Multi-targeted gene silencing strategies inhibit replication of Canine morbillivirus
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RESEARCH ARTICLE
Open Access
Multi-targeted gene silencing strategies inhibit replication of Canine morbillivirus Otávio Valério de Carvalho1,2, Marcus Rebouças Santos1, Juliana Lopes Rangel Fietto3, Mauro Pires Moraes1, Márcia Rogéria de Almeida3, Gustavo Costa Bressan3, Lindomar José Pena2* and Abelardo Silva-Júnior2*
Abstract Background: Canine morbilivirus (canine distemper virus, CDV) is a highly contagious pathogen associated with high morbidity and mortality in susceptible carnivores. 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. Results: 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. Conclusions: 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. Keywords: Gene therapy, RNA interference, Adenoviral vector, Morbillivirus
Background Canine morbilivirus (canine distemper virus, CDV) is a highly contagious and widespread virus that produces severe multisystemic disease in a broad range of wild and domestic carnivores [1]. It is the causative agent of canine distemper (CD) is an enveloped single-stranded * Correspondence: [email protected]; [email protected] 2 Department of Virology and Experimental Therapy, Oswaldo Cruz Foundation (FIOCRUZ), Aggeu Magalhães Research Center, Av. Moraes Rego, s/n, Campus UFPE, Cidade Universitária, Recife, PE 50670-420, Brazil Full list of author information is available at the end of the article
negative-sense RNA virus and member of the genus Mo
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