HCV Strains Research Articles

Upregulation of endogenous intrahepatic interferon stimulated genes during chronic hepatitis C virus infection.

The success of interferon-alpha and ribavirin combination therapy for the treatment of chronic hepatitis C viral infection differs between patients. In an attempt to identify predictors of host response to therapy, the levels of mRNA for interferon (IFN) stimulated genes: MxA, PKR, 2'5' OAS, ISG15, and interleukin 8 (IL-8), were examined in liver by real-time RT-PCR prior to commencement of therapy. The levels of intrahepatic classical IFN stimulated genes, but not IL-8, in chronic HCV disease (n = 44) were found to be significantly upregulated (P < 0.001) compared to the control cohort (n = 12). The genotype of the infecting HCV strain did not influence IFN stimulated gene expression. These results suggest that the endogenous type 1 IFN antiviral effector pathway is broadly activated during chronic HCV disease, although the levels of mRNA for any of the IFN-stimulated genes tested did not predict the outcome of combination therapy.

Expression of hepatitis C virus hypervariable region 1 and its clinical significance.

To explore the properties of hypervariable region 1 (HVR1) in the envelope 2 gene of hepatitis C virus by analyzing the reactivity of HVR1 fusion proteins from different Chinese HCV strains with sera of patients with chronic hepatitis C and by comparing their reactivity between interferon therapy responders and non-responders. Gene fragments of HVR1 of four HCV strains (three genotype 1b and one genotype 2a) were amplified from pGEMT-E2 plasmids and sub-cloned into pQE40 vectors respectively to construct recombinant expression plasmids which expressed HVR1 fused downstream to DHFR in Escherichia coli strain TG1. The purified DHFR- HVR1 proteins were then used to detect the anti-HVR1 antibodies in 70 serum samples of patients with chronic hepatitis C. Four DHFR- HVR1 fusion proteins were successfully expressed in E.coli (320-800 ug fusion proteins per 100 ml culture). Each fusion protein (SH1b, BJ1b, SD1b and SD2a) reacted with 72.8 % (51/70), 60 % (42/70), 48.6 % (34/70), and 58.6 % (41/30) of the anti-HCV positive patients' sera respectively by ELISA. 57 % (4/7) of non responders reacted with all four HVR1 fusion proteins, while only 15.3 % (2/13) of responders reacted with all of them. The O.D. values of sera from IFN therapy responders were significantly higher than those of non responders (P<0.05). The selected HVR1 fusion proteins expressed in E. coli can broadly react with HCV-infected patients' sera. The intensity and/or quality of the immune response against HCV may be a critical factor determining the response to interferon treatment. With the evolution of virus strains, anti-HVR1 antibodies can not neutralize all the quasispecies. A polyvalent and high immunogenic vaccine comprising a mixture of several HVR1 sequences that cover the reactivity of most HCV isolates may be useful.

Multiple infections with different HCV genotypes: prevalence and clinical impact.

In a HCV genotype 3a-infected patient, viremia with a different genotype (1b) was detected after 16 weeks of ineffective therapy. Serological typing revealed that this genotype had already been present prior to therapy. To investigate the epidemiology of multiple HCV infections and the therapeutical consequences for patients superinfected with a new HCV strain. Sera of 600 patients were screened for infection with multiple genotypes by using sequencing and a serological assay in parallel. Infection with two different HCV types was detected in 13 patients. The prevailing strain was genotyped by sequencing. From two of these patients additional sera were available which had been drawn up to 24 and 28 months prior to the current sample, respectively. Those early samples showed viremia with a HCV subtype that could not be detected by PCR afterwards. Only antibodies to the initial strain were detectable in the later samples. In patients serially infected by different HCV strains, one strain will prevail as the viremic virus. Under antiviral therapy, the displaced strain may become viremic again and may influence the outcome of therapy. Detection of inferior strains by serological assays before antiviral therapy may be important for choosing the adequate regimen.

Outbreak of nosocomial hepatitis C virus infection resolved by genetic analysis of HCV RNA.

In July 2000, symptomatic acute hepatitis C was diagnosed in five patients who had attended the emergency room of a municipal hospital on the same day, about 6 weeks before. Investigation of the remaining 65 patients visited at the emergency room on that day disclosed that 8 patients had a positive anti-hepatitis C virus (anti-HCV) test and 4 of them had biochemical evidence of acute anicteric hepatitis. HCV RNA was detected in 12 of the 13 anti-HCV-positive patients. Phylogenetic analysis of the nonstructural 5A (NS5A) and E2 regions showed that 10 patients, including all 9 with acute hepatitis, were infected with a closely related HCV strain, while the remaining 2 patients harbored unrelated strains. Flushing of intravenous catheters with heparin retrieved from a multidose heparin solution in saline was carried out for all the patients involved in the hepatitis outbreak but in only 1 of 23 (4%) matched controls recruited among HCV-noninfected patients attending the emergency room on the same day, and this was the only significant difference concerning risk factors for HCV infection between patients and controls. Thus, accidental contamination of a multidose heparin solution with blood from an unrecognized HCV carrier was identified as the source of this nosocomial outbreak of hepatitis C.

