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Until recently, the majority of laboratory-based investigations into the biology of HCV have focused on the genotype 2 isolate, JFH-1, involving replicons and infectious cell culture systems. However, genotype 2 is one of eight major genotypes of HCV and there is great sequence variation among these genotypes (&gt;30\u200a% nucleotide divergence). In this regard, genotype 3 is the second most common genotype and accounts for 30\u200a% of global HCV cases. Further, genotype 3 is associated with both high levels of inherent resistance to direct-acting antiviral (DAA) therapy, and a more rapid progression to chronic liver diseases. Neither of these two attributes are fully understood, thus robust genotype 3 culture systems to unravel viral replication are required. Here we describe the generation of robust genotype 3 sub-genomic replicons (SGRs) based on the adapted HCV NS3-NS5B replicase from the DBN3a cell culture infectious clone. Such infectious cell culture-adaptive mutations could potentially promote the development of robust SGRs for other HCV strains and genotypes. The novel genotype 3 SGRs have been used both transiently and to establish stable SGR-harbouring cell lines. We show that these resources can be used to investigate aspects of genotype 3 biology, including NS5A function and DAA resistance. They will be useful tools for these studies, circumventing the need to work under the biosafety level 3 (BSL3) containment required in many countries.<\/jats:p>","DOI":"10.1099\/jgv.0.001486","type":"journal-article","created":{"date-parts":[[2020,9,8]],"date-time":"2020-09-08T14:17:54Z","timestamp":1599574674000},"page":"1182-1190","source":"Crossref","is-referenced-by-count":9,"title":["Insights into the unique characteristics of hepatitis C virus genotype 3 revealed by development of a robust sub-genomic DBN3a replicon"],"prefix":"10.1099","volume":"101","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9867-393X","authenticated-orcid":false,"given":"Joseph C.","family":"Ward","sequence":"first","affiliation":[{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, 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Campos","sequence":"additional","affiliation":[{"name":"Present address: S\u00e3o Paulo State University, Institute of Biosciences, Languages and Exact Sciences, Crist\u00f3v\u00e3o Colombo Street, 2265, Post Code 15054-000, S\u00e3o Jos\u00e9 do Rio Preto, S\u00e3o Paulo State, Brazil"},{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3699-1814","authenticated-orcid":false,"given":"Santseharay","family":"Ramirez","sequence":"additional","affiliation":[{"name":"Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark"},{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Ketteg\u00e5rd All\u00e9 30, DK-2650 Hvidovre, 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