Penetration of polymeric nanoparticles loaded with an HIV-1 inhibitor peptide derived from GB virus C in a vaginal mucosa model.

Despite the great effort to decrease the HIV infectivity rate, current antiretroviral therapy has several weaknesses; poor bioavailability, development of drug resistance and poor ability to access tissues. However, molecules such as peptides have emerged asa new expectative to HIV eradication. The vaginal mucosa is the main spreading point of HIV. There are natural barriers such as the vaginal fluid which protects the vaginal epithelium from any foreign agents reaching it. This work has developed and characterized Nanoparticles (NPs) coated with glycol chitosan (GC), loaded with an HIV-1 inhibitor peptide (E2). In vitro release and ex vivo studies were carried out using the vaginal mucosa of swine and the peptide was determined by HPLC MS/MS validated method. Moreover, the peptide was labeled with 5(6)-carboxyfluoresceine and entrapped into the NPs to carried out in vivo studies and to evaluate the NPs penetration and toxicity in the vaginal mucosa of the swine. The mean size of the NPs, ξ and the loading percentage were fundamental features for to reach the vaginal tissue and to release the peptide within intercellular space. The obtained results suggesting that the fusion inhibitor peptides loaded into the NPs coated with GC might be a new way to fight the HIV-1, due to the formulation might reach the human epithelial mucosa and release peptide without any side effects.

Pace of Coreceptor Tropism Switch in HIV-1-Infected Individuals after Recent Infection.

HIV-1 entry into target cells influences several aspects of HIV-1 pathogenesis, including viral tropism, HIV-1 transmission and disease progression, and response to entry inhibitors. The evolution from CCR5- to CXCR4-using strains in a given human host is still unpredictable. Here we analyzed timing and predictors for coreceptor evolution among recently HIV-1-infected individuals. Proviral DNA was longitudinally evaluated in 66 individuals using Geno2pheno[coreceptor] Demographics, viral load, CD4+ and CD8+ T cell counts, CCR5Δ32 polymorphisms, GB virus C (GBV-C) coinfection, and HLA profiles were also evaluated. Ultradeep sequencing was performed on initial samples from 11 selected individuals. A tropism switch from CCR5- to CXCR4-using strains was identified in 9/49 (18.4%) individuals. Only a low baseline false-positive rate (FPR) was found to be a significant tropism switch predictor. No minor CXCR4-using variants were identified in initial samples of 4 of 5 R5/non-R5 switchers. Logistic regression analysis showed that patients with an FPR of >40.6% at baseline presented a stable FPR over time whereas lower FPRs tend to progressively decay, leading to emergence of CXCR4-using strains, with a mean evolution time of 27.29 months (range, 8.90 to 64.62). An FPR threshold above 40.6% determined by logistic regression analysis may make it unnecessary to further determine tropism for prediction of disease progression related to emergence of X4 strains or use of CCR5 antagonists. The detection of variants with intermediate FPRs and progressive FPR decay over time not only strengthens the power of Geno2pheno in predicting HIV tropism but also indirectly confirms a continuous evolution from earlier R5 variants toward CXCR4-using strains.IMPORTANCE The introduction of CCR5 antagonists in the antiretroviral arsenal has sparked interest in coreceptors utilized by HIV-1. Despite concentrated efforts, viral and human host features predicting tropism switch are still poorly understood. Limited longitudinal data are available to assess the influence that these factors have on predicting tropism switch and disease progression. The present study describes longitudinal tropism evolution in a group of recently HIV-infected individuals to determine the prevalence and potential correlates of tropism switch. We demonstrated here that a low baseline FPR determined by the Geno2pheno[coreceptor] algorithm can predict tropism evolution from CCR5 to CXCR4 coreceptor use.

[Cloning, Sequence Analysis and Expression of Recombinant E2 Protein of GB Virus C Genotype 7].