Expression and purification of recombinant full-length NS3 protease–helicase from a new variant of Hepatitis C virus

Viral mRNA extracted from the serum of a patient infected with HCV strain 1a was used for cloning, expression, and purification of full-length Hepatitis C NS3 protein. Sequencing of the protease gene identified the virus to be a new variant closely related to strain H77, differing in 15 out of 631 amino acids in the NS3 protein, none of which were predicted to be directly involved in catalysis, binding of substrate, or cofactor. A pBAD expression system was used to express the enzyme with an N-terminal tag in Escherichia coli. Purification from the soluble cellular fraction was achieved by Ni 2+-IMAC and PolyU Sepharose affinity chromatography. The dependence of the proteolytic activity of the full-length NS3 protein on ionic strength, glycerol concentration, and a peptide corresponding to the activating region of NS4A was analyzed and used to design an activity assay that is suitable for inhibition studies. The kinetic constants ( k cat and K M) for catalysis and the inhibitory potencies (IC 50 and K i) of five product-based hexapeptide inhibitors were comparable to those reported for the truncated NS3 protein. Detailed kinetic and inhibition studies using this variant of full-length NS3 can increase the understanding of the enzymatic characteristics of NS3, reveal the importance of the substituted amino acids and the significance of the genetic variability for design of effective inhibitors of the virus, and is thus of relevance for drug discovery.

Genetic diversity and response to IFN of the NS3 protease gene from clinical strains of the hepatitis C virus

The N-terminal one-third of the hepatitis C virus nonstructural gene 3 (NS3) codes for a serine protease. To investigate natural genetic diversity of this enzyme a nested PCR reaction was developed to obtain NS3 protease sequence data directly from patient strains. This data was used to determine genetic diversity, phylogenetic and evolutionary rates, and selection of variants by interferon therapy. The potential effect of genetic diversity on enzyme structure using molecular modeling was also attempted. Results show significant variability in clinical HCV strains at both the nucleotide (30.2% for 1a and 25.8% for 1b) and amino acid sequences (11.0% for 1a and 9.9% for 1b). Phylogenic analysis shows two distinct clades with two HCV isolates grouping as a sister clade to 1b. Structural analysis reveals that most mutations lie in the N-terminus of the enzyme. When strains were sorted as to whether or not the patient had received antiviral therapy, no difference was found in the number or locations of mutations in 1a strains. However, 1b strains demonstrated an overall drop in the number of positions that were mutated. This study demonstrates significant differences among natural strains that may pose a problem for structure based drug development.

A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg.

Hepatitis C virus (HCV) evolution is thought to proceed by mutations within the six genotypes. Here, we report on a viable spontaneous HCV recombinant and we show that recombination may play a role in the evolution of this virus. Previously, 149 HCV strains from St. Petersburg had been subtyped by limited sequencing within the NS5B region. In the present study, the core regions of 41 of these strains were sequenced to investigate the concordance of HCV genotyping for these two genomic regions. Two phylogenetically related HCV strains were found to belong to different subtypes, 2k and 1b, according to sequence analysis of the 5' untranslated region (5'UTR)-core and the NS5B regions, respectively. By sequencing of the E2-p7-NS2 region, the crossover point was mapped within the NS2 region, probably between positions 3175 and 3176 (according to the numbering system for strain pj6CF). Sequencing of the 5'UTR-core regions of four other HCV strains, phylogenetically related to the above-mentioned two strains (based on analysis within the NS5B region), revealed that these four strains were also recombinants. Since a nonrecombinant 2k strain was found in St. Petersburg, the recombination may have taken place there around a decade ago. Since the frequency of this recombinant is now high enough to allow the detection of the recombinant in a fraction of the city's population, it seems to be actively spreading there. The reported recombinant is tentatively designated RF1-2k/1b, in agreement with the nomenclature used for HIV recombinants. Recombination between HCV genotypes must now be considered in the classification, laboratory diagnosis, and treatment of HCV infection.

