Homologous recombination in GB virus C/hepatitis G virus

Analysis of 33 GB virus C/hepatitis G virus (GBV-C/HGV) full or nearly full genome sequences revealed several putative inter- and intrasubtype recombinants. The breakpoints of the recombinant regions were mapped using a maximum-likelihood method, and the statistical significance for each region was tested using Monte Carlo simulation. The results were highly significant and provided evidence for the existence of complex mosaic genomes showing as many as nine recombination events, with breakpoints in the 5' UTR and in all of the coding regions except the short NS4b gene. Recombination was confirmed by separate phylogenetic analysis of the various recombinant regions and by Sawyer's runs test. Taken together, these findings demonstrate for the first time that recombination is common in natural populations of GBV-C and that it takes place both within and between subtypes. The wide-ranging implications of such nonclonal history for reconstructing the spread and timescale of GBV-C evolution are discussed.     

Prevalence of GB virus C/hepatitis G virus in Hungary

In 1995 a new flavivirus, GB virus C/hepatitis G virus (GBV-C/HGV), was discovered. The aim of this study was to determine the prevalence of the virus in healthy persons and hepatitis patients in Hungary. The sera of 408 healthy persons older than 60 years were tested for the presence of GBV-C/HGV antibodies, and 113 were positive (28%). Eight of the 71 healthy persons younger than 60 years and twenty of the 51 sera (39%) taken from patients suffering from hepatitis of unknown origin proved to be positive for GBV-C/HGV antibodies. Ten of the 124 sera (8%) of healthy persons and 36 of the 247 sera (14.6%) of hepatitis patients proved to be positive for GBV-C/HGV RNA. Eleven PCR products were sequenced, and the sequences were found to be different from each other and from the previously published ones. However, three sequences taken from the same patient at different times were identical. These results show that GBV-C/HGV is present in Hungary and cannot be considered rare.     

Mutation patterns for two flaviviruses: hepatitis C virus and GB virus C/hepatitis G virus

We studied the mutation patterns of hepatitis C virus (HCV) and GB virus C/hepatitis G virus (HGV). Although the mutation patterns of the two viruses were similar to each other, they were quite different from that of HIV. In particular, the similarity of the patterns between HCV or HGV and human nuclear pseudogenes was statistically significant whereas there was no similarity between HIV and human nuclear pseudogenes. This finding suggests that the mutation patterns of HCV and HGV are similar to the patterns of spontaneous substitution mutations of human genes, implying that nucleotide analogues which are effective against HCV and HGV may have a side effect on the normal cells of humans.     

Prevalence and genotypes of GB virus C/hepatitis G virus among blood donors in Central Brazil

A survey was conducted in a blood donor population of Central Brazil aiming to investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) infection and also to analyze the virus genotypes distribution. A total of 241 voluntary blood donors were interviewed at the State Blood Bank in Goiania, State of Goiás, Brazil. Blood samples were collected and serum samples tested for GBV-C/HGV RNA by polymerase chain reaction. Genotypes were determined by restriction fragment length polymorphism (RFLP) analysis. Seventeen samples were GBV-C/HGV RNA-positive, resulting in a prevalence of 7.1% (95% CI: 4.2-11.1). A significant trend of GBV-C/HGV RNA positivity in relation to age was observed, with the highest prevalence in donors between 29-39 years old. Ten infected individuals were characterized by reporting parenteral (30%), sexual (18%), both (6%) and intrafamiliar (6%) transmission. However, 7 (40%) GBV-C/HGV RNA-positive donors did not mention any potential transmission route. RFLP analysis revealed the presence of genotypes 1 and 2 of GBV-C/HGV; more precisely, 10 (58.9%) samples were found belonging to the 2b subtype, 4 (23.5%) to the 2a subtype, and 3 (17.6%) to genotype 1. The present data indicate an intermediate endemicity of GBV-C/HGV infection among this blood donor population, and a predominant circulation of genotype 2 (subtype 2b) in Central Brazil.     