The purpose of this study was to explore the potential of the GB virus C(GBV-C)genotype 7E2 protein as a detection antigen for ELISA kit development. In this study, analyses of antigen epitopes, space structures and the linear B cell epitopes from the GBV-C genotype 7E2 protein were performed using an online analysis program. To establish a more reliable detection method for GBV-C studies, a 945bp gene fragment from GBV-C E2 was amplified by RT-PCR and ligated into the pET-32 a prokaryotic expression vector, which was then transformed into E. coli BL21 cells for protein expression. A protein with a molecular weight of 55 kDa was detected by 12% SDS-PAGE. The protein was found in inclusion bodies, and the His-tagged protein was detected by western blotting. The results showed that the cloned E2 gene sequence was 945 bp, and that the GBV-C E2 protein sequence had multiple antigenic epitopes. The recombinant protein formed inclusion bodies, which was consistent with expectations. These findings may provide the foundation for the development of a GBV-C detection kit.

A novel T cell evasion mechanism in persistent RNA virus infection.

Hepatitis C virus (HCV) and GB virus type C (GBV-C) are associated with impaired T cell function despite the fact that HCV replicates in hepatocytes and GBV-C in a small proportion of lymphocytes. Recently, we showed that HCV and GBV-C E2-envelope proteins reduce T cell activation via the T cell receptor (TCR) by competing for phosphorylation with a critical kinase in the TCR signaling cascade (Lck). E2 interfered with TCR signaling in E2 expressing cells and in bystander cells. The bystander effect was mediated by virus particles and extracellular microvesicular particles (exosomes). Multiple kinase substrate sites are predicted to reside on viral structural proteins and based on bioinformatic predictions, many RNA virus pathogens may interfere with TCR signaling via a similar mechanism. Identification of T cell inhibitory effects of virus structural proteins may provide novel approaches to enhance the immunogenicity and memory of viral vaccines.

HCV NS5A co-operates with PKR in modulating HCV IRES-dependent translation.

Translation initiation of the Hepatitis C virus (HCV) genome is driven by an internal ribosome entry site (IRES), located within the 5' non-coding region. Several studies have suggested that different cellular non canonical proteins or viral proteins can regulate the HCV IRES activity. However, the role of the viral proteins on HCV translation remains controversial. In this report, we confirmed previous studies showing that NS5A down-regulates IRES activity in HepG2 but not in Huh7 cells suggesting that the NS5A effect on HCV IRES is cell-type dependent. Additionally, we provide strong evidence that activated PKR up-regulates the IRES activity while silencing of endogenous PKR had the opposite effect. Furthermore, we present data indicating that the NS5A-mediated inhibitory effect on IRES-dependent translation could be linked with the PKR inactivation. Finally, we show that NS5A from GBV-C but not from GBV-B down-regulates HCV IRES activity in the absence or the presence of PKR over expression. Notably, HCV and GBV-C but not GBV-B NS5A contains a previously identified PKR interacting protein domain.

Modification of FP-HIV activity by peptide sequences of GB virus C: a biophysical approach.

Three synthetic peptide sequences of 18 amino acid each, corresponding to different fragments of the E2 capsid protein of GB virus C (GBV-C): SDRDTVVELSEWGVPCAT (P45), GSVRFPFHRCGAGPKLTK (P58) and RFPFHRCGAGPKLTKDLE (P59) have been characterized in order to find a relationship between their physicochemical properties and the results obtained in cellular models. Experiments were performed in presence and absence of the HIV fusion peptide (FP-HIV) due to the evidences that GBV-C inhibits AIDS progression. P45 peptide showed lower surface activity and less extent of penetration into 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) (3:2, mol/mol) lipid monolayers than P58 and P59. However, P45 peptide presented higher capacity to inhibit FP-HIV induced cell-cell fusion than the other two sequences. These results were supported by fluorescence anisotropy measurements which indicated that P45 had a significant effect on the inhibition of FP-HIV perturbation of liposomes of the same lipid composition. Finally, atomic force microscopy (AFM) studies have evidenced the modification of the changes induced by the FP-HIV in the morphology of lipid bilayers when P45 was present in the medium.

AIDS alters the commensal plasma virome.

