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.

Keywords: Adenoviral vector; Gene therapy; Morbillivirus; RNA interference.

Fig. 1

Inhibition of CDV replication by N-targeted shRNA plasmid vectors. Real-time qRT-PCR (a, c, e) and TCID₅₀ infectivity titration (b, d, f) of the three N-specific plasmid-based shRNA transfections (72 h). Right vertical axis presents the percentage of virus inhibition highlighted on the markers (♦). The error bars represent standard deviations. Values given are the mean ± standard error obtained from three independent experiments. Values followed by the same lower case letters do not differ by Tukey’s test (p < 0.01). BLD, below limit of detection (20 TCID₅₀/mL)

Fig. 2

Inhibition of CDV replication by L-targeted shRNA plasmid vectors. Real-time qRT-PCR (a, c, e) and TCID₅₀ infectivity titration (b, d, f) of the three L-specific plasmid-based shRNA transfections (72 h). Right vertical axis presents the percentage of virus inhibition highlighted on the markers (♦). The error bars represent standard deviations. Values given are the mean ± standard error obtained from three independent experiments. Values followed by the same lower case letters do not differ by Tukey’s test (p < 0.01). BLD, below limit of detection (20 TCID₅₀/mL)

Fig. 3

Co-transfection of shRNA-expressing plasmids targeting nucleoprotein gene. VerodogSLAM cells were first infected with CDV MOI 0.01 and then transfected with 1.2 μg of single or combinated pENTR/U6-Nis (0.4 μg each). Combination of the three N-specific shRNA plasmid vectors exhibited markedly reduced N transcript levels (a), infectious virus titers (b) and CPE (c). Average and standard deviations represent three independent transfections. Right vertical axis presents the percentage of virus inhibition highlighted on the markers (♦). More evident cytopathic effects are indicated by arrows. Values followed by the same lower case letters do not differ by Tukey’s test (p < 0.01). BLD, below limit of detection (20 TCID₅₀/mL). All figure panels (100X total magnification): representative data from at least three independent experiments using biological replicates

Fig. 4

Antiviral effects by pre-treating or post-treating CDV-infected cells with multi-shRNA Ad5 construct at various doses. Cell+SUP were collected 72 hpi for detection of N mRNA levels (a, c) and viral titers (b, d). Ad5SCR was used as negative control. Values are the mean ± standard error obtained from three independent experiments. Right vertical axis presents the percentage of virus inhibition highlighted on markers (♦). Values followed by the same lower case letters do not differ by Tukey’s test (p < 0.01). BLD, below limit of detection (20 TCID₅₀/mL)

Fig. 5

Transduction of VerodogSLAM cells with Ad5Ni(1–3) vector blocks CDV plaque formation. Samples of Ad5 construct pre- and post-treatments were added to cells and after 2-h incubation, the inoculums were removed, cells were overlaid with CMC complete medium and incubated for 72 h. Plaque reduction values of Ad5 pre-treating (a, b) and post-treating (c, d) assays represent the mean ± standard error from three independent experiments compared with untreated infected cells. Ad5SCR was used as negative control. (b, d) CDV-plaque crystal violet-staining method

Fig. 6

Ad5Ni(1–3) post-treatment at various time intervals. Multi-shRNA Ad5 construct was added at MOI of 2.5 and 5 to infected cells at 2, 12 and 24 hpi. Ad5 silencing efficiency was measured by viral RNA levels (a), infectious titers (b), and CDV plaque count (c) at time-specific treatments (** p < 0.01; *** p < 0.001). CDV infectivity inhibition was shown through reduction of CPE (D, 100X total magnification) and infectious plaque number (e) compared to controls

Fig. 7

Schematic representation of multiple shRNA-expressing adenovirus construct. (a) The multiple shRNA-expressing cassette are transcribed under the control of U6 promoters. The cassette also includes polyT transcription termination signals (T) and spacer blocks (b) of 100 nt between shRNA-coding regions to prevent potential cross-interference. (b) Multi-shRNA expression cassette was cloned into the E1 region of an E1/E3-deleted Ad5 vector plasmid by restriction enzyme sites (Clal-Xbal). A recombinant adenovirus with scrambled shRNA sequence (Ad5SCR) was generated and used as a negative control. (c, d) Ad5-vector particles were produced by transfecting Pacl-linearized pAd5Ni(1–3) clone. HEK293 cells were transfected with pAd5Ni(1–3) and after 7–10 days, isolated plaques of recombinant Ad5 were picked to obtain the monoclonal virus, and the Ad5Ni(1–3) virus was further identified by PCR and sequencing. (d) Ad5Ni(1–3) transduction and cytopathic effects observed at days 7 and 10 (40X total magnification)