GBV-C与艾滋病毒的相互作用及其机制

GBV-C（GB Virus C)是20世纪90年代中期发现的一种单股正链RNA病毒,属黄病毒科Pegivirus属,全基因组长约9.4 kb,编码约2 900个氨基酸序列.早期认为,该病毒与肝炎有关,但随后的研究发现,该病毒对人类无致病作用.最近的研究表明,GBV-C与艾滋病毒（人类免疫缺陷病毒,human immunodeficiency virus,HIV）共感染情况下可抑制HIV的增殖、提高机体免疫、延缓HIV 患者疾病进程.进一步研究GBV-C与HIV的相互作用及其机制可能会为艾滋病（acquired immune deficiency syndrome,AIDS）治疗提供新思路. 本文就GBV-C与HIV-1相互作用的两种主要类型--间接和直接的作用以及其机制进行了综述.     

Deep sequencing for the detection of virus-like sequences in the brains of patients with multiple sclerosis: detection of GBV-C in human brain

Multiple sclerosis (MS) is a demyelinating disease of unknown origin that affects the central nervous system of an estimated 400,000 Americans. GBV-C or hepatitis G is a flavivirus that is found in the serum of 1-2% of blood donors. It was originally associated with hepatitis, but is now believed to be a relatively non-pathogenic lymphotropic virus. Fifty frozen specimens from the brains of deceased persons affected by MS were obtained along with 15 normal control brain specimens. RNA was extracted and ribosomal RNAs were depleted before sequencing on the Illumina GAII. These 36 bp reads were compared with a non-redundant database derived from the 600,000+ viral sequences in GenBank organized into 4080 taxa. An individual read successfully aligned to the viral database was considered to be a "hit". Normalized MS specimen hit rates for each viral taxon were compared to the distribution of hits in the normal controls. Seventeen MS and 11 control brain extracts were sequenced, yielding 4-10 million sequences ("reads") each. Over-representation of sequence from at least one of 12 viral taxa was observed in 7 of the 17 MS samples. Sequences resembling other viruses previously implicated in the pathogenesis of MS were not significantly enriched in any of the diseased brain specimens. Sequences from GB virus C (GBV-C), a flavivirus not previously isolated from brain, were enriched in one of the MS samples. GBV-C in this brain specimen was confirmed by specific amplification in this single MS brain specimen, but not in the 30 other MS brain samples available. The entire 9.4 kb sequence of this GBV-C isolate is reported here. This study shows the feasibility of deep sequencing for the detection of occult viral infections in the brains of deceased persons with MS. The first isolation of GBV-C from human brain is reported here.     

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.     

Assessment of synthetic chimeric multiple antigenic peptides for diagnosis of GB virus C infection

The use of synthetic peptides of both structural and nonstructural proteins of GB virus C (GBV-C) has been studied for the development of new systems to diagnose infection caused by this virus. In an attempt to increase the antigenicity of linear peptide sequences, chimeric multiple antigenic peptides (MAPs) containing epitopes from E2, NS4, and NS5 GBV-C proteins have been synthesized. The synthetic constructs were evaluated by ELISA to establish whether the epitopes in chimeric branched peptides are more efficiently recognized by the specific antibodies compared to the monomeric linear sequences. Moreover, we have investigated the application of a commercial biosensor instrument for the detection of antibodies against the GBV-C in human serum samples. The results of the immunoassays reported in this work highlight the usefulness of synthetic tetrameric branched peptides containing sequences from envelope and nonstructural GBV-C proteins for the diagnosis of GBV-C infection. The potential clinical value of the MAP₄(E2-NS5a) for the serodiagnosis of GBV-C infection was demonstrated, thus providing the basis for performing prevalence studies of the infection among the hemodialyzed and hepatitis C virus (HCV)-infected population.     

GB virus-C/hepatitis G virus infection in Taiwan: A virus that fails to cause a disease?

