Induced RNA Interference Can Suppress Persistent Drosophila A Virus (DAV) Infection in Cultured Drosophila Melanogaster S2 Cells

Background: The RNA interference (RNAi) pathway is a highly conserved antiviral mechanism in eukaryotes, including insects such as fruit flies. Many viruses have therefore evolved mechanisms that protect their transcripts and/or genomes; how effectively RNAi can act is thus a question that must be answered case-by-case. Methods: We demonstrate that the RNAi machinery can successfully combat Drosophila A virus (DAV) in persistently infected Drosophila melanogaster S2 cell lines, but only when supported with exogenous triggers. Since such an infection poses a challenge to the fitness of cells and the reproducibility of results obtained with them, our approach provides a convenient method to recover precious experimentally modified cell lines from infection through the application of exogenous double-stranded RNA (dsRNA) targeting the DAV sequence. The in vitro transcribed dsRNAs were applied to infected S2 cells via direct “soaking,” utilizing the cells’ natural endocytic uptake. Results: We monitored treatment efficacy via RT-PCR-based detection of viral sequences, and our results demonstrate that continued application of DAV-specific dsRNAs led to a reduction in viral abundance and all eight tested cell lines appeared DAV-negative within several weeks of treatment. While a set of cell lines exhibited viral recurrence several weeks after the cessation of treatment, others remained virus-free for at least 16 weeks, suggesting that a permanent “cure” can indeed be achieved. Notably, while RNAi activation effectively cleared DAV in some cell lines, no beneficial effect was observed for a concomitantly applied Drosophila X-virus (DXV) treatment in the co-infected cultures. Conclusions: This suggests that DXV has more efficient evasion mechanisms but cannot protect DAV in trans. Our protocol thus provides a robust, scalable approach for clearing persistent DAV infections, but may not be effective against all viruses known to infect Drosophila cell cultures.