Characterization of a Novel Hepatitis C Subtype, 6xj, and Its Consequences for Direct-Acting Antiviral Treatment in Yunnan, China

Abstract

Hepatitis C virus (HCV) has a high rate of genetic variability, with eight genotypes and 91 subtypes. The genetic diversity of HCV genotype 6 (HCV-6) is the highest with 31 subtypes, and this genotype is prevalent in Southeast Asia. In this study, we investigated 160 individuals with chronic hepatitis C in Yunnan Province, China. Using reverse transcription (RT)-PCR and Sanger sequencing, 147 cases were successfully amplified and genotyped as 3b (4.9%), 3a (19.73%), 6n (12.24%), 1b (7.48%), 2a (6.12%), 6a (2.04%), 1a (0.68%), 6v (0.68%), and 6xa (0.68%), with eight sequences remaining unclassified. Subsequently, the eight nearly full-length genomes were successfully amplified and analyzed. The eight complete coding sequences formed a phylogenetic group that was distinct from the previously assigned HCV-6 subtypes and clustered with two previously unnamed HCV-6 sequences. Furthermore, Simplot analysis showed no recombination and the p-distance was more than 15% in comparison to the 6a to 6xi subtypes. Taken together, we identified a new HCV-6 subtype, 6xj, which originated approximately in 1775 according to Bayesian analyses. Moreover, all eight individuals received follow-up assessments at 44 weeks from the beginning of their 12-week treatments of sofosbuvir/velpatasvir (after-treatment week 32). One case relapsed at after-treatment week 32. Next-generation sequencing (NGS) was conducted and showed that the treatment failure case had two suspected antiviral resistance mutations, NS5A V28M (a change of V to M at position 28) and NS5B A442V, compared with the baseline. Overall, this newly identified 6xj subtype further confirmed the high diversity of the HCV-6 genotype. The newly identified resistance-associated amino acid substitutions may help inform future clinical treatments. IMPORTANCE This study investigated the genetic diversity of hepatitis C virus (HCV), particularly in relation to genotype 6, which is prevalent in Yunnan, China, and is often difficult to treat successfully. We identified a new HCV-6 subtype, 6xj, which is an ancient strain. Moreover, all eight individuals with the novel subtype received follow-up assessments at 44 weeks from the beginning of their treatments. One case relapsed after 8 months of withdrawal. NGS was conducted and showed that the isolate from the treatment failure case had two suspected antiviral resistance mutations, NS5A V28M and NS5B A442V, compared with the baseline. Overall, this newly identified 6xj subtype further confirmed the high diversity of the HCV-6 genotype. The newly identified resistance-associated amino acid substitutions may help inform future clinical treatments. We believe that our study makes a significant contribution to the literature based on the results described above.

Keywords: DAA; RAS; follow-up; hepatitis C virus; novel subtype; resistance-associated substitutions.

FIG 1

Map of the study region and phylogenetic tree analysis and genotypic distribution of HCV isolates among individuals with chronic hepatitis C in Yunnan, China. (A) Map of the study region. The Yunnan province of southwestern China is marked by shading. MYA, Myanmar; THA, Thailand; LAO, Laos; VIE, Vietnam. (B) Phylogenetic tree based on the partial NS5B sequences amplified from 147 HCV-infected individuals. The different subtypes are shown in different colors. (C) Comparison of HCV subtype distribution rates among individuals with chronic hepatitis C in Yunnan, China.

FIG 2

Phylogenetic tree and analyses of the nucleotide divergence between 6xj and the other known genotype 6 subtypes (6a to 6xi) and recombinant based on full-length HCV genome sequences. (A) Previously reported HCV subtype 6 reference sequences (6a to 6xi) were used. Phylogenetic analysis was performed by the maximum-likelihood method, based on the GTR+G+I substitution model, with 1,000 bootstrap replicates, using MEGA version 6. The HCV 6xj sequences are marked in red. (B) Pairwise comparisons of nucleotide similarities between 3 HCV 6xi strains and 30 reference genotype 6 sequences. (C) Analysis using the SimPlot program demonstrates the genetic distance to the reference strain in different parts of the genome. (D) Bootscan plot using Simplot 3.5.1 software based on 100 replicates with a 300-bp sliding window moving in steps of 50 bases demonstrates the phylogenetic relationship to the reference strain.

FIG 3

Maximum clade credibility (MCC) tree of HCV full-length coding region sequences and Bayesian skyline plot (BSP) estimating the past population dynamics of HCV 6xj. (A) The MCC tree was constructed by Bayesian Markov chain Monte Carlo (MCMC) analysis based on HCV complete coding region sequences. HCV 6xj strains from Yunnan are highlighted in red. The node of the HCV subtype 6xj clade is shown by the black dot. The node age with 95% confidence interval is shown at top left. (B) The BSP was reconstructed using Tracer version 1.7.1. The y axis represents the estimates of the effective numbers of HCV 6xj strains, and the x axis represents time. The solid line represents the median estimates, and the shaded area represents the 95% confidence intervals. The pink area represents a period of exponential growth.

FIG 4

Changes of HCV RNA levels and resistance-associated substitutions (RASs) among the eight chronic hepatitis C patients infected by the new HCV 6xj subtype during sofosbuvir/velpatasvir (SOF/VEL) treatment. (A) Patients infected with HCV subtype 6xj received SOF/VEL (Epclusa) (FDC, 400/100 mg per tablet; Gilead Sciences) at 1 tablet once daily for 12 weeks. Serum HCV RNA levels were assessed at on-treatment weeks 0, 4, and 12 and at after-treatment weeks 12, 24, and 32 (follow-up weeks 24, 36, and 44). The lowest detection limit for HCV RNA was 1.2 log₁₀ IU/ml (15 IU/ml). (B) RASs of the eight baseline sequences (week 0 [0W]) and one treatment failure sequence at after-treatment week 32 (follow-up week 44 [44W]) analyzed in the NS3, NS5A, and NS5B regions by next-generation sequencing (NGS).