Recently, an RNA virus designated GB virus-C or hepatitis G virus (GBV-C/HGV) was identified; however, its clinical significance remains uncertain. This discovery prompted us to investigate the virological, epidemiological and clinical implications of GBV-C/HGV infection in Taiwan where chronic liver diseases and liver cancer are endemic. Our results showed that genetic heterogeneity of GBV-C/HGV isolates exists, and primers from the highly conserved 5 untranslated region of viral genome can efficiently detect GBV-C/HGV RNA. Epidemiological surveys showed that GBV-C/HGV infection is common in high-risk groups in Taiwan, and its coinfection does not aggravate the course of chronic hepatitis B or C. A prospective study of transfusion-transmitted GBV-C/HGV infection also showed GBV-C/HGV does not cause classic hepatitis in most patients. In addition, GBV-C/HGV plays a minimal role in causing fulminant hepatitis. Like hepatitis C virus, sexual transmission of GBV-C/HGV exists. The risk increases with prolonged duration of exposure. In addition, high-titered maternal viremia and mode of delivery are associated with the mother-to-infant transmission of GBV-C/HGV. Interestingly, we found that GBV-C/HGV exerts no suppression on levels of chronic hepatitis B or hepatitis C viremia, and GBV-C/HGV responds to interferon; however, ribavirin plus interferon does not induce a higher sustained response. As to the replication sites of GBV-C/HGV, our preliminary results showed liver and peripheral blood mononuclear cells are not the major sites for GBV-C/HGV replication, and thus GBV-C/HGV is not a primary hepatotropic virus. In conclusion, transfusion and exchange of body fluids indeed can transmit GBV-C/HGV; however, current lines of evidence suggest that GBV-C/HGV fails to cause a disease.     

Translation initiation in GB viruses A and C: evidence for internal ribosome entry and implications for genome organization.

GB viruses A and C (GBV-A and GBV-C) are two recently described RNA viruses which appear to be members of the Flaviviridae. Although both viruses appear to contain long 5' nontranslated regions, the sites of polyprotein initiation and the presence of core-like proteins remain to be determined. Translation studies were undertaken to determine the mechanism and sites of polyprotein initiation in GBV-A and GBV-C. Rabbit reticulocyte lysates programmed with monocistronic RNAs containing 5' ends of GBV-A or GBV-C fused in-frame with the chloramphenicol acetyltransferase (CAT) open reading frame generated GBV-CAT fusion proteins in vitro. Site-specific mutagenesis and N-terminal sequencing located the sites of translation initiation immediately upstream of the putative signal sequence for the GBV E1 envelope glycoproteins. Efficient translation of the monocistronic GBV-CAT RNAs required the inclusion of GBV coding sequences. This, coupled with the presence of at least 523 nucleotides of 5' nontranslated RNA containing multiple AUG codons, suggests that translation initiation of these RNAs did not utilize a ribosome scanning mechanism. Translation of bicistronic RNAs containing 5' nontranslated sequences within the intercistronic space was consistent with the presence of a weakly active internal ribosome entry site in both GBV-A and GBV-C. Secondary structure predictions indicate that the 5' ends of these viruses assume similar complex structures distinct from those identified in the internal ribosome entry site-containing picornaviruses, pestiviruses, and hepatitis C viruses. The data indicate that GBV-A and GBV-C are unique members of the Flaviviridae that do not contain core-like proteins at the N termini of their putative polyproteins.     

Genomic variability of hepatitis G virus/GBV-C at the NS3 region: clinical implications

A new hepatitis virus, named GBV-C or hepatitis G virus (HGV), closely related to the hepatitis C virus (HCV), was identified in 1994. The existence of quasispecies in HCV is very important. In this work polymerase chain reaction amplification of the NS3 region of the genome of GBV-C/HGV and heteroduplex mobility assay (HMA) were combined to investigate the presence of quasispecies in patients with chronic infection by GBV-C/HGV. Patients with chronic infection by HCV were used to validate the method. The HMA was also used to investigate the similarity between the cited genomic region of GBV-C/HGV in different infected patients. A high degree of heterogeneity was found for HGV existing as quasispecies and as differences between samples. This is of extreme importance because of the intrinsic clinical and pathogenic implications of quasispecies of a virus capable of producing disease, and is in accord with other studies which report on the genomic variability of the NS3 region.     

