Next-generation sequencing studies on the E1-HVR1 region of hepatitis C virus (HCV) from non-high-risk HCV patients living in Punjab and Khyber Pakhtunkhwa, Pakistan.

The incidence rate of hepatitis C virus (HCV) infection in Pakistan is very high. In this study, we evaluated the genetic heterogeneity of HCV hypervariable region 1 (HVR1) from the HCV-infected Pakistani population and compare the isolated genotypes with representative sequences from internationally diverse geographic regions. We also investigated potential transmission events in non-high-risk HCV patients. Next-generation sequencing (NGS) data from the E1-HVR1 region from 30 HCV patients were used for phylogenetic analysis. Reference sequences were retrieved from the Los Alamos HCV and GenBank databases. NGS data were analyzed to examine HCV HVR1 sequence diversity and identify transmission links among HCV-infected individuals using Global Hepatitis Outbreak and Surveillance Technology (GHOST). Phylogenetic analysis showed the predominance of HCV genotype 3a (86.6%), followed by 1a (6.6%), 1b (3.3%), and 3b (3.3%). NGS of HVR1 displayed significant genetic heterogeneity of HCV populations within each patient. The average nucleotide sequence diversity for HVR1 was 0.055. JR781281 was found to be the most diverse (0.14) of the specimens. Phylogenetic analysis demonstrated that all HCV specimens sequenced in this study were more similar to each other and showed variations from the representative sequences. The GHOST results suggested genetic relatedness between two (6.6%) HCV cases, possibly defining an incipient outbreak in a non-high-risk population. We urge rigorous countrywide investigation of outbreaks to identify transmission clusters and their sources to incorporate preventive measures for disease control.