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The replication fidelity of RNA viruses can be modulated by subtle amino acid changes in the viral RNA-dependent RNA polymerase (RdRp). In our study, two novel amino acid substitutions (V218I and P386S) in the RdRp of PRRSV were identified under the ribavirin selection. A series of mutant viruses with single or double amino acid replacements were generated from high-fidelity PRRSV NJ-Rb and wild-type NJ-a P80 infectious cDNA clones. Subsequently, we evaluated the genetic stability, ribavirin sensitivity, and biological characteristics of the recombinant viruses. Our findings indicated that the mutation frequencies of the recombinant mutants (vI218V, vS386P, and vVP) based on NJ-Rb were significantly increased and that these recombinant viruses exhibited a loss of ribavirin resistance. The high-fidelity virus NJ-Rb was undetectable using a virus titration assay in porcine alveolar macrophages (PAMs). Our in vivo experiments demonstrated that NJ-Rb was nearly incapable of establishing infection and replicating in the lungs. The recombinant mutants vV218I, vP386S, and vIS, based on NJ-a P80, significantly increased replication fidelity and ribavirin resistance. These results indicated that PRRSV RdRp (NSP9) contained fidelity checkpoints. Furthermore, Val218 and Pro386 were identified as critical sites that determined PRRSV\u2019s genetic stability and ribavirin resistance. These findings contribute to understanding how RdRp affects PRRSV\u2019s genetic stability and ribavirin sensitivity and provide a theoretical basis for designing a genetically stable high-fidelity PRRSV vaccine.<\/jats:p>","DOI":"10.1186\/s13567-025-01517-9","type":"journal-article","created":{"date-parts":[[2025,4,19]],"date-time":"2025-04-19T05:14:56Z","timestamp":1745039696000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Contribution of critical amino acid residues in the RNA-dependent RNA polymerase to the replication fidelity and viral ribavirin sensitivity of porcine reproductive and respiratory syndrome virus"],"prefix":"10.1186","volume":"56","author":[{"given":"Xiaoyan","family":"Zhang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziyin","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhibang","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhisheng","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yipeng","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taotao","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinxiang","family":"Gong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kang","family":"Feng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junping","family":"He","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qisheng","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jibo","family":"Hou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1643-7701","authenticated-orcid":false,"given":"Pengcheng","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,4,19]]},"reference":[{"key":"1517_CR1","doi-asserted-by":"publisher","first-page":"361","DOI":"10.1016\/0378-1135(94)90067-1","volume":"42","author":"M Bloemraad","year":"1994","unstructured":"Bloemraad M, de Kluijver EP, Petersen A, Burkhardt GE, Wensvoort G (1994) Porcine reproductive and respiratory syndrome: temperature and pH stability of Lelystad virus and its survival in tissue specimens from viraemic pigs. 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All animal experiments were conducted per the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"The authors declare that they have no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"83"}}