Whole genome sequencing of Hepatitis C virus subtype 6xa and NS5A resistance mutations in a genotype 3b relapse case after direct-acting antiviral therapy in India

Introduction and Objectives: Pangenotypic direct-acting antivirals (DAA) are effective against highly prevalent Hepatitis C virus (HCV) subtypes, but have been clinically validated almost exclusively in high-income countries. Unusual HCV subtypes may carry natural polymorphisms, potentially impacting DAA susceptibility. We conducted full-genome characterization and resistance analysis of unusual HCV subtypes in patients receiving DAA treatment. Patients and methods: In this prospective hospital-based study, eligible patients were screened for anti-HCV antibodies and active infection was confirmed by diagnostic 5’NCR-based HCV RNA detection. Genotyping was performed by core region sequencing, and viral load quantified by real-time PCR. For whole genome sequencing, multiplex primers were designed using alignments of global reference sequences. Sequencing was carried out using the Oxford Nanopore Technology platform. Phylogenetic analysis used multiple sequence alignment and the HCV-GLUE resource for resistance-associated substitution (RAS) analysis. Results: Predominant genotype was genotype 3 in 64.3% (n=45); genotype 6 in 21.4% (n=15); and genotype 1 in 14.2% (n=10). Unusual HCV subtype 6xa was detected in two patients and showed no NS5A resistance mutations. One genotype 3b patient relapsed at 24 weeks post-DAA treatment completion and carried NS5A resistance-associated substitutions 30K and 31M both at baseline and at relapse, conferring high-level resistance to NS5A inhibitors. Conclusion: This is the first report from India of whole genome sequencing of HCV subtype 6xa. The identification of NS5A resistance mutations in the 3b relapse case underscores challenges for global HCV elimination strategies .