Abu-Baker Sulaiman Ismael
University of Manchester, UK
Posters & Accepted Abstracts: J Antivir Antiretrovir
New antiviral drugs that inhibit the HCV polymerase enzyme are beginning to be used in the treatment of hepatitis C virus infection, particularly in the hard to treat non-genotype 2 and 3 infected patients. The efficacy of these new antivirals is limited by the presence of those mutations that give rise to amino-acid substitutions within the targeted protein and affect the viral sensitivity to these compounds. The continued emergence of HCV antiviral resistance to polymerase inhibitors reinforces the urgent requirement for development of novel amplification (PCR) and sequencing method for NS5B gene which is responsible for replication of HCV. This project developed a method for amplifying and sequencing the HCV polymerase gene in order to determine antiviral drug resistance in different isolates of genotype 1 hepatitis C infected patients. The sensitivity of one step and two-step reverse transcriptase polymerase chain reactions was compared in order to optimize amplification of genotype 1 HCV NS5B region. Higher sensitivity was observed in the two-step RT-PCR using random hexamers than the one step RT-PCR using NS5B genespecific primers. Moreover, a combination of four In-House designed primers was compared with published sets of primers for amplification of HCV genotype 1a and 1a variants, using the same optimized protocol and PCR conditions. After optimization of the whole assay a total of 20 sera specimens were tested and we found that the two-step RT-PCR is more sensitive than the one step reaction. In addition, the sensitivity of the In-House primers was significantly higher than the published primers using the two step- RTPCR method. Overall, 19 samples were sequenced and analyzed for determination of resistance mutations to different nucleoside and non-nucleoside inhibitors. After the analysis, C316N and V499A which are natural polymorphisms in the HCV genotype 1 NS5B region and may be associated with antiviral resistance were found in 5.25% and 84.2% of the sequenced samples, respectively.
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