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Although there are CDV vaccines available, the disease poses a huge threat to dogs and wildlife hosts due to vaccine failures and lack of effective treatment. Thus, the development of therapeutics is an urgent need to achieve rapid outbreak control and reduce mortality in target species. Gene silencing by RNA interference has emerged as a promising therapeutic approach against different human and animal viruses. In this study, plasmid-based short hairpin RNAs (shRNAs) against three different regions in either CDV nucleoprotein (N), or large polymerase (L) genes and recombinant adenovirus-expressing N-specific multi-shRNAs were generated. Viral cytopathic effect, virus titration, plaque-forming unit reduction, and real-time quantitative RT-PCR analysis were used to check the efficiency of constructs against CDV.<\/jats:p>\n<\/jats:sec><jats:sec>\n<jats:title>Results<\/jats:title>\n<jats:p>In CDV-infected VerodogSLAM cells, shRNA-expressing plasmids targeting the N gene markedly inhibited the CDV replication in a dose-dependent manner, with viral genomes and titers being decreased by over 99%. Transfection of plasmid-based shRNAs against the L gene displayed weaker inhibition of viral RNA level and virus yield as compared to CDV N shRNAs. A combination of shRNAs targeting three sites in the N gene considerably reduced CDV RNA and viral titers, but their effect was not synergistic. Recombinant adenovirus-expressing multiple shRNAs against CDV N gene achieved a highly efficient knockdown of CDV N mRNAs and successful inhibition of CDV replication.<\/jats:p>\n<\/jats:sec><jats:sec>\n<jats:title>Conclusions<\/jats:title>\n<jats:p>We found that this strategy had strong silencing effects on CDV replication in vitro. Our findings indicate that the delivery of shRNAs using plasmid or adenovirus vectors potently inhibits CDV replication and provides a basis for the development of therapeutic strategies for clinical trials.<\/jats:p>\n<\/jats:sec>","DOI":"10.1186\/s12917-020-02671-2","type":"journal-article","created":{"date-parts":[[2020,11,19]],"date-time":"2020-11-19T22:45:47Z","timestamp":1605825947000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Multi-targeted gene silencing strategies inhibit replication of Canine morbillivirus"],"prefix":"10.1186","volume":"16","author":[{"given":"Ot\u00e1vio Val\u00e9rio","family":"de Carvalho","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marcus","family":"Rebou\u00e7as Santos","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juliana","family":"Lopes Rangel Fietto","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mauro","family":"Pires Moraes","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M\u00e1rcia Rog\u00e9ria","family":"de Almeida","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gustavo","family":"Costa Bressan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lindomar","family":"Jos\u00e9 Pena","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Abelardo","family":"Silva-J\u00fanior","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2020,11,19]]},"reference":[{"key":"2671_CR1","doi-asserted-by":"publisher","unstructured":"Deem SL, Spelman LH, Yates RA, Montali RJ. 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