Spouse-to-Spouse Transmission and Evolution of Hypervariable Region 1 and 5’ Untranslated Region of Hepatitis C Virus Analyzed by Next-Generation Sequencing

Abstract

Hepatitis C virus (HCV) transmission between spouses remains poorly characterized, largely due to the limited availability of samples from the early stage of infection, as well as methodological constraints. A fifty-eight year-old male developed acute hepatitis C infection and his 53-year old spouse has been HCV-positive for over 10 years. Serum samples were collected from both at the time of acute hepatitis C diagnosis in male (baseline) and then at 9 and 13 months. Hypervariable region 1 (HVR1) and 5’ untranslated region (5’UTR) sequences were amplified and subjected to next generation sequencing (NGS) using a pyrosequencing platform. Genetic variants were inferred by Shorah reconstruction method and compared by phylogenetic and sequence diversity analysis. As the sequencing error of the procedure was previously determined to be ≤ 1.5%, the analysis was conducted with and without the 1.5% cut-off with regard to the frequency of variants. No identical HVR1 variants were identified in spouses at baseline and follow-up samples regardless whether the cut-off was applied or not. However, there was high similarity (98.3%) between a minor baseline donor variant (1.7% frequency) and the most abundant baseline recipient variant (62.5% frequency). Furthermore, donor and recipient strains clustered together when compared to 10 control subjects from the same area and infected with the same HCV subtype. There was an increase in HVR1 complexity (number of genetic variants) over time in both spouses. In contrast, the 5'UTR region was stable and of low complexity throughout the study. In conclusion, intrafamilial HCV transmission may be established by a very minor variant and investigation of this phenomenon requires high-sensitivity assays, such as NGS.

Next-generation sequencing analysis of a cluster of hepatitis C virus infections in a haematology and oncology center

“…The complexity of variant populations, reflected by the number of nucleotide variants, was very low (mean 3.4 variants) which is in contrast to high diversity of HVR1 displayed during chronic infection. For example, in our previous study, the average number of HVR1 variants during chronic HCV 1b infection was 30–40 [ 16 , 24 ]. This low complexity probably reflects the bottleneck effect at the time of transmission and suggests that the infection has been initiated by a single variant, so called “founder”, which must have been very similar or even identical to the consensus sequence inferred from all patients’ sequences [ 3 , 4 , 27 ].…”

“…Furthermore, the structure of HVR1 population was “narrow” in the majority of cases (as one predominant variant was accompanied by minor variants at <10% frequency), which is also compatible with a recent single variant infection. Previous studies showed that in chronic hepatitis C viral population tends to become more “flat” in terms of frequency structure, with higher predominance of moderate and low frequency variants [ 24 ]. Alternatively, the structure of the populations could have been affected by the presence of immunosuppression due to immunosuppressive drugs and the underlying disease.…”

“…Phylogenetic trees were constructed according to the Maximum Likelihood method based on the Tamura-Nei model [ 23 ] using MEGA 6.0. We used the same approach in our previous studies [ 20 , 24 ] and the superiority of Tamura-Nei model for the analysis of HVR1 was reported by others [ 25 ]. The robustness of tree topology was estimated by the bootstrapping method (resampling 1000 data sets) using MEGA 6.0.…”

Evidence of bottleneck effect on hepatitis C virus transmission between a couple under interferon based therapy

“…Factors as the host's immune genetic characteristics could be the cause of the different treatment outcomes, but, considering the importance of the genetic diversity the bottleneck effect can be at least partially responsible for that difference. Despite published data already supporting spouse-to-spouse transmission, (Caraballo Cortes et al, 2016;Njouom et al, 2011;Tahan et al, 2005) our work is the first to report the transmission of a HCV population from two patients with different treatment responses and phylogenetic evidence of a bottleneck event.…”