Analysis of the NS3 protease gene across hepatitis C viral genotypes in treatment-naive individuals

Abstract

In the world, approximately 71 million people are infected with hepatitis C virus (HCV) and Egypt has the highest prevalence. Estimates of 27% hepatitis C cases are reported in sub-Saharan Africa. South Africa has approximately 0.91% HCV cases. This raises concerns as many of the infected individuals have no knowledge of their HCV status. With the rise in the various new direct acting antiviral drugs (DAAs), targeting different regions of the viral genome, it is important to subtype strains and to identify the type of mutations found within circulating strains in South Africa. Identification of resistance mutations to drug therapy will help in the policy and treatment guidelines specific to the South African population. The aim of this study was to identify and describe mutations in the non-structural (NS3) gene across HCV genotypes circulating in South Africa. In order to achieve the aim, the objectives were as follows; to determine whether naturally occurring variants in the NS3 region across different genotypes occur, to molecularly characterize naturally occurring NS3 variants across different genotypes, to compare genotype 5a NS3 variants to that of other genotypes (1 & 5a) and map epitope rich areas on the NS3 protein. This was a retrospective, descriptive study using stored HCV-RNA positive plasma. Several techniques including RNA extraction, cDNA synthesis, reverse transcriptase polymerase chain reaction (RT-PCR), polymerase chain reaction (PCR), cloning, Sanger and next generation sequencing (NGS) were executed. Phylogenetic and mutational analyses were performed using MEGA 7 and BioEdit 7.1.3 software packages. A web-based prediction server Immune Epitope Database (IEDB) was used to map epitope-rich regions of the HCV NS3 protein. The NS3 gene was amplified for all the samples using an in-house optimised recombinant Thermococcus kodakaraensis (KOD) PCR protocol with published primers as well as a commercial assay (DeepChek) PCR protocol. A total of 39 positive amplicons were gel purified and sequenced. Sanger sequencing results were compared to NGS. Phylogenetic analyses indicated that the majority of the study isolates subtyped as 1a or 1b, clustered with strains from the USA, Australia and France. Both subtypes are globally distributed. Genotype 5a appeared to be clustering with the USA strains. By NGS, we found resistance-associated mutations (I18V, S122A/G, A170V and D168Y) to NS3 proteases for viral genotypes 1b or 5a. Mutation D168Y was observed in both subtype 1b and subtype 5a. Infected individuals with this mutation have shown reduced susceptibility to protease inhibitors, Faldaprevir, Simeprevir and Vaniprevir. The well-characterized Q80K polymorphism, frequently observed in subtype 1a treatment naïve individuals elsewhere, was not observed in this study. Other known resistance mutations reported for genotype 1 (V36A, T54S and A156T) were observed in this study by NGS but not with Sanger sequencing. NGS was opted in order to characterize the minor mutations. Results from the NGS reported mutations V36L/G and S122R that were not observed by Sanger sequencing. NGS results were obtained at 5% frequency supporting the specificity of the NGS method as compared to the conventional sequencing 20% Majority of the observed epitopes showed high binding affinity to the HLA A2*01 across the HCV genotypes. In conclusion, we found resistance-associated mutations to NS3 proteases for viral genotypes 1b and 5a. However, the well-published resistance mutation for genotype 1 (Q80K) was rarely observed. Although NGS has reduced cost and time, navigating through the mega dataset could be challenging. The high copy number variant might lead to less accurate sequencing by the NGS because of inherent statistical methods used for assembly. Sanger sequencing is still a preferred method for clinical application. Therefore, more studies to screen for mutations need to be conducted to foster the understanding of the impact of mutations on drug resistance and progression of disease. The development of a vaccine capable of preventing chronic HCV infections remains the most feasible and realistic method of managing HCV infection globally.