GENETIC DIVERSITY OF HEPATITIS C VIRUS AMONG BLOOD DONORS AND SYMPTOMATIC PATIENTS IN SOUTHWESTERN NIGERIA

Abstract

Hepatitis C virus (HCV) infection results in liver cirrhosis, hepatic failure or hepatocellular carcinoma (HCC). The virus exhibits extreme sequence variability, which has resulted in emergence of variants that are distributed differently. There is dearth of information on HCV diversity and its association with disease progression. This study was designed to investigate HCV strains and disease outcomes in blood donors and patients in Nigeria. Three hundred and one anti-HCV positive blood samples were collected from 99 symptomatic hepatitis patients, 125 HIV-infected individuals and 77 asymptomatic blood donors, over a period of three years (2014-2017) in Ibadan, Nigeria. Viral RNA was extracted from samples and reverse–transcribed to complimentary DNA and HCV nonstructural gene 5b (NS5B) amplified by PCR, using specific primers. The amplified products were sequenced by Sanger’s method and edited on CLC workbench and BioEdit programs. Basic Local Alignment Search Tool (BLAST) was used to compare gene sequences with those from GenBank and HCV databases. Sequence alignment was done on MEGA 7.0 and phylogenetic trees constructed using Neighbor-Joining method with bootstrap of 1000 replicates. Viral proteins were analysed on Netphos 3.1 software and data analysis was done using Fisher’s Exact test at Chiᴧ2 = 0.05. A total of 301 samples from individuals aged 3 months to 81 years were analysed. The NS5B genes were amplified by PCR in 60 (20%) of the samples, of which the gene was successfully sequenced in 42 with 97- 99% homology to HCV prototype H77 and sequences from around the world. Of the 42 isolates, 12 (28.6%), 5 (11.9%), 4 (9.5%), 1 (2.4%), 1 (2.4%) and 19 (45.2%) were subtypes 1a, 1b, 2b, 2c, 3a and 5a respectively, with 5a and 1a as predominant strains. Subtypes 1b and 2b were found only in patients with hepatitis, while subtypes 1a and 5a were predominant in blood donors and individuals with HIV respectively (p=0.0004). A 10% nucleotide variation was observed within isolates and 20% when compared with H77 prototype, while amino acid substitution varied among isolates with highest substitutions observed in symptomatic patients. Major clinically-important resistance mutations detected were S15G in 28 isolates, Q47H (35 isolates), T7N (24 isolates), S54L (22 isolates), G61R (23 isolates), and T79M (12 isolates). Analyses of viral proteins for conformational modifications revealed Serine/Threonine phosphorylated residues at positions 52 and 73, which are viral markers for disease progression. Four (30.8%) phosphorylated residues were found in hepatitis patients; 2 (15.4%) in blood donors and 7 (53.8%) in HIV-infected individuals (p=0.0400). Multiple hepatitis C virus genotypes circulate in Nigeria with predominance of genotype 5, found for the first time in West Africa. The observed variations in hepatitis C virus NS5B gene have implications for therapy. Routine survey of hepatitis C virus circulating in Nigeria is recommended.