Sequencing Analysis of NS3/4A, NS5A, and NS5B Genes from Patients Infected with Hepatitis C Virus Genotypes 5 and 6

Abstract

Direct-acting antivirals (DAAs) with activity against multiple genotypes of the hepatitis C virus (HCV) were recently developed and approved for standard-of-care treatment. However, sequencing assays to support HCV genotype 5 and 6 analysis are not widely available. Here, we describe the development of a sequencing assay for the NS3/4A, NS5A, and NS5B genes from HCV genotype 5 and 6 patient isolates. Genotype-and subtype-specific primers were designed to target NS3/4A, NS5A, and NS5B for cDNA synthesis and nested PCR amplification. Amplification was successfully performed for a panel of 32 plasma samples from HCV-infected genotype 5 and 6 patients with sequencing data obtained for all attempted samples. LiPA 2.0 (Versant HCV genotype 2.0) is a reverse hybridization line probe assay that is commonly used for genotyping HCV-infected patients enrolled in clinical studies. Using NS3/4A, NS5A, and NS5B consensus sequences, HCV subtypes were determined that were not available for the initial LiPA 2.0 result for genotype 6 samples. Samples amplified here included the following HCV subtypes: 5a, 6a, 6e, 6f, 6j, 6i, 6l, 6n, 6o, and 6p. The sequencing data generated allowed for the determination of the presence of variants at amino acid positions previously characterized as associated with resistance to DAAs. The simple and robust sequencing assay for genotypes 5 and 6 presented here may lead to a better understanding of HCV genetic diversity and prevalence of resistance-associated variants. Globally, 130 to 150 million people have chronic hepatitis C virus (HCV) infection. Without treatment, chronic hepatitis may lead to liver cirrhosis and hepatocellular carcinoma and the possible need for a liver transplantation (1-3). Hepatitis C virus has high genetic diversity and is classified into seven genotypes and at least 67 subtypes among the genotypes (4, 5). At the nucleotide level for the open reading frame, there is a 31 to 33% difference between genotypes and 20 to 25% difference between subtypes (6).HCV genotype 1 is the most prevalent genotype and is widely distributed across the globe. Genotype 1 accounts for the majority of the HCV cases in North America, northern and western Europe, South America, Asia, and Australia (7,8). Similarly, genotypes 2 and 3 have broad geographical distribution. In contrast, genotypes 4, 5, and 6 are common only in specific regions. Genotype 4 and genotype 5 are endemic to Egypt and South Africa, respectively. Genotype 6 has the highest prevalence in Southeast Asia and southern China. Although genotypes 5 and 6 are not widespread, there are an estimated 9.8 million and 1.4 million people infected with genotypes 5 and 6, respectively, worldwide (9-13). Better treatment options are needed for genotype 5-and 6-infected patients.Due to the high prevalence and broad geographical distribution of genotype 1, early HCV research and development efforts were focused on this genotype. With the recent approval of HCV direct-acting antivirals (DAAs) active against multiple genotypes, such as Sovaldi (...

Sequencing of hepatitis C virus for detection of resistance to direct‐acting antiviral therapy: A systematic review

“…The HCV genotype 4‐specific method, Ref084, covered all key DAA RASs and reported the most in‐depth validation of all genotype 4‐specific methods identified. The HCV subtype 5a‐specific method, Ref035, covered all key DAA RASs and was the only method specific for genotype 5 that was identified. No methods covering all DAA RASs in NS5A designed to amplify genotype 6 were identified.…”

Efficacy and safety of direct-acting antiviral therapy for chronic hepatitis C genotype 6

“…A total of 1026 records were excluded after scanning titles and abstracts. As a result, 49 full-text articles were subjected to detailed evaluation, of which, in one study, patients were coinfected with HIV [7] ; 4 papers were review articles [8–11] ; 2 studies did not include patients infected with genotype 6 HCV [12,13] ; 1 study had a smaller sample size than the other study from the same region with the same topic [14] ; 10 studies did not have relevant outcomes [13,15–23] ; 6 studies were repeat reports [24–28] ; 8 studies included <10 patients infected with genotype 6 HCV. [29–35] Finally, 7 randomized-controlled trials and 10 cohorts were chosen for inclusion in the meta-analysis, which comprised a total of 3343 patients.…”

Systematic review: epidemiology and response to direct‐acting antiviral therapy in genotype 6 chronic hepatitis C virus infection

“…A LDV/SOF trial by Gane et al found NS5A baseline polymorphism in all GT6 patients but negative in a patient who experienced virologic relapse, who instead, had NS5B variant S282T (polymorphism that is associated with resistance to SOF) detected at the time of relapse. Both NS3 and NS5A RASs were found in a patient who experienced virologic breakthrough in a study by Asselah et al (T93A in NS3 sequence and A156M in NS5A sequence, which were both associated with resistance to PIB and GLE respectively), yet high SVR rates were noted in these trials . Thus, based on the limited data available, baseline RASs seems to be common but minimally impact treatment outcome in GT6 patients.…”