Prevalence and genotypes of hepatitis G virus/GB virus C in a multirisk group in Hungary

HGV/GBV-C is a mainly parenterally transmitted Flavivirus that causes a persistent infection. So far no disease has been associated with HGV/GBV-C infection, but its beneficial role in co-infection with the human immunodeficiency virus has been shown in many recent studies. The aim of our study was to determine the frequency of ongoing HGV/GBV-C infections among a sociologically unique group of the Hungarian population, who are at great risk for parenterally transmitted diseases. Viral RNA was detected in 75 serum samples by an RT-PCR method specific for the NS5 region. Nine (12%) samples were positive for HGV/GBV-C RNA. All nine PCR products were sequenced and a phylogenetic analysis was performed to identify the genotypes and subtypes of the detected viruses. All nine isolates proved to be genotype 2, eight of them were classified as subtype 2a, and one as subtype 2b.     

GB virus type C/hepatitis G virus: a non-pathogenic flavivirus associated with prolonged survival in HIV-infected individuals

GB virus C (GBV-C) is a common virus that does not appear to cause disease. GBV-C persists in up to 50% of exposed individuals, and virus replication appears to be in lymphocytes including CD4+ T cells. GBV-C infection is common among HIV-positive people, and several studies have found that HIV-positive individuals co-infected with GBV-C survive for statistically significantly longer periods of time than people without GBV-C. In vitro studies suggest that GBV-C inhibits HIV replication and alters cytokine, chemokine and chemokine receptor expression. Thus, GBV-C may be a major factor influencing the natural history of HIV disease.     

GB virus C/hepatitis G virus infection in dialysis patients and kidney transplant recipients in Central Brazil

In order to investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) infection in dialysis patients and kidney transplant recipients in Central Brazil and also to analyze the virus genotypes distribution, a total of 123 patients including 98 on hemodialysis, 13 on continuous ambulatory peritoneal dialysis treatment, and 12 who received kidney transplantation were interviewed in one unit of dialysis treatment in Goiania city. Blood samples were collected and serum samples tested for GBV-C/HGV RNA by polymerase chain reaction. Genotypes were determined by restriction fragment length polymorphism (RFLP) analysis. Eighteen samples were GBV-C/HGV RNA-positive, resulting in an overall prevalence of 14.6% (95% CI: 9.2-21.7). A high positivity for GBV-C/HGV RNA was observed in patients who had received kidney transplant (16.7%), followed by those on hemodialysis (15.3%), and peritoneal dialysis (7.7%). RFLP analysis revealed the presence of genotypes 1, 2, and 3 of GBV-C/HGV; more precisely, 9 (50%) samples were found belonging to the 2b subtype, 4 (22%) to the 2a subtype, 3 (17%) to genotype 1, and 2 (11%) to genotype 3. The present data indicate an intermediate prevalence of GBV-C/HGV infection among dialysis patients and kidney transplant recipients in Central Brazil. Genotype 2 (subtype 2b) seems to be the most prevalent GBV-C/HGV genotype in our region.     

Transmissibility of intra-host hepatitis C virus variants

Background

Intra-host hepatitis C virus (HCV) populations are genetically heterogeneous and organized in subpopulations. With the exception of blood transfusions, transmission of HCV occurs via a small number of genetic variants, the effect of which is frequently described as a bottleneck. Stochasticity of transmission associated with the bottleneck is usually used to explain genetic differences among HCV populations identified in the source and recipient cases, which may be further exacerbated by intra-host HCV evolution and differential biological capacity of HCV variants to successfully establish a population in a new host.

Results

Transmissibility was formulated as a property that can be measured from experimental Ultra-Deep Sequencing (UDS) data. The UDS data were obtained from one large hepatitis C outbreak involving an epidemiologically defined source and 18 recipient cases. k-Step networks of HCV variants were constructed and used to identify a potential association between transmissibility and network centrality of individual HCV variants from the source. An additional dataset obtained from nine other HCV outbreaks with known directionality of transmission was used for validation.Transmissibility was not found to be dependent on high frequency of variants in the source, supporting the earlier observations of transmission of minority variants. Among all tested measures of centrality, the highest correlation of transmissibility was found with Hamming centrality (r?=?0.720; p?=?1.57 E-71). Correlation between genetic distances and differences in transmissibility among HCV variants from the source was found to be 0.3276 (Mantel Test, p?=?9.99 E-5), indicating association between genetic proximity and transmissibility. A strong correlation ranging from 0.565–0.947 was observed between Hamming centrality and transmissibility in 7 of the 9 additional transmission clusters (p?<?0.05).

Conclusions

Transmission is not an exclusively stochastic process. Transmissibility, as formally measured in this study, is associated with certain biological properties that also define location of variants in the genetic space occupied by the HCV strain from the source. The measure may also be applicable to other highly heterogeneous viruses. Besides improving accuracy of outbreak investigations, this finding helps with the understanding of molecular mechanisms contributing to establishment of chronic HCV infection.