We compared the plasma viromes of HIV-infected subjects with low versus high CD4(+) T cell counts from the United States and Uganda by using deep sequencing and detected HIV, hepatitis C virus, hepatitis B virus, GB virus C, anellovirus, and human endogenous retrovirus (HERV) reads. An increase in the proportion of reads for anelloviruses, a family of highly prevalent and genetically diverse human viruses, was seen in subjects with AIDS from both countries. The proportion of endogenous human retrovirus reads was increased in AIDS subjects from Uganda but not the United States. Progression to AIDS is therefore associated with changes in the plasma concentration of commensal viruses.

GB virus C particles inhibit T cell activation via envelope E2 protein-mediated inhibition of TCR signaling.

Viruses enter into complex interactions within human hosts, leading to facilitation or suppression of each other's replication. Upon coinfection, GB virus C (GBV-C) suppresses HIV-1 replication in vivo and in vitro, and GBV-C coinfection is associated with prolonged survival in HIV-infected people. GBV-C is a lymphotropic virus capable of persistent infection. GBV-C infection is associated with reduced T cell activation in HIV-infected humans, and immune activation is a critical component of HIV disease pathogenesis. We demonstrate that serum GBV-C particles inhibited activation of primary human T cells. T cell activation inhibition was mediated by the envelope glycoprotein E2, because expression of E2 inhibited TCR-mediated activation of Lck. The region on the E2 protein was characterized and revealed a highly conserved peptide motif sufficient to inhibit TCR-mediated signaling. The E2 region contained a predicted Lck substrate site, and substitution of an alanine or histidine for the tyrosine reversed TCR-signaling inhibition. GBV-C E2 protein and a synthetic peptide representing the inhibitory amino acid sequence were phosphorylated by Lck in vitro. The synthetic peptide also inhibited TCR-mediated activation of primary human CD4(+) and CD8(+) T cells. Extracellular microvesicles from GBV-C E2-expressing cells contained E2 protein and inhibited TCR signaling in bystander T cells not expressing E2. Thus, GBV-C reduced global T cell activation via competition between its envelope protein E2 and Lck following TCR engagement. This novel inhibitory mechanism of T cell activation may provide new approaches for HIV and immunoactivation therapy.

HIV-1 fusion is blocked through binding of GB Virus C E2-derived peptides to the HIV-1 gp41 disulfide loop [corrected].

A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors.

GB virus type C E2 protein inhibits human immunodeficiency virus type 1 assembly through interference with HIV-1 gag plasma membrane targeting.

GB virus type C (GBV-C) is a single-stranded positive-sense RNA virus classified in the Flaviviridae family. Persistent coinfection with GBV-C is associated with lower human immunodeficiency virus type 1 (HIV-1) load, higher CD4(+) T-cell count, and prolonged survival in HIV-1 coinfected patients. The GBV-C envelope glycoprotein E2 has been reported to interfere with HIV-1 entry. In this study, we showed that the expression of GBV-C E2 inhibited HIV-1 Gag assembly and release. Expression of glycosylated GBV-C E2 inhibited HIV-1 Gag precursor processing, resulting in lower production of CAp24 and MAp17, while the overall expression level of the Gag precursor Pr55 remained unchanged. Membrane floatation gradient and indirect immunofluorescence confocal microscopy analysis showed that glycosylated E2 disrupted HIV-1 Gag trafficking to the plasma membrane, resulting in Gag accumulation in subcellular compartments. This interference in HIV-1 Gag trafficking led to diminished HIV-1 particle production, which is a critical step for HIV-1 to infect new host cells. These findings shed light on a novel mechanism used by GBV-C E2 to inhibit HIV-1 replication and may provide insight into new approaches for suppressing HIV-1 replication.

Characterization of a peptide domain within the GB virus C envelope glycoprotein (E2) that inhibits HIV replication.

