Ultradeep Pyrosequencing of Hepatitis C Virus Hypervariable Region 1 in Quasispecies Analysis

Abstract

Genetic variability of hepatitis C virus (HCV) determines pathogenesis of infection, including viral persistence and resistance to treatment. The aim of the present study was to characterize HCV genetic heterogeneity within a hypervariable region 1 (HVR1) of a chronically infected patient by ultradeep 454 sequencing strategy. Three independent sequencing error correction methods were applied. First correction method (Method I) implemented cut-off for genetic variants present in less than 1%. In the second method (Method II), a condition to call a variant was bidirectional coverage of sequencing reads. Third method (Method III) used Short Read Assembly into Haplotypes (ShoRAH) program. After the application of these three different algorithms, HVR1 population consisted of 8, 40, and 186 genetic haplotypes. The most sensitive method was ShoRAH, allowing to reconstruct haplotypes constituting as little as 0.013% of the population. The most abundant genetic variant constituted only 10.5%. Seventeen haplotypes were present in a frequency above 1%, and there was wide dispersion of the population into very sparse haplotypes. Our results indicate that HCV HVR1 heterogeneity and quasispecies population structure may be reconstructed by ultradeep sequencing. However, credible analysis requires proper reconstruction methods, which would distinguish sequencing error from real variability in vivo.

Effects of ribavirin monotherapy on the viral population in patients with chronic hepatitis C genotype 1: Direct sequencing and pyrosequencing of the HCV regions

“…Previous studies have sought to characterise the mutagenic effect of RBV using conventional sequencing methods, and have revealed that HCV infection comprises a cloud of closely related sequence variants differing by as little as one nucleotide from a population average sequence. Pyrosequencing techniques make it possible to detect all the genomic variants of the viral population and offer a new and useful approach in HCV research [Caraballo Cortes et al, ]. In order to confirm previous research results using direct sequencing, in the present study, the complete NS5A region of the baseline sample was pyrosequenced, at four weeks after ribavirin monotherapy, in a patient chosen at random.…”

“…The newly available pyrosequencing technique opens up a new approach to the analysis of complex viral genomes, by enabling the detection of rare molecular variants and thus a better understanding of genetic variability. This approach has been applied successfully in studies of the human immunodeficiency virus, but very few studies have used it with respect to HCV [Nasu et al, ; Caraballo Cortes et al, ].…”

Inter and intra-host variability of hepatitis C virus genotype 1a hypervariable envelope coding domains followed for a 4–11 year of human immunodeficiency virus coinfection and highly active antiretroviral therapy

“…At intra-host level, HCV exists as quasispecies defined by several closely related yet genetically distinct populations mainly because of low viral RNA polymerase fidelity (Bittar et al, 2013;Caraballo Cortes et al, 2013). The mechanisms driving HCV quasispecies diversification are not completely understood.…”

Bayesian Coalescent Analysis of the Intra-Host Evolution of Hepatitis C Virus: Memory Genomes and Clinical Implications

“…With nextgeneration sequencing (NGS) platforms, it is now possible to investigate viral quasispecies at much greater detail. Their high throughput allows for generation of millions of reads in a single sequencing run, facilitating indepth sequencing [22]. These new methods can detect variants at low frequencies, which would go undetected by standard sequencing methods [23].…”

“…In order to gain insight into these matters, we have performed a Bayesian coalescent analysis of HVR1 sequences of a HCV quasispecies population circulating in a chronic patient infected with HCV genotype 1b recently obtained by ultra-deep sequencing [22]. The results of these studies highlight the importance of minority genomes in HCV population history and evolution, the mutant clouds as reservoirs of phenotypic and genetic variants for virus adaptability, as well as the roll of the mutant spectra to overcome selective constraints.…”