     

Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.     

Co-infections with hepatitis G and TT virus in patients with chronic hepatitis C in Hungary

The significance of co-infections with novel hepatitis viruses Hepatitis G (GBV-C, HGV) and TT virus (TTV) in chronic hepatitis C is not clear. We determined the prevalence of HGV RNA and TTV DNA in chronic hepatitis C patients and in asymptomatic hepatitis C virus (HCV) carriers, and assessed the influence of these agents on the course of HCV infection. Seventy-seven patients with chronic hepatitis C--50 of them treated with interferon (IFN)--and 33 HCV carriers with normal alanine aminotransferase have been investigated. Previous HBV infection was detected by testing serum HBsAg and aHBc. HGV RNA and TTV DNA were detected by PCR. In the healthy population, the prevalence of anti-HCV was 0.3%, HGV RNA 8.0% and TTV DNA 18.5%. In chronic hepatitis C HGV RNA occurred in 9.09% and TTV DNA in 40.25% of cases. In IFN-treated patients with sustained remission, the frequency of TTV was 20% vs. 45.7% found in non-responders. Among asymptomatic HCV-carriers, the prevalence of HGV RNA was 9.09% and TTV DNA 75.7%. Neither HGV RNA nor TTV DNA had apparent effect on the HCV infection. TTV was detected with the lowest frequency in persons with sustained remission due to IFN, suggesting antiviral effect of IFN on TTV.     

RNA-dependent RNA polymerases from different hepatitis C virus genotypes reveal distinct biochemical properties and drug susceptibilities

The RNA-dependent RNA polymerase of the hepatitis C virus (HCV) is the key enzyme for viral replication, recognized as one of the promising targets for antiviral intervention. Several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GTs). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. This work gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1-6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg(2+) were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a>2a, 3a, 4a, 5a>1a. Furthermore, the optimized assay conditions for genotypic profiling were confirmed by testing the inhibitory activity of 4 known prototype NNIs addressing the NNI binding sites 1 to 4.     

GB virus B and hepatitis C virus NS3 serine proteases share substrate specificity.

GB virus B (GBV-B) is a recently discovered virus responsible for hepatitis in tamarins (Saguinus species). GBV-B belongs to the Flaviviridae family and is closely related to the human pathogen hepatitis C virus (HCV). Nonstructural protein 3 (NS3) of HCV has been shown to encompass a serine protease domain required for viral maturation. GBV-B and HCV share only about 30% of the amino acid sequence within the NS3 protease domain. The catalytic triad is conserved, and the residue Phe-154, presumed to be a crucial amino acid for determining the S1 specificity pocket of the HCV NS3 protease, is also conserved. We have expressed a synthetic gene encoding the GBV-B NS3 protease domain in Escherichia coli and have characterized the purified recombinant protein for its activity on HCV substrates. We have shown that the NS3 region of the GBV-B genome actually encodes a serine protease that, despite the low sequence homology, shares substrate specificity with the HCV NS3 protease.     

Slow Evolutionary Rate of GB Virus C/Hepatitis G Virus

With the aim of elucidating evolutionary features of GB virus C/hepatitis G virus (GBV-C/HGV), molecular evolutionary analyses were conducted using the entire coding region of this virus. In particular, the rate of nucleotide substitution for this virus was estimated to be less than 9.0 × 10⁻⁶ per site per year, which was much slower than those for other RNA viruses. The phylogenetic tree reconstructed for GBV-C/HGV, by using GB virus A (GBV-A) as outgroup, indicated that there were three major clusters (the HG, GB, and Asian types) in GBV-C/HGV, and the divergence between the ancestor of GB- and Asian-type strains and that of HG-type strains first took place more than 7000–10,000 years ago. The slow evolutionary rate for GBV-C/HGV suggested that this virus cannot escape from the immune response of the host by means of producing escape mutants, implying that it may have evolved other systems for persistent infection. Received: 2 June 1998 / Accepted: 8 August 1998     

Two hepatitis C virus glycoprotein E2 products with different C termini.

