CONFERENCE ON RETROVIRUSES
AND OPPORTUNISTIC INFECTIONS

Boston, Massachusetts
March 8–11, 2020

 

Conference Dates and Location: 
February 13–16, 2017 | Seattle, Washington
Abstract Number: 
140

IDENTIFICATION OF HCV INTER-SUBTYPE RECOMBINANT STRAINS WITH BREAKPOINTS IN NS5A

Author(s): 

Suqin Cai1, Alicia Newton1, Wei Huang1, Jeannette Whitcomb1

1Monogram BioScis, South San Francisco, USA

Abstract Body: 

Hepatitis C virus (HCV) is classified into seven major genotypes (GT) and numerous subtypes. HCV GT and subtype are associated with differences in clinical outcomes, response to treatment and epidemiology. A better understanding of recombination in HCV is of practical importance and evolutionary interest.

Recombinant HCV strains were identified during routine drug resistance testing of clinical specimens. HCV RNA was isolated from plasma and NS5A amplicons were generated by RT-PCR and sequenced on a next-generation sequencing platform. Sequencing reads were analyzed using a proprietary analysis pipeline with a subtyping routine that aligns a subset of reads to a library of reference HCV strains. Samples with a large proportion of reads that aligned to two different references were evaluated as potential recombinant viruses. Consensus sequences were compared to subtype reference sequences (Bootscan, Simplot) to define recombination breakpoints. Phylogenetic analysis was performed (Clustal) to determine relatedness of independently collected HCV specimens.

Ten 1a/1b recombinant viruses were identified from diverse geographic collection centers. All 10 had 5' NS5A 1a sequences and 3' NS5A 1b sequences. The recombination breakpoint for 7 of these viruses was consistently at amino acid (aa) 386 and for 2 of the viruses at aa 366 of NS5A relative to the H77 reference. All 9 of these 1a/1b recombinants had the same recombination locus in 1b at aa 348 relative to the Con1 reference. A single 1a/1b recombinant had a 1a breakpoint at aa 277 (H77) and a 1b breakpoint at aa 214 (Con1). The recombinant NS5As were longer than H77 and Con1 NS5A due to duplication of sequences at the site of recombination. Phylogenetic analysis demonstrated that the 9 1a/1b recombinant viruses with breakpoints at 366/386 in 1a and 348 in 1b were all closely related suggesting that this may represent a circulating recombinant form of HCV. We also identified one inter-genotype recombinant (1a/4), with a breakpoint in NS5A at aa 349 of H77 and at aa 339 of the GT4 ED43 reference.

HCV recombination is thought to be rare. However, the prevalence of recombinant forms may have been underestimated due to the use of genotyping methods, such as single-locus sequencing, that are unlikely to detect recombination. The independent identification of a similar recombinant strain from multiple samples and diverse geographic collection areas suggests that the strain exists as a circulating recombinant form.

Session Number: 
O-9
Session Title: 
HEPATITIS C: PROBLEMS AND PROGRESS
Presenting Author: 
Suqin Cai
Presenter Institution: 
Monogram Biosciences