Hepatitis C virus genotype 3a with phylogenetically distinct origin is circulating in Pakistan.
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ABSTRACT:
SUBMITTER: Rehman IU
PROVIDER: S-EPMC3023652 | biostudies-literature | 2011 Jan
REPOSITORIES: biostudies-literature
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Hepatitis C virus genotype 3a with phylogenetically distinct origin is circulating in Pakistan.
Rehman Irshad-Ur IU Idrees Muhammad M Ali Muhammad M Ali Liaqat L Butt Sadia S Hussain Abrar A Akbar Haji H Afzal Samia S
Genetic vaccines and therapy 20110106 1
<h4>Background</h4>Hepatitis C virus (HCV) is one of the leading causes of viral hepatitis worldwide and its genotype 3a is predominant in vast areas of Pakistan.<h4>Findings</h4>The present study reports the first full sequence of HCV 3a isolate PK-1 from Pakistan. This nucleotide sequence was compared with six other HCV genotype 3a full length sequences from different regions of the world by using statistical methods of phylogenetic analysis.<h4>Conclusion</h4>The nucleotide difference of thes ...[more]
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Project description:AimTo assess the association between chronic hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC) in Pakistan, and the genotype distribution among these HCC patients.MethodsOne hundred and sixty-one subjects with HCC were included in this study. Liver biopsy was performed on 145 of the patients; sixteen were excluded because they failed to fulfill the inclusion criteria. Qualitative polymerase chain reaction (PCR) was performed for hepatitis B virus and HCV. Samples positive for HCV RNA were genotyped using genotype-specific PCR and confirmed by HCV 5' noncoding region sequencing analysis.ResultsChronic HCV infection was identified a major risk factor (63.44% of tested HCC patients) for the development of HCC. The time from HCV infection to appearance of cancer was 10-50 years. In the HCC patient population, broader distributions of genotypes were present with genotype 3a as the predominant genotype. Using the type-specific genotyping method, we found HCV genotype 3a in 40.96%, 3b in 15.66%, 1a in 9.63%, and 1b in 2.40% of HCC tissue samples. About 28% of cases were found with mixed genotypes. Two cases were unable to be genotyped because of low viral load. Sixty-six percent of treated patients with cirrhosis had an end of treatment response, but unfortunately they relapsed quickly when the treatment was discontinued, and HCC developed during a median 3.8 years.ConclusionThere was a strong association between chronic HCV infection and HCC in Pakistan, and between HCV genotype 3a and HCC.
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Project description:BACKGROUND: Hepatitis C virus (HCV) is a common and leading cause for liver cirrhosis and hepatocellular carcinoma. Current therapies to treat HCV infection are shown to be partially effective and poorly tolerated. Therefore, ample efforts are underway to rationally design therapies targeting the HCV non-structural proteins. Most of the work carried out in this direction has been focusing mainly on HCV genotype 1. Two direct-acting antiviral agents (DAAs) Telaprevir and Boceprevir are being used against genotype 1a infection in combination therapy with interferon and ribavirin. Unfortunately these DAAs are not effective against genotype 3a. Considering the wide spread infection by HCV genotype 3a in developing countries especially South Asia, we have focused on the recombinant production of antiviral drug targets NS3 and NS5A from HCV genotype 3a. These protein targets are to be used for screening of inhibitors. RESULTS: High-level expression of NS3 and NS5A was achieved at 25°C, using ~1 and 0.5 mM Isopropyl ?-D-1-thiogalactopyranoside (IPTG), respectively. Yields of the purified NS3 and NS5A were 4 and 1 mg per liter culture volume, respectively. Although similar amounts of purified NS3 were obtained at 25 and 14°C, specificity constant (Kcat/Km) was somewhat higher at expression temperature of 25°C. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy revealed that both NS3 and NS5A contain a mixture of alpha-helix and beta-sheet secondary structures. For NS3 protein, percentages of secondary structures were similar to the values predicted from homology modeling. CONCLUSIONS: NS3 and NS5A were over-expressed and using Nickel-affinity method both proteins were purified to?~?95% purity. Yield of the purified NS3 obtained is four fold higher than previous reports. CD spectroscopy revealed that difference in activity of NS3 expressed at various temperatures is not related to changes in global structural features of the protein. Moreover, CD and FT-IR analysis showed that NS3 and NS5A contain both alpha-helical and beta-sheet structures and for NS5A, the proportion is almost equal. The production of NS3 and NS5A in milligram quantities will allow their characterization by biophysical and biochemical means that will help in designing new strategies to fight against HCV infection.
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Project description:Background & aimsSofosbuvir is a frequently used pan-genotype inhibitor of hepatitis C virus (HCV) polymerase. This drug eliminates most chronic HCV infections, and resistance-associated substitutions in the polymerase are rare. However, HCV genotype 3 responds slightly less well to sofosbuvir-based therapies than other genotypes. We collected data from England's National Health Service Early Access Program to search for virus factors associated with sofosbuvir treatment failure.MethodsWe collected patient serum samples and used the capture-fusion assay to assess viral sensitivity to sofosbuvir in 14 HCV genotype 3 samples. We identified polymorphisms associated with reduced response and created modified forms of HCV and replicons containing the substitutions of interest and tested their sensitivity to sofosbuvir and ribavirin. We examined the effects of these polymorphisms by performing logistic regression multivariate analysis on their association with sustained virologic response in a separate cohort of 411 patients with chronic HCV genotype 3 infection who had been treated with sofosbuvir and ribavirin, with or without pegylated interferon.ResultsWe identified a substitution in the HCV genotype 3a NS5b polymerase at amino acid 150 (alanine [A] to valine [V]), V at position 150 was observed in 42% of patients) with a reduced response to sofosbuvir in virus replication assays. In patients treated with sofosbuvir-containing regimens, the A150V variant was associated with a reduced response to treatment with sofosbuvir and ribavirin, with or without pegylated interferon. In 326 patients with V at position 150, 71% achieved an sustained virologic response compared to 88% with A at position 150. In cells, V at position 150 reduced the response to sofosbuvir 7-fold. We found that another rare substitution, glutamic acid (E) at position 206, significantly reduced the response to sofosbuvir (8.34-fold reduction); the combinations of V at position 150 and E at position 206 reduced the virus response to sofosbuvir 35.77-fold. Additionally, in a single patient, we identified 5 rare polymorphisms that reduced sensitivity to sofosbuvir our cell system.ConclusionsA common polymorphism, V at position 150 in the HCV genotype 3a NS5b polymerase, combined with other variants, reduces the virus response to sofosbuvir. Clinically, infection with HCV genotype 3 containing this variant reduces odds of sustained virologic response. In addition, we identified rare combinations of variants in HCV genotype 3 that reduce response to sofosbuvir.
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