Shift in predominating subtype of HCV from 1b to 3a in St. Petersburg mediated by increase in injecting drug use*

The genotypes of 149 HCV strains from St. Petersburg were determined by limited sequencing and phylogenetic analysis within the NS5B region. One hundred two strains derived from patients that attended infectious disease clinics, of whom 48 admitted injecting drug use, and 47 derived from dialysis patients. Subtype 3a was predominant in the patients from infectious disease clinics, both in patients that admitted injecting drug use (56%) and in those with unknown source of infection (46%). However, 89% of the strains from dialysis patients belonged to subtype 1b. Eleven of twelve characterised strains from recent cases of hepatitis C at these units were at phylogenetic analysis shown to be related to strains already circulating there, demonstrating that within the dialysis units nosocomial transmission is the most important route of HCV infection. The predominance of subtype 1b strains in dialysis patients indicates that these strains have been circulating for a long time in dialysis units. The predominance of subtype 3a also among patients who did not admit drug use and that their strains were intermixed with the strains from injecting drug users in the phylogenetic analysis shows that the increase in injecting drug use is the major factor that explains the recent spread of HCV in the St. Petersburg population. This supports the concept that injecting drug use remains the major route for HCV infection in developed countries and that the control of drug abuse is the most important measure to prevent its spread.

Competition and selection among different HCV strains

Competition and selection among different HCV strains

Competition and selection among different HCV strains

Competition and selection among different HCV strains

Molecular analysis of HCV type 1 to 5 envelope gene: application to investigations of posttransfusion transmission of HCV.

Until 1990, HCV infection was common in transfused patients, resulting in more than 200,000 cases of posttransfusion hepatitis C in France alone. A molecular method that permits the investigation of posttransfusion hepatitis C infections is presented. Viral sequences in the envelope region of HCV were obtained for 12 pairs of blood recipients and their respective blood donors. The HCV strains studied belonged to types 1 (subtypes 1a and 1b), 2, 3, 4, and 5. Genetic distances and mutation rates were determined, and sequences were submitted to phylogenetic analysis along with sequences retrieved from nucleotide databases. Pairwise distances in the donor-recipient pairs were found to be less than 0.05 mutation per site, which corresponds to a mutation rate ranging from 0.6 x 10(-3) to 2.1 x 10(-3) per site per year. Sequences obtained from the 12 donor-recipient pairs clustered in 12 monophyletic nests. The genetic analysis of the envelope region of HCV can be used for the forensic evaluation of virus transmission. It permits the refutation of a link between blood transfusion and HCV transmission, rather than proof of the existence of such a link.

Role of the specific T-cell response for clearance and control of hepatitis C virus.

T cells are believed to be the main players in antiviral defence. To investigate the role of the specific CD4+ T cell response for clearance and control of the hepatitis C virus we studied patients with acute hepatitis C (AHC) during the phase of spontaneous viral clearance and during follow up after elimination of the virus and resolution of disease. Symptomatic AHC has a self-limited course in 50% of patients, whereas the other half show virus persistence and develop chronic course of disease. Patients who were able to mount a vigorous, polyclonal, multispecific, TH1 lymphokine dominated CD4+ T-cell response showed viral clearance and a self-limited course of disease. In contrast, absence of this T-cell response in patients with AHC invariably led to viral persistence and chronic hepatitis. The characteristics of the T-cell response were as follows: it was mainly directed against nonstructural proteins of the virus, it was multispecific and demonstrated immunodominant epitopes, and the majority of T-cell clones established from our patients responded to a single peptide (NS3 amino acid 1248-1261) within the helicase region of HCV. Presentation of the peptide was HLA DR specific, the peptide showed promiscuous binding, and it had high binding affinity to 10 of the most common 13 HLA DR alleles, thus patients with diverse HLA DR backgrounds could mount an immune response. Furthermore, the epitope was conserved in 100% of 33 HCV strains published in databases. This strong initial CD4+ T-cell response is not sufficient for a definitive recovery from AHC, it has to be maintained to control the hepatitis C virus. Loss of the response after initial resolution of disease is followed by relapse. Even 20 years after an episode of self-limited AHC with elimination of HCV, we have observed a significant virus-specific CD4+ T-cell response. Our data indicate the decisive role of the virus-specific CD4+ T-cell response for clearance and control of HCV, and contribute to our understanding of immune mechanisms by which the host defends the HCV virus. This is a prerequisite for the development of new strategies to efficiently defend the virus by manipulating or modulating the immune response.