GB virus C (GBV-C) infection is associated with prolonged survival in HIV-infected cohorts, and GBV-C E2 protein inhibits HIV entry when added to CD4+ T cells. To further characterize E2 effects on HIV replication, stably transfected Jurkat cell lines expressing GBV-C E2 or control sequences were infected with HIV and replication was measured. HIV replication (all 6 isolates studied) was inhibited in all cell lines expressing a region of 17 amino acids of GBV-C E2, but not in cell lines expressing E2 without this region. In contrast, mumps and yellow fever virus replication was not inhibited by E2 protein expression. Synthetic GBV-C E2 17mer peptides did not inhibit HIV replication unless they were fused to a tat-protein-transduction-domain (TAT) for cellular uptake. These data identify the region of GBV-C E2 protein involved in HIV inhibition, and suggest that this GBV-C E2 peptide must gain entry into the cell to inhibit HIV.

Biophysical investigations of GBV-C E1 peptides as potential inhibitors of HIV-1 fusion peptide.

Five peptide sequences corresponding to the E1 protein of GBV-C [NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18), and AQLVGELGSLYGPLSVSA (P22)] were synthesized because they were capable of interfering with the HIV-1 fusion peptide (HIV-1 FP)-vesicle interaction. In this work the interaction of these peptides with the HIV-1 FP, as well as with membrane models, was analyzed to corroborate their inhibition ability and to understand if the interaction with the fusion peptide takes place in solution or at the membrane level. Several studies were carried out on aggregation and membrane fusion, surface Plasmon resonance, and conformational analysis by circular dichroism. Moreover, in vitro toxicity assays, including cytotoxicity studies in 3T3 fibroblasts and hemolysis assays in human red blood cells, were performed to evaluate if these peptides could be potentially used in anti-HIV-1 therapy. Results show that P10 is not capable of inhibiting membrane fusion caused by HIV-1 and it aggregates liposomes and fuses membranes, thus we decided to discard it for futures studies. P18 and P22 do not inhibit membrane fusion, but they inhibit the ability of HIV-1 FP to form pores in bilayers, thus we have not discarded them yet. P7 and P8 were selected as the best candidates for future studies because they are capable of inhibiting membrane fusion and the interaction of HIV-1 FP with bilayers. Therefore, these peptides could be potentially used in future anti-HIV-1 research.

Effect of E1(64-81) hepatitis G peptide on the in vitro interaction of HIV-1 fusion peptide with membrane models.

One way to gain information about the fusogenic potential of virus-derived synthetic peptides is to examine their interfacial properties and subsequently to study them in monolayers and bilayers. Here, we characterize the physicochemical surface properties of the peptide E1(64-81), whose sequence is AQLVGELGSLYGPLSVSA. This peptide is derived from the E1 structural protein of GBV-C/HGV which was previously shown to inhibit leakage of vesicular contents caused by the HIV-1 fusion peptide (HIV-1 FP). Mixed isotherms of E1(64-81) and HIV-1 FP were obtained and their Brewster angle microscopy (BAM) and atomic force microscopy (AFM) images showed that the peptide mixture forms a different structure that is not present in the pure peptide images. Studies with lipid monolayers (1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) and 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG)) show that both peptides interact with all the lipids assayed but the effect that HIV-1 FP has on the monolayers is reduced in the presence of E1(64-81). Moreover, differential scanning calorimetry (DSC) experiments show the capacity of HIV-1 FP to modify the properties of the bilayer structure and the capacity of E1(64-81) to inhibit these modifications. Our results indicate that E1(64-81) interacts with HIV-1 FP to form a new structure, and that this may be the cause of the previously observed inhibition of the activity of HIV-1 FP by E1(64-81).

Study of the inhibition capacity of an 18-mer peptide domain of GBV-C virus on gp41-FP HIV-1 activity.

