Simian hepatitis A virus (HAV) strain AGM-27 : comparison of genome structure and growth in cell culture with other HAV strains

Fragments of cDNA representing greater than 99% of the entire genome of wild-type hepatitis A virus (HAV) strain AGM-27, isolated from an African green monkey, were obtained by the polymerase chain reaction and sequenced. Comparison with other HAV isolates revealed differences in the predicted amino acid sequence in functionally critical parts of the genome. Comparison of the biological properties of AGM-27 with those of human wild-type and cell culture-adapted HM-175 strains revealed that AGM-27 grew in cell culture significantly better than did wild-type HM-175, but not as well as cell culture-adapted HM-175. AGM-27 and cell culture-adapted HM-175 were distinguishable by their differential growth in CV-1, FRhK-4 and primary AGMK cells.

Clinical and Epidemiological Relevance of Quantitating Hepatitis E Virus-Specific Immunoglobulin M

ABSTRACT Diagnosis of acute hepatitis E by detection of hepatitis E virus (HEV)-specific immunoglobulin M (IgM) is an established procedure. We investigated whether quantitation of HEV IgM and its ratio to HEV total Ig furnished more information than conventional IgM tests that are interpreted as positive or negative. A previously described indirect immunoassay for total Ig against a baculovirus-expressed HEV capsid protein was modified to quantitate HEV-specific IgM in Walter Reed (WR) antibody units by using a reference antiserum and the four-parameter logistic model. A receiver-operating characteristics curve derived from 197 true-positive specimens and 449 true-negative specimens identified 30 WR units/ml as an optimum cut point. The median HEV IgM level in 36 patients with acute hepatitis E fell from 3,000 to 100 WR units/ml over 6 months, suggesting that 100 WR units/ml would be a more appropriate cut point for distinguishing recent from remote IgM responses. Among three hepatitis E case series, determination of the HEV IgM-to-total-Ig ratio in acute-phase serum revealed that most patients had high ratios consistent with primary infections whereas a few had low ratios, suggesting that they had sustained reinfections that elicited anamnestic antibody responses. The diagnostic utility of the new IgM test was similar to that of a commercially available test that uses different HEV antigens. In conclusion, we found that HEV IgM can be detected specifically in >95% of acute hepatitis E cases defined by detection of the virus genome in serum and that quantitation of HEV IgM and its ratio to total Ig provides insight into infection timing and prior immunity.

Phylogenetic analysis suggests only one serotype of Japanese encephalitis virus.

Phylogenetic analysis was performed for different genome regions of Japanese encephalitis virus (JEV). Similar genetic groupings were identified for all analyzed genome regions including complete genomes. More extensive analysis was performed for 92 isolates (complete envelope sequences) available in the GenBank. Results of phylogenetic analysis were compared with those performed for human positive strand RNA viruses with well characterized serotypes - poliovirus (PV) and dengue virus (DEN). The observed level of the JEV inter-genotype diversity was much less than that observed across PV and DEN serotypes and was consistent with the genetic diversity observed within PV or DEN serotypes. This genetic analysis supports the contention that all known JEV isolates comprise a single serotype.

Role of immune serum globulins in pregnant women during an epidemic of hepatitis E

The efficacy of an Indian preparation of immune serum globulins (ISG) was evaluated among pregnant women during an epidemic of hepatitis E in Karad, Western India from January to March 1993. Ten of 55 women receiving ISG developed immunoglobulin M (IgM) antibodies to hepatitis E virus (anti‐HEV) during the 1 month of follow‐up compared with 18 out of 53 control subjects. Although the total number of recent HEV infections was significantly less in the ISG‐treated group, no significant difference could be shown in the proportion of clinical hepatitis E cases because of the very small numbers of patients who developed clinical disease. The observed marginal beneficial effect of ISG might be the result of a low immunoglobulin G (IgG) anti‐HEV IgG titre (1:500) of the ISG preparation used. Preparation and testing of high‐titred ISG should be a high priority for protecting pregnant women during epidemics of hepatitis E.