Suresh M. Desai

Sequence and genomic organization of GBV-C: A novel member of the flaviviridae associated with human non-A-E hepatitis

Recently, sequences from a novel virus, termed GB virus C (GBV‐C), were identified in serum from several patients with cryptogenic hepatitis. In the present study, the nucleotide sequence of this virus has been extended to near‐genome length. GBV‐C encodes a putative single large polyprotein in which the structural proteins are positioned at the N‐terminal end, with the nonstructural proteins located at the C‐terminal end. Amino acid sequence analysis of this large polyprotein reveals the presence of protease, helicase, and replicase motifs. Sequence alignments of the polyprotein followed by phylogenetic analyses suggest that GBV‐C is a member of the Flaviviridae, most closely related to the recently described GB virus A.

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. J. Med. Virol. 57:243–251, 1999.

IMPROVED DETECTION SYSTEMS FOR TT VIRUS REVEAL HIGH PREVALENCE IN HUMANS, NON-HUMAN PRIMATES AND FARM ANIMALS

TT virus is a newly described agent infecting humans. Initially isolated from a patient (initials T.T.) with unexplained hepatitis, the virus has since been found in both normal and diseased individuals. In the present study, we utilized genomic-length sequences from distinct genotypes of TT virus to design PCR-based assays using conserved oligonucleotide primers from three independent regions of the virus genome. Each of the three assays was found to be superior to the PCR-based assays previously published. The most sensitive of the new assays was utilized to demonstrate the prevalence of TT virus to be at least 34.1% in volunteer blood donors, 39.6% in commercial blood donors, 59.6% in non-A-GB hepatitis cases, 81.7% in injectable drug users and 95.9% in haemophiliacs. In an attempt to identify a possible source of human infection, we found TT virus sequences to be present in 19% of chickens, 20% of pigs, 25% of cows and 30% of sheep. Sequence determination and phylogenetic analyses demonstrated that isolates from farm animals were not genetically distinct from those found in humans. This study clearly demonstrates that previously reported PCR assays dramatically underestimate the true prevalence of TT virus within the human population. Due to the high rate of infection in both blood donors and those with non-A-GB hepatitis, these results question the causal role of TT virus in cases of unexplained hepatitis. Further, it is possible that domesticated farm animals serve as a source of human infection.

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.

A hepatitis E virus variant from the United States: molecular characterization and transmission in cynomolgus macaques.

The partial sequence of a hepatitis E virus (HEV-US1) isolated from a patient in the United States (US), suffering from acute viral hepatitis with no known risk factors for acquiring HEV, has been reported. These sequences were significantly different from previously characterized HEV isolates, alluding to the existence of a distinct human variant. In this paper, we report the near full-length sequences of HEV-US1 and a second US isolate (HEV-US2). HEV-US2 was identified in a US patient suffering from acute viral hepatitis. These sequences verify the presence of a new HEV strain in North America and provide information as to the degree of variability between variants. The HEV-US nucleotide sequences are 92% identical to each other and only 74% identical to the Burmese and Mexican strains. Amino acid and phylogenetic analyses also demonstrate that the US isolates are genetically distinct, suggesting the presence of three genotypes of HEV. Serum from the second US patient induced hepatitis following inoculation into a cynomolgus macaque. Within 2-4 weeks, HEV-US2 RNA was detectable in both the serum and faecal material coinciding with elevated serum alanine transaminase levels. Infection resolved as antibody titres increased 8 weeks post-inoculation.

