{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:03:18Z","timestamp":1785423798846,"version":"3.56.0"},"reference-count":20,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T00:00:00Z","timestamp":1785369600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["392687528"],"award-info":[{"award-number":["392687528"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"award":["392687528"],"award-info":[{"award-number":["392687528"]}],"id":[{"id":"https:\/\/ror.org\/018mejw64","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Non-Coding RNA","ncRNA"],"abstract":"<jats:p>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 \u201csoaking,\u201d utilizing the cells\u2019 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 \u201ccure\u201d 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.<\/jats:p>","DOI":"10.3390\/ncrna12040028","type":"journal-article","created":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:55:11Z","timestamp":1785419711000},"page":"28","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Induced RNA Interference Can Suppress Persistent Drosophila A Virus (DAV) Infection in Cultured Drosophila Melanogaster S2 Cells"],"prefix":"10.3390","volume":"12","author":[{"given":"Wiebke","family":"Kochend\u00f6rfer","sequence":"first","affiliation":[{"name":"Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universit\u00e4t M\u00fcnchen, Feodor-Lynen-Strasse 25, 81377 M\u00fcnchen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Klaus","family":"F\u00f6rstemann","sequence":"additional","affiliation":[{"name":"Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universit\u00e4t M\u00fcnchen, Feodor-Lynen-Strasse 25, 81377 M\u00fcnchen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"804","DOI":"10.3855\/jidc.4187","article-title":"Antiviral innate immune response of RNA interference","volume":"8","author":"Sidahmed","year":"2014","journal-title":"J. Infect. Dev. Ctries."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"806","DOI":"10.1038\/35888","article-title":"Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans","volume":"391","author":"Fire","year":"1998","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Koeppe, S., Kawchuk, L., and Kalischuk, M. (2023). RNA Interference Past and Future Applications in Plants. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24119755"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Ishchuk, O.P., Ahmad, K.M., Koruza, K., Bojanovi\u010d, K., Sprenger, M., Kasper, L., Brunke, S., Hube, B., S\u00e4ll, T., and Hellmark, T. (2019). RNAi as a Tool to Study Virulence in the Pathogenic Yeast Candida glabrata. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.01679"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Ebenezer, O., Oyebamiji, A.K., Olanlokun, J.O., Tuszynski, J.A., and Wong, G.K.-S. (2025). Recent Update on siRNA Therapeutics. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26083456"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Giansanti, M.G., and Piergentili, R. (2025). Drosophila: A Versatile Model in Biology and Medicine. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26115032"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1015","DOI":"10.1007\/BF02616216","article-title":"Endogenous viruses of Drosophila melanogaster cell lines: Their frequency, identification and origin","volume":"14","author":"Plus","year":"1978","journal-title":"Vitro"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2985","DOI":"10.1101\/gad.1482006","article-title":"The RNA silencing endonuclease Argonaute 2 mediates specific antiviral immunity in Drosophila melanogaster","volume":"20","author":"Saleh","year":"2006","journal-title":"Genes Dev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"5938","DOI":"10.1073\/pnas.1400378111","article-title":"Host adaptation to viruses relies on few genes with different cross-resistance properties","volume":"111","author":"Martins","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Magwire, M.M., Fabian, D.K., Schweyen, H., Cao, C., Longdon, B., Bayer, F., and Jiggins, F.M. (2012). Genome-wide association studies reveal a simple genetic basis of resistance to naturally coevolving viruses in Drosophila melanogaster. PLoS Genet., 8.","DOI":"10.1371\/journal.pgen.1003057"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e2122026119","DOI":"10.1073\/pnas.2122026119","article-title":"A novel transposable element-mediated mechanism causes antiviral resistance in Drosophila through truncating the Veneno protein","volume":"119","author":"Brosh","year":"2022","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1038\/ncb1439","article-title":"The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing","volume":"8","author":"Saleh","year":"2006","journal-title":"Nat. Cell Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1016\/j.bbagrm.2008.06.008","article-title":"Monitoring miRNA-mediated silencing in Drosophila melanogaster S2-cells","volume":"1779","author":"Shah","year":"2008","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Webster, C.L., Waldron, F.M., Robertson, S., Crowson, D., Ferrari, G., Quintana, J.F., Brouqui, J.-M., Bayne, E.H., Longdon, B., and Buck, A.H. (2015). The Discovery, Distribution, and Evolution of Viruses Associated with Drosophila melanogaster. PLoS Biol., 13.","DOI":"10.1101\/021154"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"vez061","DOI":"10.1093\/ve\/vez061","article-title":"A new lineage of segmented RNA viruses infecting animals","volume":"6","author":"Obbard","year":"2020","journal-title":"Virus Evol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e89","DOI":"10.1093\/nar\/gku289","article-title":"Efficient chromosomal gene modification with CRISPR\/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells","volume":"42","author":"Bottcher","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Merk, K., Breinig, M., B\u00f6ttcher, R., Krebs, S., Blum, H., Boutros, M., and F\u00f6rstemann, K. (2017). Splicing stimulates siRNA formation at Drosophila DNA double-strand breaks. PLoS Genet., 13.","DOI":"10.1371\/journal.pgen.1006861"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"R25","DOI":"10.1186\/gb-2009-10-3-r25","article-title":"Ultrafast and memory-efficient alignment of short DNA sequences to the human genome","volume":"10","author":"Langmead","year":"2009","journal-title":"Genome Biol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Bottcher, R., Frohlich, T., and Forstemann, K. (2026). Low-toxic and organic solvent-free isolation of RNA. PLoS ONE, 21.","DOI":"10.1371\/journal.pone.0345312"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1016\/S1046-2023(03)00052-5","article-title":"In vitro analysis of RNA interference in Drosophila melanogaster","volume":"30","author":"Haley","year":"2003","journal-title":"Methods"}],"container-title":["Non-Coding RNA"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2311-553X\/12\/4\/28\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:11:39Z","timestamp":1785420699000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2311-553X\/12\/4\/28"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,30]]},"references-count":20,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["ncrna12040028"],"URL":"https:\/\/doi.org\/10.3390\/ncrna12040028","relation":{},"ISSN":["2311-553X"],"issn-type":[{"value":"2311-553X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,30]]}}}