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ARS Home » Plains Area » Manhattan, Kansas » Center for Grain and Animal Health Research » ABADRU » Research » Publications at this Location » Publication #407209
Research Project: Ecology of Hemorrhagic Orbiviruses in North America

Location: Arthropod-borne Animal Diseases Research

Title: RISC-y business: Impact of siRNA knockdown of nonstructural protein 1 on bluetongue virus replication in cells of its insect vector

Author
item ROSEN, DANNY - Kansas State University Agricultural Research Center-Hays
item Scroggs, Stacey
item Steele, Taylor
item Gutierrez, Jessica
item Drolet, Barbara

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 8/15/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Bluetongue virus (BTV, Reoviridae, Orbivirus) causes hemorrhagic disease in domestic and wild ruminants. BTV is transmitted by Culicoides biting midges. Disease severity varies by host species, but sheep experience the highest mortality rate. There are at least 26 serotypes of BTV worldwide, with 17 serotypes either established or reported in the US. RNA interference (RNAi) is an immune response in these vector insects that degrades intracellular viral RNA through the RISC protein assemblage. We investigated if inducing an RNAi response with a small interfering RNA (siRNA) targeting the BTV protein synthesis regulator, nonstructural protein 1 (NS1), would suppress the replication of BTV in a Culicoides cell line (W8). W8 cells were transfected with either NS1 or luciferase control siRNAs, then infected at 24 h with BTV at a low (0.2) then high (3) multiplicity of infection (MOI). Virus was collected from the cells and media at 1, 24, 48, and 96 hours post infection (hpi) in the first experiment (MOI: 0.2, slow infection dynamic) and 1, 3, 6, 9, and 96 hpi in the second (MOI: 3, rapid synchronized infection). Infectious virus was quantified via plaque assay on Vero cells and viral RNA was quantitated by reverse transcription real time quantitative PCR (RT-qPCR). Viral RNA showed down regulation by NS1 dsRNA very early (1 hpi) by RT-qPCR and had lower infectious titers at 24 and 48 hpi by plaque assay. Both assays showed viral recovery by 96 hpi. Unexpectedly, infectious virus was detected earlier for both siRNA treatments compared to the untreated control. This may suggest that experimental introduction of small RNAs distract the innate immune system, as has been reported for other insect-transmitted viruses. These preliminary data highlight the complexity of developing RNAi-based control measures to decrease viral replication. Further research will be needed to evaluate RNAi as a means of controlling BTV in Culicoides biting midges or if another knockdown target would prove successful.