Hepatitis C Virus (HCV) NS3 Sequence Diversity and Antiviral Resistance-Associated Variant Frequency in HCV/HIV Coinfection

Abstract

HIV coinfection accelerates disease progression in chronic hepatitis C and reduces sustained antiviral responses (SVR) to interferon-based therapy. New direct-acting antivirals (DAAs) promise higher SVR rates, but the selection of preexisting resistanceassociated variants (RAVs) may lead to virologic breakthrough or relapse. Thus, pretreatment frequencies of RAVs are likely determinants of treatment outcome but typically are below levels at which the viral sequence can be accurately resolved. Moreover, it is not known how HIV coinfection influences RAV frequency. We adopted an accurate high-throughput sequencing strategy to compare nucleotide diversity in HCV NS3 protease-coding sequences in 20 monoinfected and 20 coinfected subjects with well-controlled HIV infection. Differences in mean pairwise nucleotide diversity (), Tajima's D statistic, and Shannon entropy index suggested that the genetic diversity of HCV is reduced in coinfection. Among coinfected subjects, diversity correlated positively with increases in CD4 ؉ T cells on antiretroviral therapy, suggesting T cell responses are important determinants of diversity. At a median sequencing depth of 0.084%, preexisting RAVs were readily identified. Q80K, which negatively impacts clinical responses to simeprevir, was encoded by more than 99% of viral RNAs in 17 of the 40 subjects. RAVs other than Q80K were identified in 39 of 40 subjects, mostly at frequencies near 0.1%. RAV frequency did not differ significantly between monoinfected and coinfected subjects. We conclude that HCV genetic diversity is reduced in patients with well-controlled HIV infection, likely reflecting impaired T cell immunity. However, RAV frequency is not increased and should not adversely influence the outcome of DAA therapy.

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

“…Using a fixed cutoff can retain a large proportion of offspring sequences, introducing skewing from rerepresentation of templates with the abundant Primer ID reads as the apparent templates of offspring Primer IDs. Second, previous studies used the Roche 454 platform, which has a much lower throughput compared to the MiSeq platform used in the present study (21,24,25), which, along with the Ion Torrent platform, has a high error rate at homopolymer runs (26,27) that is not a feature of MiSeq platform. When the number of raw sequence reads per template is not sufficient (due either to low capacity or too much multiplexing), the peak of the Primer ID read distribution will be shifted toward the low-abundance error end, making template recovery significantly less than optimal (Fig.…”

“…The technology of using a degenerate block of nucleotides as indexing tags for NGS has been introduced in both DNA template sequencing (20) and RNA virus sequencing (6,21). However, adoption of this technology has been slowed by several con- cerns.…”

Development and Validation of Two Screening Assays for the Hepatitis C Virus NS3 Q80K Polymorphism Associated with Reduced Response to Combination Treatment Regimens Containing Simeprevir

“…The MiSeq assay could potentially be used in cases in which the detection of minority resistance variants is important, although the performance of the assay in that context has not fully been evaluated. However, given the absence of minority variants carrying the Q80K polymorphism detected in this and previous studies (23,24) and that the population prevalence of Q80K is likely due to a founder effect rather than active selection (15), deep sequencing specifically for the NS3 Q80K polymorphism may be unnecessary.…”

“…Previous studies have examined the prevalence of the Q80K polymorphism in treatment-naive and treatment-experienced populations and have demonstrated a substantial disparity in the prevalence of the Q80K polymorphism in GT1-infected populations in North America versus Europe (11,14,(21)(22)(23)(24). We have demonstrated that both Sanger sequencing of a 1,200-bp and/or 564-bp fragment of NS3 and next-generation sequencing (MiSeq) of a near-full-genome product can be used to accurately screen for the NS3 Q80K polymorphism.…”

Similarities between Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Genetic and Phenotypic Protease Quasispecies Diversity

“…Previous studies performed with the HCV NS3 protease of genotype 1 also showed an equivalent intrasample mean pairwise nucleotide diversity, ranging from 0.0133 to 0.0174 in HCV monoinfected patients (31) (32) and between 0.0075 and 0.0120 in HCV/HIV-1-coinfected patients (18,31,33,34). A more recent study analyzed, by deep sequencing and primer ID, the HCV NS3 protease of genotype 1 from 20 HCV monoinfected patients and 20 HCV/HIV-1 coinfected patients (35). A lower intrasample mean pairwise nucleotide diversity was observed in HCV/HIV-1-coinfected patients (0.0095 Ϯ 0051) than in HCV monoinfected patients (0.0143 Ϯ 0.0069).…”