Reconstructing the origins of human hepatitis viruses

Infections with hepatitis B and C viruses (HBV, HCV) are widespread in human populations throughout the world, and are major causes of chronic liver disease and liver cancer. HBV, HCV and the related hepatitis G virus or GB virus C (referred to here as HGV/GBV-C) are capable of establishing persistent, frequently lifelong infections characterized by high levels of continuous replication. All three viruses show substantial genetic heterogeneity, which has allowed each to be classified into a number of distinct genotypes that have different geographical distributions and associations with different risk groups for infection. Information on their past transmission and epidemiology might be obtained by estimation of the time of divergence of the different genotypes of HCV, HBV and HGV/GBV-C using knowledge of their rates of sequence change. While information on the latter is limited to short observation periods and is therefore subject to considerable error and uncertainty, the relatively recent times of origin for genotype of each virus predicted by this method (HCV, 500-2000 years; HBV, 3000 years; HGV/GBV-C, 200 years) are quite incompatible with their epidemiological distributions in human populations. They also cannot easily be reconciled with the recent evidence for species-associated variants of HBV and HGV/GBV-C in a range of non-human primates. The apparent conservatism of viruses over long periods implied by their epidemiological distributions instead suggests that nucleotide sequence change may be subject to constraints peculiar to viruses with single-stranded genomes, or with overlapping reading frames that defy attempts to reconstruct evolution according to the principles of the 'molecular clock'. Large population sizes and intense selection pressures that optimize fitness may be additional factors that set virus evolution apart from that of their hosts.     

Hepatitis B Virus with X Gene Mutation Is Associated with the Majority of Serologically “Silent” Non-B,Non-C Chronic Hepatitis

Hepatitis B virus (HBV) with X gene mutations has been a putative pathogen of chronic hepatitis without serological markers of known hepatitis viruses. The aim of this study was to reconfirm whether the HBV with the X gene mutation is associated with these serologically “silent” non-B, non-C (NBNC) chronic hepatitis, alcoholic liver disease (ALD) and autoimmune hepatitis (AIH). HBV DNA was amplified from serum and sequenced in 30 patients with NBNC chronic hepatitis in comparison with 20 patients with ALD and 5 patients with AIH. HBV DNA was identified in 21 patients (70%) in NBNC chronic hepatitis by nested polymerase chain reaction while only one patient (5%) in ALD and none in AIH showed HBV DNA. Eighteen (85.7%) of the 21 identified HBV DNAs had an identical 8-nucleotide deletion mutation at the distal part of the X region. This mutation affected the core promoter and the enhancer II sequence of HBV DNA and created a translational stop codon which truncated the X protein by 20 amino acids from the C-terminal end. All the HBV DNAs had a precore mutation at the 83rd nucleotide resulting in disruption of HBe antigen synthesis. These results indicate that HBV mutants are closely associated with the majority of serologically “silent” NBNC chronic hepatitis cases and the population of such mutant HBV DNAs is not uniform.     

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.     

Detection of HCV and GBV-CHGV RNA in peripheral blood mononuclear cells of patients with chronic type C hepatitis

Hepatitis C virus (HCV) and hepatitis G virus (HGV) viraemia were investigated by RT-PCR protocols in peripheral blood mononuclear cells (PBMC) of 22 patients with chronic type C hepatitis. Samplings were at basal and 4-8 months after a 12 month period of treatment with interferon-alpha. A plus strand of HCV in PBMC was detected in 8 of 21 patients (38%) (p <0.05; chi2 test) with a lack of response to therapy; a minus strand was detected in 10% of chronic type C hepatitis and 25% of the patients harboured HCV RNA in PBMC. The association with a response was nearly significant (p <0.1; chi2 test). GBV-C/HGV RNA was detected in the serum of 9 of 21 (43%) patients and in PBMC of 20% of the patients viraemic. Genomic sequences of GBVC/HGV in PBMC were found, but further investigation is needed to assess the findings reported for HCV.     

Structural Constraints on RNA Virus Evolution

The recently discovered hepatitis G virus (HGV) or GB virus C (GBV-C) is widely distributed in human populations, and homologues such as HGV/GBV-CCPZ and GBV-A are found in a variety of different primate species. Both epidemiological and phylogenetic analyses support the hypothesis that GB viruses coevolved with their primate hosts, although their degree of sequence similarity appears incompatible with the high rate of sequence change of HGV/GBV-C over short observation periods. Comparison of complete coding sequences (8,500 bases) of different genotypes of HGV/GBV-C showed an excess of invariant synonymous sites (at 23% of all codons) compared with the frequency expected by chance (10%). To investigate the hypothesis that RNA secondary-structure formation through internal base pairing limited sequence variability at these sites, an algorithm was developed to detect covariant sites among HGV/GBV-C sequences of different genotypes. At least 35 covariant sites that were spatially associated with potential stem-loop structures were detected, whose positions correlated with positions in the genome that showed reductions in synonymous variability. Although the functional roles of the predicted secondary structures remain unclear, the restriction of sequence change imposed by secondary-structure formation provides a mechanism for differences in net rate of accumulation of nucleotide substitutions at different sites. However, the resulting disparity between short- and long-term rates of sequence change of HGV/GBV-C violates the assumptions of the "molecular clock." This places a major restriction on the use of nucleotide or amino acid sequence comparisons to calculate times of divergence of other viruses evolving under the same structural constraints as GB viruses.     

