Single-molecular real-time deep sequencing reveals the dynamics of multi-drug resistant haplotypes and structural variations in the hepatitis C virus genome

Abstract

While direct-acting antivirals (DAAs) for hepatitis c virus (HcV) have dramatically progressed, patients still suffer from treatment failures. For the radical eradication of HCV, a deeper understanding of multiple resistance-associated substitutions (RASs) at the single-clone level is essential. To understand HcV quasispecies and their dynamics during DAA treatment, we applied single-molecule real-time (SMRT) deep sequencing on sera from 12 patients with genotype-1b HCV infections with DAA treatment failures, both pre-and post-treatment. We identified >3.2 kbp sequences between NS3 and NS5A genes of 187,539 clones in total, classifying into haplotype codes based on the linkage of seven RAS loci. The number of haplotype codes during the treatment, per sample, significantly decreased from 14.67 ± 9.12 to 6.58 ± 7.1, while the number of nonsynonymous codons on the seven RAS loci, per clone, significantly increased from 1.50 ± 0.92 to 3.64 ± 0.75. In five cases, the minority multi-drug resistant haplotypes at pre-treatment were identical to the major haplotypes at relapse. Moreover, various structural variations (SVs) were detected and their dynamics analysed. These results suggest that SMRt deep sequencing is useful for detecting minority haplotypes and SVs, and to evaluate the dynamics of viral genomes at the single-clone level. The hepatitis C virus (HCV) has approximately 9.6 kb of a single-stranded RNA genome. After the approval of oral direct-acting antivirals (DAAs), after drastic HCV treatment, levels of HCV-RNA remain undetectable (sustained virological response; SVR) in most patients chronically infected by HCV or suffering from HCV-related diseases 1-5. In some patients, however, DAAs cannot completely eradicate HCV 1-4. One of the major causes of HCV survival during DAA treatment is thought to be a mutation of its genome. Mutations are likely to occur in the HCV genome due to the fact that RNA-dependent RNA polymerase lacks a proofreading function. Therefore, during HCV infection, the population of HCV includes similar but slightly different clones, and HCV is therefore known as a quasispecies 6,7. Some quasispecies have resistance-associated substitutions (RASs) and make the DAAs ineffective. For example, Y93H on the NS5A gene is associated with the resistance of NS5A inhibitors 8-11 , while the inframe deletion of the NS5A-P32 codon leads to the failure of glecaprevir and pibrentasvir treatments 12-15. Besides these resistance mutations, Q80/D168 on the NS3 gene and R30/ L31/Q54 on the NS5A gene are associated with RASs in HCV 16,17. The antiviral treatment of patients after liver transplantation, or of those with liver cirrhosis, is still challenging, and to achieve SVR in some of these cases,

SARS-CoV-2 Quasispecies Provides an Advantage Mutation Pool for the Epidemic Variants

“…CCSs with a pass number of ≥5 were not only close to the best results of gap correction ( Fig. 2 ) but also revealed a low substitute error of ∼0.023% ( 36 ). Under this threshold, the SMRT platform produced an average of ∼20,000 qualified CCSs for each sample.…”

“…The clone (or haplotype) number was much more than that produced from traditional Sanger methods, such as the study of SARS-CoV quasispecies that obtained 28 full spike clones for 19 patients ( 37 ). Under the same SMRT platform and threshold (pass number ≥ 5), a recent study of HCV quasispecies produced an average 8,130 CCSs for each sample ( 36 ). These indicated that a sufficient and high-quality data set was obtained in the present study.…”

Long-read sequencing reveals the evolutionary drivers of intra-host diversity across natural RNA mycovirus infections

“…Recently, intra-host viral structural variants have also begun to be classified, opening up a new component of virus diversity to be explored (e.g. hepatitis C virus, Yamashita et al. 2020 ).…”

SARS-CoV-2 Quasispecies provides insight into its genetic dynamics during infection

“…We obtained an average 27,000 quasispecies sequences for each sample, and the number was more than the SARS-Cov quasispecies study using traditional sanger methods (28 spike sequences from 19 patients) 28 and results from the recent HCV quasispecies study using the same SMRT platform (average 8,130 CCS for each samples with pass > 5) 29 . More than 80,000 cumulative SNVs facilitated us to calculate the evolutionary rate at quasispecies level.…”