Processing of the boundary region between the putative structural and nonstructural regions of the hepatitis C virus precursor polyprotein was analyzed by in vitro translation using reticulocyte lysate in the presence of canine microsomal membranes. At this boundary in the precursor polyprotein, the most carboxy-terminal of the structural proteins, gp70 (E2), is proximal to the amino terminal of the nonstructural protein p21 (NS2). The presence of a novel microsomal membrane-dependent cleavage site was observed at the region upstream of the amino-terminal end of p21 (NS2) in the precursor polyprotein. The cleavage site was assigned to amino acid residues 746/747 in the hepatitis C virus precursor polyprotein. Inefficient cleavage of this site resulted in the production of two forms of E2 products with different sizes of peptide backbones. Translation and cleavage of various C-terminal deletion constructs established the significance of the C-terminal hydrophobic amino acid sequences of E2 products in membrane anchoring.     

Antibodies to different virus antigens in patients with chronic hepatitis C

A total of 176 hospital patients with chronic hepatitis C (CHC), among them 110 males and 66 females, were examined. The spectrum of antibodies to four hepatitis C virus (HCV) proteins (core, NS3, NS4, NS5) and in 142 patients --IgM antibodies to HCV (anti-HCV IgM) were determined. In 92% of the CHC patients antibodies to core, NS3 and NS4 proteins were simultaneously detected. Differences in the detection of antibodies to HCV in males and females were not statistically reliable. In CHC patients aged up to 20 years anti-NS4 and anti-NS5 were less frequently detected. Among males of different age groups reliable differences in the detection rate of anti-NS5 were registered, while among females of different age groups no such differences were observed. With the increase of age these antibodies were detected somewhat more often. In females over 60 years anti-HCV IgM occurred more often than in males of the same age. The levels of alanine aminotransferase (ALT) were higher in persons with the presence of anti-NS5 and anti-HCV IgM than in persons with their absence. In all groups of CHC patients with biochemical activity and liver cirrhosis the detection rate of anti-HCV IgM was significantly higher than in patients with normal ALT activity. The antibody spectrum with the simultaneous absence of HCV IgM and anti-NS5, while found to contain antibodies to other HCV antigens, was registered significantly less frequently in patients with moderate and high CHC activity and the liver cirrhosis induced by HCV infection.     

High expression of APOBEC3G in patients infected with hepatitis C virus

APOBEC3G (an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a member of the APOBEC family, which possesses cytidine deaminase activity that causes C/G to T/A transition mutations in virus genomes such as human immunodeficiency virus 1 and hepatitis B virus, is reported to play an important role in host-defense mechanisms. However, APOBEC3G expression in patients infected with chronic hepatitis C virus (HCV), of which there are currently more than 170 million worldwide, has not yet been well studied. We investigated this issue herein, and demonstrated an increased expression of APOBEC3G in both hepatocytes and lymphocytes of chronic hepatitis patients infected with HCV. Transfection of the NS5A gene, but not any other non-structural protein genes of HCV tested, to the hepatocellular carcinoma cell line enhanced APOBEC3G expression. Incubation of the cells with interferon also resulted in the augmentation. These results may provide new insight into the pathogenesis of chronic HCV infection.     

Identification of distinct gamma-secretase complexes with different APH-1 variants

The gamma-secretase complex catalyzes the final intramembraneous cleavage of the beta-amyloid precursor protein, liberating the neurotoxic amyloid beta-peptide implicated in Alzheimer's disease. Apart from the catalytic subunit presenilin (PS), three additional subunits, nicastrin, APH-1, and PEN-2, have been identified. In mammals, two PS homologues, PS1 and PS2, which are part of distinct gamma-secretase complexes, exist. Likewise, two APH-1 homologues, APH-1a and APH-1b, have been identified. Furthermore, two APH-1a splice forms, APH-1aS and APH-1aL, have been reported. Here we show that both APH-1a splice forms and APH-1b are expressed in peripheral and neuronal cells. APH-1aS, APH-1aL, and APH-1b form separate, proteolytically active gamma-secretase complexes containing either one of the two PSs. Deficiency of APH-1a caused a decrease in nicastrin, PS, and PEN-2 levels and an increase in the levels of APH-1b, whereas deficiency of APH-1b did not affect the levels of APH-1a or the other complex components. Consistent with this finding, we found that deficiency of APH-1a was associated with reduced gamma-secretase activity, whereas deficiency of APH-1b was not. Thus, APH-1b gamma-secretase complexes may fulfill redundant functions. Taken together, our results suggest that, dependent on the tissue expression of the individual subunits, six distinct gamma-secretase complexes composed of the known subunits can exist in human cells.