Partial nucleotide sequencing of the NS3/helicase region of hepatitis G virus to prove vertical transmission

To study non-parental transmission of hepatitis G virus and/or GB virus C (HGV/GBV-C), we sequenced and compared the NS3/helicase region of the virus for five HGV/GBV-C RNA-positive mothers and their 11 children who had experienced neither blood transfusion nor overt hepatitis and were negative for HBV, HCV and HIV, except in one mother coinfected with HCV. The nucleotide sequences of the familial HGV/GBV-C isolates showed high similarity of 99-100% (mean 99.8%, 100% at the deduced amino acid level) between mother and her child(ren) in each family. These findings strongly suggest the spontaneous occurrence of mother-to-child transmission of HGV/GBV-C as reported previously. They also suggest that nucleotide sequence analysis on the NS3/helicase region of HGV/GBV-C may be a useful tool to study HGV/GBV-C transmission.     

Autoantibody profiling of Chinese patients with autoimmune hepatitis using immunoproteomic analysis

In the present study, immunoproteomic analysis was utilized to systemically characterize global autoantibody profiles in autoimmune hepatitis (AIH). Sera from 21 patients with AIH and 15 healthy controls were analyzed for the antibody reactivity against the protein antigens of HepG2, a human hepatoma cell line. The lysates of HepG2 cells were separated by two-dimensional electrophoresis and then immunoblotted with each serum sample. Matrix-assisted laser desorption/ionization mass spectrometry or/and nanoelectrospray ionization MS/MS were then used to identify antigens, among which a bifunctional enzyme in mitochondrial, fumarate hydratase (FH), was further analyzed by ELISA using recombinant FH as a coating antigen. A total of 18 immunoreactive spots were identified as 13 proteins, 8 of which have not been reported in AIH. Immune reactivity to FH was detected in 66.67% of patients with AIH, 19.35% of patients with primary biliary cirrhosis (PBC), 12.31% of patients with chronic hepatitis B (CHB), 6.35% of patients with chronic hepatitis C (CHC), 11.32% of patients with systemic lupus erythematosus (SLE), and 3.57% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P<0.001). These data demonstrate the serological heterogeneity in AIH and suggest the diversity of the mechanisms underlying AIH. FH, recognized mainly in AIH rather than in viral hepatitis and other autoimmune diseases, may have utility in improved diagnosis of AIH.     

Characterization of GBV-C infection in HIV-1 infected patients

BACKGROUND: GB virus C, a positive-stranded RNA virus, is classified in the family Flaviviridae. It is currently believed that persistent infection occurs in 25-50% of infected individuals, however, it still remains an "orphan" virus in search of a role in human pathology. Molecular epidemiological studies have demonstrated that GBV-C infection is present in about 1-1.4% of the healthy population in developed countries, that it shares routes of transmission with HIV and HCV and that the prevalence of GBV-C in these populations is higher than in blood donors. On the basis of the sequence variation among the isolates, GBV-C is classified into at least four major genotypes. Preliminary evidence has suggested that GBV-C is a lymphotropic virus that replicates mainly in the spleen and bone marrow. Recently, several reports have investigated the possible beneficial effect of GBV-C co-infection on HIV disease progression to AIDS, reduced mortality in HIV infected individuals and lower HIV viral loads, not leading to a definitive conclusion yet. AIM: To investigate the role of GBV virus C co-infection in two different subsets of HIV-infected patients, and to evaluate the prevalence of GBV-C genotypes in Northern Italy. METHODS: A total of 86 HIV positive patients were examined for GBV-C viremia (years after HIV sera conversion: 12 +/- 5). Control population (Group A): 46 patients (mean age 42 years) with <200CD4/ml during the observation period. Longterm non progressor population (Group B): 40 patients, (mean age 40 years) with >500 CD4/ml for at least 8 years and never treated with HAART. After extraction of viral RNA from plasma samples, amplification of a highly conserved region of 5'UTR was performed by nested RT-PCR. All positive samples were genotyped by sequencing, alignment with published sequences and phylogenetic analysis. CD4 cell count, HIV plasma levels were also evaluated. RESULTS: 9 out of 46 (19.56%) in Group A and 15 out of 40 (37.5%) in Group B had detectable GBV-C viremia (p=0.064, OR 2.47, percent confidence interval 0.94 to 6.51). No statistical difference was observed when disease stage was evaluated between the two groups. In Group B, after regression analysis for CD4 cell count decrease over the period observed, no significant difference was detected between GBV-C positive and negative patients. No significant difference was observed in Group B in HIV viremia and CD4 cell count at time of GBV-C detection between GBV-C infected patients and GBV-C negative patients. All Italian patients were genotype 2, the only African patient carried GBV-C genotype 1. CONCLUSIONS: Although previous results suggest that GBV-C virus may be a favorable marker for long term non progression of HIV disease, whether it plays a direct anti-HIV role or just takes advantage of non progessors' higher CD4 cell count to replicate more efficiently, still remains to be answered. Follow up of untreated patients and further evaluation of virological interactions, between the viruses and the host immune system, will be helpful to shed some light on these observations, offering new prognostic and eventually therapeutical tools for the management of HIV patients.     