The peptide sequence (175-192) RFPFHRCGAGPKLTKDLE (P59) of the E2 envelope protein of GB virus C (GBV-C) has been proved to decrease cellular membrane fusion and interfere with the HIV-1 infectivity in a dose-dependent manner. Based on these previous results, the main objective of this study was to deepen in the physicochemical aspects involved in this interaction. First, we analyzed the surface activity of P59 at the air-water interface as well as its interaction with zwitterionic or negatively charged lipid monolayers. Then we performed the same experiments with mixtures of P59/gp41-FP. Studies on lipid monolayers helped us to understand the lipid-peptide interaction and the influence of phospholipids on peptide penetration into lipid media. On another hand, studies with lipid bilayers showed that P59 decreased gp41-FP binding to anionic Large Unilamellar Vesicles. Results can be attributed to the differences in morphology of the peptides, as observed by Atomic Force Microscopy. When P59 and gp41-FP were incubated together, annular structures of about 200 nm in diameter appeared on the mica surface, thus indicating a peptide-peptide interaction. All these results confirm the gp41-FP-P59 interaction and thus support the hypothesis that gp41-FP is inhibited by P59.

Down-regulation of intra-hepatic T-cell signaling associated with GB virus C in a HCV/HIV co-infected group with reduced liver disease.

BACKGROUND & AIMS: Studies have shown that GB virus C (GBV-C) infection leads to reduced liver disease in hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infection. Considering that the underlying mechanism(s) are unknown, we aim to identify differential gene and protein expression associated with GBV-C in HCV/HIV co-infection that may be responsible for reduced liver disease. METHODS: Liver, peripheral blood mononuclear cells (PBMCs), and plasma samples were collected from 43 HCV/HIV patients. Plasma was tested for GBV-C RNA by RT-PCR with NS5B gene primers. A microarray was performed on the liver and RT-qPCRs on the liver/PBMC samples. Hepatic protein expression was measured by immunohistochemistry. RESULTS: Sixteen out of 43 patients had GBV-C RNA. GBV-C was associated with reduced hepatic fibrosis (p=0.005) and inflammation (p=0.007). The microarray analysis of the liver samples (n=10) showed down-regulation of genes critical to intra-hepatic T-cell signaling associated with GBV-C. Quantitative RT-PCR of the liver samples (n=13) confirmed the down-regulation of lymphocyte-specific protein tyrosine kinase (LCK) (p=0.02) and docking protein 2 (DOK2) (p=0.04). No differences in the expression levels of these genes were observed in PBMCs (n=22) according to the GBV-C status. The hepatic expression of the LCK protein, measured by immunohistochemistry (n=36), was decreased in CD3-positive T-cells within portal tracts associated with GBV-C (p=0.003). This remained significant in multivariate analysis controlling for hepatic fibrosis and inflammation (p=0.027). No differences were observed in plasma cytokine concentrations (n=25) or ex-vivo peripheral T-cell responses (n=13) versus GBV-C status. CONCLUSIONS: GBV-C infection is associated with down-regulation of critical genes involved in intra-hepatic T-cell signaling in HCV/HIV co-infection. This may be relevant to the pathogenesis of reduced HCV-related liver disease in HIV co-infection.

Capture of enveloped viruses using polymer tentacles containing magnetic latex particles.

A new rapid and efficient method for enveloped viruses' capture, purification and concentration has been developed. The approach is based on the use of functionalized magnetic latex particles as a carrier for capturing of the biological samples. After the capturing step, the magnetic particles are rapidly separated from the supernatant via simple permanent magnet. The nucleic acids are then directly extracted from the captured viruses on the magnetic particles using a commercial kit. The concentration methodology of viruses was first optimized for low-concentrated samples without loss of viral activity and infectivity (detected by HIV-1 p24 antigen production assay). Sensitivity of the capture assay was determined using PCR or RT-PCR method and the detection limit was found to be 10 and 16 copies for HGV and HBV respectively. The capture assay has been further used in the subtraction hybridization technique for identification of differentially expressed sequences. Finally, the lack of the tester and driver samples from the same patient by "viral positivation" of a negative serum has been circumvented using viral capture and transfer.

GB virus type C interactions with HIV: the role of envelope glycoproteins.