Prevalence studies of GB virus-C infection using reverse transcriptase-polymerase chain reaction

Among the three recently described GB viruses (GBV‐A, GBV‐B, and GBV‐C), only GBV‐C has been linked to cryptogenic hepatitis in man. Because of the limited utility of currently available research tests to determine antibody response to GBV‐C proteins, the prevalence of GBV‐C RNA in human sera was studied using reverse transcription‐polymerase chain reaction (RT‐PCR). The prevalence of GBV‐C is higher among volunteer blood donors with elevated serum alanine aminotransferase (ALT) levels (3.9%) than among volunteer blood donors with normal ALT levels (0.8%). Higher rates were also noted among commercial blood donors (12.9%) and intravenous drug users (16.0%). GBV‐C was frequently detected in residents of West Africa, where the prevalence was >10% in most age groups. Approximately 20% of patients diagnosed with either acute or chronic hepatitis C virus (HCV) were found to be positive for GBV‐C RNA. In addition, GBV‐C RNA sequences were detected in individuals diagnosed with non‐A‐E hepatitis, with clinical courses ranging from mild disease to fulminant hepatitis. Fourteen of sixteen subjects with or without clinically apparent hepatitis were positive for GBV‐C RNA more than 1 year after the initial positive result.

Prevalence of TT virus infection in US blood donors and populations at risk for acquiring parenterally transmitted viruses.

Two overlapping sets of TT virus (TTV)-specific polymerase chain reaction primers were used to test for presence of TTV, which was found in approximately 10% of US volunteer blood donors, 13% of commercial blood donors, and 17% of intravenous drug abusers. The rate of TTV infection among US non-A, non-B, non-C, non-D, non-E hepatitis patients was only 2%. Among commercial blood donors and intravenous drug abusers, only 1%-3% of the TTV-positive individuals were coinfected with GB virus C (GBV-C), a parenterally transmitted virus. This suggests that GBV-C and TTV may have different routes of transmission. Comparison of the sensitivities of 2 TTV polymerase chain reaction (PCR) primer sets showed that the majority of samples were detected with only 1 of the 2 sets. Therefore, previous studies in which only a single PCR primer pair was used may have significantly underestimated the true prevalence of TTV.

Rapid detection of Hepatitis E virus RNA by reverse transcription-polymerase chain reaction using universal oligonucleotide primers.

A rapid reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of Hepatitis E virus (HEV) RNA in serum is described. Total nucleic acids are extracted from a small volume of human serum and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR employing degenerate HEV consensus primers. These primers are designed to sequences conserved between the Burma, Mexico, and US HEV strains, generating amplicons within each of the three open reading frames. Reactions are analyzed by agarose gel electrophoresis and samples showing an ethidium bromide stained band of the appropriate size in the first and second amplification, or in the second amplification only, are designated as positive. This protocol allows for the rapid and sensitive detection of HEV infection in human serum.

Analyses of TT virus full-length genomic sequences

Since the identification of TT virus, only one full-length and two near full-length sequences representing a single subtype of the virus have been reported. In order to understand further the nature of the TT virus genome, nine of the most divergent TT virus sequences have been extended to full-length or near full-length. Phylogenetic analysis demonstrated that these sequences represent three distinct TT virus genotypes and two subtypes. A high degree of nucleotide sequence variability (approximately 30%) was observed across the genomes with several significantly more divergent regions. Three conserved ORFs were identified, none of which shared significant amino acid sequence identity to sequences present in public databases. Additionally, sequence motifs, such as those necessary for protein translation and for rolling circle replication, were found to be partially conserved between all TT virus isolates.

Experimental Infection of Rhesus Macaques with West Nile Virus: Level and Duration of Viremia and Kinetics of the Antibody Response after Infection

Reports of transfusion-associated cases of West Nile virus (WNV) infection indicate the need for sensitive screening methods to identify WNV-infected blood products. We experimentally infected 5 rhesus macaques with WNV, to determine the level and duration of viremia, the kinetics of the humoral immune response, and the sensitivity of various assay systems for detecting WNV in blood. All macaques developed subclinical infections with low levels of viremia; nested reverse-transcription polymerase chain reaction was the most sensitive method for detecting virus or viral RNA in blood. Specific WNV antibodies appeared during the second week of infection; the results of an IgM enzyme-linked immunosorbent assay became positive on the ninth or tenth day after infection, followed in 1-2 days by hemagglutination-inhibiting and neutralizing antibodies. Our results suggest that both nucleic acid and serological testing may be needed to determine exposure to WNV and to identify potentially infected blood donors.