Informational gene phylogenies do not support a fourth domain of life for nucleocytoplasmic large DNA viruses

Mimivirus is a nucleocytoplasmic large DNA virus (NCLDV) with a genome size (1.2 Mb) and coding capacity ( 1000 genes) comparable to that of some cellular organisms. Unlike other viruses, Mimivirus and its NCLDV relatives encode homologs of broadly conserved informational genes found in Bacteria, Archaea, and Eukaryotes, raising the possibility that they could be placed on the tree of life. A recent phylogenetic analysis of these genes showed the NCLDVs emerging as a monophyletic group branching between Eukaryotes and Archaea. These trees were interpreted as evidence for an independent "fourth domain" of life that may have contributed DNA processing genes to the ancestral eukaryote. However, the analysis of ancient evolutionary events is challenging, and tree reconstruction is susceptible to bias resulting from non-phylogenetic signals in the data. These include compositional heterogeneity and homoplasy, which can lead to the spurious grouping of compositionally-similar or fast-evolving sequences. Here, we show that these informational gene alignments contain both significant compositional heterogeneity and homoplasy, which were not adequately modelled in the original analysis. When we use more realistic evolutionary models that better fit the data, the resulting trees are unable to reject a simple null hypothesis in which these informational genes, like many other NCLDV genes, were acquired by horizontal transfer from eukaryotic hosts. Our results suggest that a fourth domain is not required to explain the available sequence data.     

Cytochrome P4502D6(193-212): a new immunodominant epitope and target of virus/self cross-reactivity in liver kidney microsomal autoantibody type 1-positive liver disease

Cytochrome P4502D6 (CYP2D6), target of liver kidney microsomal autoantibody type 1 (LKM1), characterizes autoimmune hepatitis type 2 (AIH2) but is also found in patients with chronic hepatitis C virus (HCV) infection. To provide a complete linear epitope B cell map of CYP2D6, we tested peptides spanning the entire sequence of CYP2D6. In addition to confirming previously described antigenic sites, we identified four new epitopes (193-212, 238-257, 268-287, and 478-497). CYP2D6(193-212) is immunodominant and was the target of 12 of 13 (93%) patients with AIH2 and 5 of 10 (50%) HCV/LKM1-positive patients. Because LKM1 is present in both AIH2 and a viral infection, we tested whether Abs to CYP2D6(193-212) arise through cross-reactive immunity between virus and self. We identified a hexameric sequence "RLLDLA" sharing 5 of 6 aa with "RLLDLS" of HCV(2985-2990) and all 6 aa with CMV(130-135). Of 17 CYP2D6(193-212)-reactive sera, 11 (7 AIH and 4 HCV) reacted by ELISA with the HCV homologue, 8 (5 AIH and 3 HCV) with the CMV homologue, and 8 (5 AIH and 3 HCV) showed double reactivity. Autoantibody binding to CYP2D6(193-212) was inhibited by preincubation with HCV(2977-2996) or CMV(121-140). Recombinant HCV-nonstructural protein 5 and CMV-UL98 proteins also inhibited Ab binding to CYP2D6(193-212). Affinity-purified CYP2D6(193-212)-specific Ab inhibited the metabolic activity of CYP2D6. The demonstrated similarity and cross-reactivity between CYP2D6(193-212) and two unrelated viruses suggests that multiple exposure to viruses mimicking self may represent an important pathway to the development of autoimmunity.