Maintenance of a virus specific helper T cell response 20 years after infection with one strain of HCV in healthy individuals

Maintenance of a virus specific helper T cell response 20 years after infection with one strain of HCV in healthy individuals

Markers for Transfusion- Transmissible Infections in North Indian Voluntary and Replacement Blood Donors: Prevalence and Trends 1989-1996

Background and Objectives: Twenty five to 30% of multiple-transfusion recipients in India show evidence of infection with both HBV and non-A non-B hepatitis or HCV To be licensed, blood banks must screen each donor unit for HBsAg, antibodies to HIV-1 and -2, and VDRL. Materials and Methods: Between 1989 and 1996, 132,093 voluntary and replacement donors at this centre were screened for the above markers, using commercially available kits. Some 19,531 donors were screened for HCV antibodies in 1995 and 1996 with an inhouse EIA, using a synthetic peptide from core, NS3 and NS4 region proteins of all major HCV strains. Results: Data were tabulated annually. The proportion of voluntary donors increased from 15 to 32% during the eight years. HBsAg rates remained below 2.5%, antibodies to HIV increased from 0.04% to 0.55%, and VDRL reactivity increased from 0.23 to 0.52% between 1989 and 1995. Prevalence of all three infections showed a small but significant drop in 1996, and all three were significantly less frequent in voluntary donors. HCV antibodies were detected in 1.49% of donors tested. Donors with multiple infections were uncommon. Conclusions: A change to voluntary blood transfusion service and addition of anti-HBc, anti-HCV, and possibly HIV antigen to the mandatory list for screening donor blood would reduce posttransfusion infections. Indigenous production of these assays would mitigate the financial burden, as has been the experience with the in-house HCV assay.

Hepatitis C virus in a hemodialysis unit: Molecular evidence for nosocomial transmission

Between February 1991 and January 1992, elevated alanine aminotransferase (ALT) levels were observed in several hemodialysis patients in a dialysis center in Dendermonde, Belgium. By the end of 1992, 25 out of 68 patients had seroconverted for HCV antibodies. The HCV strains from 23 of these seroconverters were genotyped and classified as genotype 1b. Sequence analysis of the HCV Core region was carried out in 12 patients, 9 of whom were infected with a strain bearing a unique sequence motif as compared with the currently known HCV 1b strains. A new 5' UR/Core line probe assay was designed to screen for such variations. Twenty patients tested positively for this special sequence motif, while the other 3 showed the regular subtype 1b sequence. Phylogenetic analysis of the Core sequences further revealed that the latter three were neither related to the main special strain of the infection, nor to each other. These three strains could be traced to two patients already infected at the time of residence in other dialysis units and to one patient who already showed ALT elevations in 1989. Epidemiological studies revealed no traceable source for this outbreak. In conclusion, molecular analysis demonstrates nosocomial transmissions by a peculiar genotype 1b strain in a dialysis center. Three other genotype 1b strains were also present in the unit, but were not responsible for the outbreak.

Hepatitis C infection in renal transplant patients: new insights and unanswered questions

The prevalence of HCV infection in a population of renal transplantation patients is mostly dependent on that preexisting before transplantation. It also has been demonstrated that HCV infection can be transmitted by the renal graft. Although grafting an HCV+ kidney does not affect survival 5 years after surgery, the risk incurred by recipients on the longer term is unclear. A suggestion has been made to reserve HCV+ kidneys for recipients who are themselves HCV+. However, it has been established that a given HCV strain has little chance of inducing immunity to a different HCV strain. This is why the use of HCV+ kidneys no longer meets consensus. It could be considered to match the recipient and the graft with regard to the HCV strain when genotype identification is routinely available, quick and reliable. Immunosuppressive therapy enhances viral replication. Its long-term effect on the course of HCV disease is unclear. In particular, no studies have compared the long-term outcome of HCV+ patients treated by haemodialysis and transplantation. The data available on the 10-year outcome of HCV+ grafted patients are nonetheless reassuring. At least they allow considering renal transplantation to non-cirrhotic HCV+ patients. Several issues related to the interaction between HCV and immunosuppressive therapy remain to be clarified. Does the viral strain play a role in the course of infection under immunosuppressive treatment? Does immunosuppressive treatment promote strain mutagenesis? Does HCV infection require modulation of the immunosuppressive treatment?

Hepatitis C vims RNA detection and HCV genotype in patients with chronic non-A, non-B hepatitis in Jakarta

Antibody response in HCV infection may be variable and the variability of the serological response could be due to the differences in HCV strains. Since the distribution of hepatitis C virus genotype has been found to be geographically dependent, it is important to determine the distribution of HCV genotype in various countries with high prevalence of chronic non-A, non-B hepatitis. In this study, serum HCV RNA was examined in 53 patients suspected of chronic non-A, non-B hepatitis with an anti-HCV test as determined by currently available assay. HCV viremia was detected in 48 patients (90.6%). These patients had elevated serum ALT level at the time of HCV RNA determination. Using specific genotype probes, all isolates were classified into three different genotypes. Double and triple infections were also noted. HCV genotype 1b is the predominant genotype found in chronic hepatitis C patients in Jakarta.

