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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&gt;G and U&gt;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&gt;G and U&gt;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.<\/jats:p>","DOI":"10.1038\/s41598-022-11151-6","type":"journal-article","created":{"date-parts":[[2022,4,30]],"date-time":"2022-04-30T10:07:43Z","timestamp":1651313263000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Mutational spectrum of hepatitis C virus in patients with chronic hepatitis C determined by single molecule real-time sequencing"],"prefix":"10.1038","volume":"12","author":[{"given":"Fumiyasu","family":"Nakamura","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haruhiko","family":"Takeda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshihide","family":"Ueda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Atsushi","family":"Takai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ken","family":"Takahashi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuji","family":"Eso","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Soichi","family":"Arasawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eriko","family":"Iguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takahiro","family":"Shimizu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masako","family":"Mishima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ken","family":"Kumagai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taiki","family":"Yamashita","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinji","family":"Uemoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nobuyuki","family":"Kato","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroyuki","family":"Marusawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akihiro","family":"Sekine","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Seno","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,4,30]]},"reference":[{"key":"11151_CR1","doi-asserted-by":"publisher","first-page":"637","DOI":"10.7326\/m16-2575","volume":"166","author":"O Falade-Nwulia","year":"2017","unstructured":"Falade-Nwulia, O. et al. 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