Recombinant HCV type 2a Nonstructural Protein 5 [GST]

Genotyping of the hepatitis C virus (HCV) RNA can be performed by a variety of methods following polymerase chain reaction amplification of a stable RNA portion of the genome. The gold standard is amplification of the RNA from the NS5 region, followed by direct sequencing and homology comparison. This method is extremely labor intensive. In this study, we compared an immunoblot serotyping technique (HCV STA) to a reverse-hybridization line-probe assay (LiPA) for genotype classification among non-alcoholic HCV infected patients. We then compared and contrasted the response in this cohort to a population of alcoholic patients with HCV infection. To validate the serotype assay, sera from 110 patients with chronic HCV infection was utilized. Serotyping (Chiron SIA) and genotyping by the LiPA (Line Probe Assay, Innogenetics) reverse-hybridization technique was performed. Additionally, both methods were compared to sequence-derived genotyping in 26 patients based on PCR amplification of the NS5 region. After the validation phase, sera from 105 alcoholic patients was genotypically classified by the serologic method. The nonalcoholic and alcoholic groups were then compared with regard to serotype, demographics, and frequency of untypable test results. Among typable pairs, the overall concordance rate between serotyping and LiPA-based genotyping was 93.75%. Patients with genotype 1 by reverse hybridization demonstrated a 95.8% concordance with serotype. Untypable samples were present for both techniques, but since they occurred in different patients, the techniques were complementary Alcoholic patients were significantly more likely to be infected with untypable serotypes than those without a pattern of alcohol abuse. These patients were also more likely to be HCV RNA negative than sera from typable patients. Serotype 1 was associated with high HCV RNA titer and poor interferon treatment response among both nonalcoholic and alcoholic patients. An immunoblot method for the evaluation of genotype classification was rapid and easily performed compared to sequence-based genotyping. There was a high degree of concordance compared to reverse-hybridization and sequence-based genotype characterization methods, Failure to detect HCV RNA in the serum is associated with a higher likelihood of classification failure. This problem was particularly prevalent in the alcoholic population HCV RNA titers and treatment outcomes were strongly associated with serotype classification results, demonstrating clinical utility of this assay technique.

AIM: To study hepatic virus C (HCV) RNA and HCV protein expression in peripheral blood mononuclear cells (PBMCs) of patients with HCV infection, and explore the relationship between the HCV RNA in the PBMCs and response to interferon (IFN) therapy. METHODS: Type-specific primers were designed and RT- nested PCR was used to detect the plus- and minus- strands of HCV RNA in PBMCs of 54 patients with HCV infection; Indirect immunofluorescence assay was applied to identify HCVNS5 protein expression in PBMCs; 6 month-, 3 MU-IFN regiment was administrated to observe the responses to IFN in 35 chronic hepatitis C patients with different HCV RNA status in PBMCs. RESULTS: HCV plus strand RNA was found in 10 of 19 (52.6 %) acute hepatitis C patients and 22 of 35 (62.9 %) chronic hepatitis C patients. HCV minus strand RNA was detected in 14 of 35 (40.0 %) chronic hepatitis C patients, but only one patient (5.3 %) with acute HCV infection was found to be minus HCV RNA positive. Though no HCV NS5 protein expression was found in the examined 10 cases of acute HCV infection, it was positive in 17 of 20 (85.0 %) chronic hepatitis C patients by indirect immunofluoresence assay. There are significant differences of positive rate of the minus-strand and HCVNS5 protein between acute and chronic hepatitis C groups(u=2.07, P<0.05and u=4.43, P<0.01 respectively). The patients with minus-strand HCV RNA showed a significantly lower 6-month sustained response (SR6) to IFN compared to those without minus-strand HCVRNA in PBMCs (biologically 14.3 % vs 42.8 %, x(2) =4.12, P<0.05 and virologically 7.1 % vs 23.9 %, x(2) =4.24, P<0.05). CONCLUSION: HCV is capable of infecting and replicating in PBMCs, and HCVNS5 protein was expressed in PBMCs. The patients with minus strand HCV RNA in PBMCs showed a significantly lower 6-month sustained response to IFN, suggesting that minus-strand HCV RNA in PBMCs may be one of the factors influencing response to IFN therapy.