GB virus C/hepatitis G virus (GBV-C/HGV) is the most closely related human virus to hepatitis C virus (HCV). GBV-C is lymphotropic and not associated with any known disease, although it is associated with improved survival in HIV-infected individuals. In peripheral blood mononuclear cells, GBV-C induces the release of soluble ligands for HIV entry receptors (RANTES, MIP-1a, MIP-1b and SDF-1), suggesting that GBV-C may interact with lymphocytes to induce a chemokine and/or cytokine milieu that is inhibitory to HIV infection. Expression of GBV-C envelope glycoprotein E2 in CD4+ T cells or addition of recombinant E2 to CD4 cells recapitulates the HIV inhibition seen with GBV-C infection. Like HCV E2, GBV-C E2 is predicted to be post-translationally processed in the endoplasmic reticulum and is involved with cell binding. The C-termini of GBV-C E1 and E2 proteins contain predicted transmembrane domains sharing features with HCV TM domains. To date, cellular receptor(s) for GBV-C E2 have not been identified. GBV-C E2-mediated HIV inhibition is dose-dependent and HIV replication is blocked at the binding and/or entry step. In addition, a putative GBV-C E2 fusion peptide interferes with HIV gp41 peptide oligomerization required for HIV-1 fusion, further suggesting that GBV-C E2 may inhibit HIV entry. Additional work is needed to identify the GBV-C E2 cellular receptor, characterize GBV-C E2 domains responsible for HIV inhibition, and to examine GBV-C E2-mediated fusion in the context of the entire envelope protein or viral-particles. Understanding the mechanisms of action may identify novel approaches to HIV therapy.

An 85-aa segment of the GB virus type C NS5A phosphoprotein inhibits HIV-1 replication in CD4+ Jurkat T cells.

GB virus type C (GBV-C) is an apparently nonpathogenic virus that replicates in T and B lymphocytes and is a common cause of persistent human infection. Among HIV-1-infected individuals, persistent coinfection with GBV-C is associated with prolonged survival, and infection of blood mononuclear cells or CD4+ T cells with GBV-C and HIV in vitro results in significantly reduced HIV-1 replication. To date, the viral protein(s) that lead to HIV inhibition have not been identified. The GBV-C nonstructural phosphoprotein (NS5A) is predicted to have pleotropic effects on cells, including interactions with the IFN-induced dsRNA-activated protein kinase (PKR). We studied GBV-C NS5A to determine whether it is involved in inhibition of HIV replication. GBV-C NS5A protein from an isolate that was cleared by IFN therapy did not inhibit PKR, whereas NS5A from an isolate that was not cleared by IFN-inhibited PKR function in a yeast genetic system. Both of these GBV-C NS5A proteins were expressed in a CD4+ T cell line (Jurkat), and both induced a potent, dose-dependent inhibition of HIV-1 replication, thus the effect was independent of PKR inhibition. NS5A induced the release of the chemokine SDF-1 and decreased surface expression of the HIV coreceptor CXCR4, potentially explaining the HIV inhibition. Deletion mapping of the NS5A protein found that an 85-aa region between amino acids 152 and 237 inhibits HIV-1 replication. Thus, GBV-C NS5A protein alters the cellular milieu necessary for HIV-1 replication and may provide a previously undescribed therapeutic approach for anti-HIV therapy.

GB virus C/hepatitis G virus infection in Indian blood donors and high-risk groups.

The hepatitis G virus (HGV) or GB virus C (GBV-C) was discovered in 1995 as a putative agent of post-transfusion, non-A-E hepatitis. The present study was carried out with the aim to find the prevalence of this virus among various subject groups at risk for parenteral transmission as well as in healthy control subjects both individually and along with other parenterally transmitted hepatitis viruses. Of the 402 subjects tested, 6.22% were positive for the HBsAg surface antigen, 7.21% were positive for HCV RNA while only 2.24% were seen to be carriers of the HGV/GBV-C RNA. All the HGV/GBV-C positive cases were either multi-transfused thalassaemic subjects or hemodialysis patients. None of the healthy control subjects showed presence of the virus. Seven of the HGV/GBV-C positive subjects showed co-infection with one or more additional virological markers. Also, of the 9 HGV/GBV-C positive subjects, 5 showed elevated ALT levels while 4 showed elevated alkaline phosphatase levels. Overall our findings seem to indicate that HGV infections generally are asymptomatic, transient and self-limiting and the virus does not seem to show a very high prevalence among the Indian population.