{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T19:58:20Z","timestamp":1784231900056,"version":"3.55.0"},"reference-count":98,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2021,5,26]],"date-time":"2021-05-26T00:00:00Z","timestamp":1621987200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003130","name":"Fonds Wetenschappelijk Onderzoek","doi-asserted-by":"publisher","award":["1S04516N"],"award-info":[{"award-number":["1S04516N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Drug resistance studies on human \u03b3-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine \u03b3-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of \u03b3-herpesviruses. In this study, we investigated the mechanism of resistance to nucleoside (ganciclovir (GCV)), nucleotide (cidofovir (CDV), HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet (PFA)) analogues and the impact of these drug resistance mutations on viral fitness. Viral fitness was determined by dual infection competition assays, where MHV-68 drug-resistant viral clones competed with the wild-type virus in the absence and presence of antivirals. Using next-generation sequencing, the composition of the viral populations was determined at the time of infection and after 5 days of growth. Antiviral drug resistance selection resulted in clones harboring mutations in the viral DNA polymerase (DP), denoted Y383SGCV, Q827RHPMP-5azaC, G302WPFA, K442TPFA, G302W+K442TPFA, C297WHPMPO-DAPy and C981YCDV. Without antiviral pressure, viral clones Q827RHPMP-5azaC, G302WPFA, K442TPFA and G302W+K442TPFA grew equal to the wild-type virus. However, in the presence of antivirals, these mutants had a growth advantage over the wild-type virus that was moderately to very strongly correlated with antiviral resistance. The Y383SGCV mutant was more fit than the wild-type virus with and without antivirals, except in the presence of brivudin. The C297W and C981Y changes were associated with a mutator phenotype and had a severely impaired viral fitness in the absence and presence of antivirals. The mutator phenotype caused by C297W in MHV-68 DP was validated by using a CRISPR\/Cas9 genome editing approach.<\/jats:p>","DOI":"10.3390\/v13060985","type":"journal-article","created":{"date-parts":[[2021,5,26]],"date-time":"2021-05-26T21:56:44Z","timestamp":1622066204000},"page":"985","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["An MHV-68 Mutator Phenotype Mutant Virus, Confirmed by CRISPR\/Cas9-Mediated Gene Editing of the Viral DNA Polymerase Gene, Shows Reduced Viral Fitness"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0249-9776","authenticated-orcid":false,"given":"Erika","family":"Trompet","sequence":"first","affiliation":[{"name":"Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3076-6317","authenticated-orcid":false,"given":"Arturo","family":"Temblador","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Gillemot","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dimitrios","family":"Topalis","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert","family":"Snoeck","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2609-4896","authenticated-orcid":false,"given":"Graciela","family":"Andrei","sequence":"additional","affiliation":[{"name":"Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,5,26]]},"reference":[{"key":"ref_1","first-page":"9530","article-title":"Distribution and effects of amino acid changes in drug-resistant alpha and beta herpesviruses DNA polymerase","volume":"44","author":"Topalis","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Renner, D.W., and Szpara, M.L. 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