Hepatitis C in liver transplantation: preliminary study of prognostic factors.

At the University of Miami liver transplantation for chronic liver disease in HCV-positive patients has shown good results, with a 92% patients survival rate (follow up 8 to 57 months, median 21). None the less, we found that a large number of patients are expected to develop serious histological graft damage and may need retransplantation, which may place a further strain on the already scarce donor resources. We have conducted a preliminary investigation on the importance of parameters which may correlate with the prognosis of HCV grafts. We found no impact of HLA match or typing. An interesting hypothesis, which deserves further investigation, is that some HCV strains could be more virulent than others and play a role as an independent risk factor. We have identified six strains among our patients and the BK serotype shows a trend to be associated with a worse outcome. We have found that patients developing and maintaining higher liver enzyme levels (ALT and GGT) after transplant and those with higher levels of viremia may be at risk to develop serious damage to their grafts.

Antigen detection in human respiratory Coronavirus infections by monoclonal time-resolved fluoroimmunoassay

Background: The diagnosis of respiratory infections by detecting viral antigens has received considerable attention using immunofluorescent assays (IFA) and enzyme immunoassays (EIA). Time-resolved fluoroimmunoassay (TR-FIA) has been developed for several viruses. Objectives: To prepare monoclonal antibodies to coronavirus strains, to incorporate them into a TR-FIA, and test the assay on clinical specimens. Study design: Monoclonal antibodies were prepared to the N nucleoprotein of the two human respiratory coronaviruses, HCV strains 229E and OC43. Monoclonals to both viruses were completely type-specific; they did not cross-react between themselves or with multiple strains of other respiratory viruses. These antibodies were configured into optimized EIA and TR-FIA tests. The all-monoclonal tests were then compared to polyclonal EIA tests in terms of their ability to detect virus in clinical specimens. Results: The all-monoclonal TR-FIA was uniformly the most sensitive, detecting virus in all 13 229E-positive specimens compared to 69% for the monoclonal EIA and 54% for the polyclonal EIA test. Similar results were obtained for 10 OC43-positive specimens: 100% in TR-FIA, 90% in monoclonal EIA, and 80% in polyclonal EIA. For 229E in TR-FIA, mean positive/negative (P/N) ratios were 143 for 229E-positive human embryonic lung fibroblast (HLF) cell culture fluids and 10 for positive nasopharyngeal aspirate specimens; for OC43 in TR-FIA, mean P/N values were 964 for OC43-positive rhabdomyosarcoma (RD) cell culture fluids and 174 for positive NPA specimens. The sensitivities of the TR-FIA were determined with purified virions to be 0.308 ng virus per well for HCV-229E and 0.098 ng virus per well for HCV-OC43. Conclusions: This rapid and sensitive test appears to be much more sensitive than traditional antigen detection assays but will require more extensive field testing on clinical specimens.

The impact of hepatitis C virus infection on renal allograft recipients

A second generation hepatitis C virus recombinant immunoblot assay (RIBA) was used to screen stored perioperative serum from 641 renal allograft recipients. One hundred and nine (17%) were anti-HCV positive at the time of transplant. RIBA positivity was found to be an independent predictor of post-transplant liver disease in a logistic regression model (P < 0.05). Moreover, RIBA positive patients were at greater risk for infectious events (P = 0.03) and rejection episodes (P = 0.002). The cumulative dose of antilymphoblast globulin administered as induction therapy was an independent predictor of post-transplant liver disease in a dose response relationship. Qualitative PCR showed that 74% of the perioperative RIBA positive patients had detectable HCV RNA in a current serum sample. Further, quantitative HCV RNA analysis with a competitive template PCR and HCV strain identification by restriction fragment length polymorphism demonstrated a large range of HCV RNA copies/ml of serum and three different HCV strains (BK, Hutch and HCV-1). Neither quantity of HCV RNA nor strain type correlated with abnormal transaminases post-transplant. As yet, there has not been an effect of anti-HCV status on actuarial patient and graft survival. This study suggests that anti-HCV is not a contraindication to renal transplantation; however, we would recommend that the pre-transplant evaluation of the anti-HCV positive patient include a liver biopsy to properly stage the disease. Close post-transplant follow-up is required in view of the increased risk for infection and rejection.