in use situations including flexing, massage (Leite-Silva et al., 2016a), occlusion (Leite-Silva et al., 2016b, repeated hourly and daily application (Mohammed et al., 2019), and now bathing. Health promotion organizations, healthcare professionals, teachers, and parents can confidently recommend the use of effective nanoparticle-based sunscreens to protect children and adults from sunburn, skin cancer, and photo-aging.

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,

Single molecular real-time (SMRT) sequencing, also called third-generation sequencing, is a novel sequencing technique capable of generating extremely long contiguous sequence reads. While conventional short-read sequencing cannot evaluate the linkage of nucleotide substitutions distant from one another, SMRT sequencing can directly demonstrate linkage of nucleotide changes over a span of more than 20 kbp, and thus can be applied to directly examine the haplotypes of viruses or bacteria whose genome structures are changing in real time. In addition, an error correction method (circular consensus sequencing) has been established and repeated sequencing of a single-molecule DNA template can result in extremely high accuracy. The advantages of long read sequencing enable accurate determination of the haplotypes of individual viral clones. SMRT sequencing has been applied in various studies of viral genomes including determination of the full-length contiguous genome sequence of hepatitis C virus (HCV), targeted deep sequencing of the HCV NS5A gene, and assessment of heterogeneity among viral populations. Recently, the emergence of multi-drug resistant HCV viruses has become a significant clinical issue and has been also demonstrated using SMRT sequencing. In this review, we introduce the novel third-generation PacBio RSII/Sequel systems, compare them with conventional next-generation sequencers, and summarize previous studies in which SMRT sequencing technology has been applied for HCV genome analysis. We also refer to another long-read sequencing platform, nanopore sequencing technology, and discuss the advantages, limitations and future perspectives in using these third-generation sequencers for HCV genome analysis.

No abstract

The emergence of hepatitis C virus (HCV) with resistance-associated substitution (RAS), produced by mutations in the HCV genome, is a major problem in direct acting antivirals (DAA) treatment. This study aimed to clarify the mutational spectrum in HCV-RNA and the substitution pattern for the emergence of RASs in patients with chronic HCV infection. HCV-RNA from two HCV replicon cell lines and the serum HCV-RNA of four non-liver transplant and four post-liver transplant patients with unsuccessful DAA treatment were analyzed using high-accuracy single-molecule real-time long-read sequencing. Transition substitutions, especially A>G and U>C, occurred prominently under DAAs in both non-transplant and post-transplant patients, with a mutational bias identical to that occurring in HCV replicon cell lines during 10-year culturing. These mutational biases were reproduced in natural courses after DAA treatment. RASs emerged via both transition and transversion substitutions. NS3-D168 and NS5A-L31 RASs resulted from transversion mutations, while NS5A-Y93 RASs was caused by transition substitutions. The fidelity of the RNA-dependent RNA polymerase, HCV-NS5B, produces mutational bias in the HCV genome, characterized by dominant transition mutations, notably A>G and U>C substitutions. However, RASs are acquired by both transition and transversion substitutions, and the RASs-positive HCV clones are selected and proliferated under DAA treatment pressure.