Multi-targeted gene silencing strategies inhibit replication of Canine morbillivirus

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.

Antiviral activity of ouabain against a Brazilian Zika virus strain

“…After 120 h of incubation at 37 °C in a 5% CO 2 atmosphere, the CPE score was evaluated using an inverted microscope (AE2000 binocular microscope, Motic, Hong Kong). The CPE scores were defined as (1) CPE < 25%, (2) CPE between 25 and 50%, (3) CPE between 51 and 75% and (4) CPE > 75%, as described by Carvalho et al 40 . After that, 50 μL of freshly prepared MTT solution (1 mg/mL) was added to each well and then the microplate was incubated for 4 h .…”

Development of HEK293T-produced recombinant receptor-Fc proteins as potential candidates against canine distemper virus

“…Synthetic substances, for example, ribavirin (RBV) and favipiravir were purine analog drug with a primary antiviral mechanism of suppressing the RNA-dependent RNA polymerase (RdRP) activity, also exhibited effective suppression of CDV replication in vitro (32,33). Short interfering RNA and A77 1726 suppressed CDV replication by inhibiting viral nucleotide synthesis (34,35). Other antiviral inhibitors including polyclonal antibody showed potential therapeutic effects against CDV infection in dogs (36).…”