Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus, belonging to the Flaviviridae family, and exhibits substantial global sequence diversity. The HCV genomic RNA consists of three distinct regions: a 5′... [ view full abstract ]
Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus, belonging to the Flaviviridae family, and exhibits substantial global sequence diversity. The HCV genomic RNA consists of three distinct regions: a 5′ untranslated region (UTR), a long open reading frame (ORF) coding precursor protein, and a short 3′ UTR. The discovery of a viable intergenotypic circulating recombinant form (CRF) HCV suggested that recombination can also lead to genetic diversity in HCV. Previously assumed to be infected by genotype 2, studies showed that RF1_2k/1b strain is common among ethnic Georgian patients. More than 70% of HCV genotype 2 patients are indeed infected with the RF1_2k/1b strain based on the discordant results from 5’UTR and 3’UTR amplification based genotyping methods. The purpose of the study was to confirm and characterize the presence of suspected HCV RF1_2k/1b strains among Georgian patients using next-generation sequencing technology.
We genotyped and analyzed two samples from chronic HCV patients enrolled in the national hepatitis C elimination program at the Infectious Diseases, AIDS and Clinical Immunology Research Center of Georgia. HCV viral load analyses was done using a Cobas Taqman (detection limit of 25 IU/ml). The initial HCV genotyping was done using an INNO line probe assay (LiPA). The next-generation sequencing of HCV was conducted at the Genome Center of National Center for Disease Control and Public Health (NCDC) Lugar Center. The RNA library preparation was conducted using NEB-Next Ultra RNA Library Prep Kit for Illumina (New England BioLabs). RNA was reverse transcribed, amplified (12 PCR cycles) using indexed primers, and then purified using Ampure XP beads (Beckman Coulter). Libraries were quantified (Qubit HS DNA Assay Kit; Invitrogen) and assessed for fragment sizes (Bioanalyzer 2100, High Sensitivity Kit; Agilent). Metagenomic (host-pathogen) RNA sequencing libraries were sequenced with MiSeq Sequencing Kit v3 (500 cycles). Low-quality bases were trimmed from demultiplexed sequences using a CLC Bio Workbench (8.5). Human sequences were excluded by map reads using the National Center for Biotechnology Information’s (NCBI’s) human genome reference. HCV-derived paired reads were assembled de novo into contigs and reads were mapped back to the assembly using CLC Bio Workbench (8.5). Genotypes 2k, 1b and recombinant RF 2k/1b, provided by NCBI, were used as a reference.
Our finding confirmed that the RF1_2k/1b recombinant strain is common among Georgian patients. The virus analyzed had a 5′ genome region that is most closely related to genetic subtype 2k, and a 3′ genome region that is most closely related to the global epidemic genetic subtype 1b and hence the virus genotype is designated as RF1_2k/1b.
Conclusion: HCV recombinant RF1_2k/1b appears to be widespread in Georgia, highlighting the need for further studies investigating recombination events in epidemiological studies. Also stressing the need for considering RF1_2k/1b HCV classification and nomenclature, as well as evaluating its diagnostic and clinical impact of the National Hepatitis C Elimination Program.
Sequencing strategies and technology advancements using the various NGS platforms , Human, non-human, and infectious disease applications
