title: Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5 cells: stability and interactions with the core RNAi machinery.
abstract: While several studies have been conducted to investigate the stability of dsRNA in the extracellular medium (hemolymph, gut content, saliva), little is known regarding the persistence of dsRNA once it has been introduced into the cell. Here, we investigate the stability of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) genomic dsRNA fragments after transfection into Bombyx-derived Bm5 cells. Using RT-PCR as a detection method, we found that dsRNA could persist for long periods (up to 8 days) in the intracellular environment. While the BmCPV genomic dsRNA was processed by the RNAi machinery, its presence had no effects on other RNAi processes, such as the silencing of a luciferase reporter by dsLuc. We also found that transfection of BmCPV genomic dsRNA could not establish a viral infection in the Bm5 cells, even when co-transfections were carried out with dsRNAs targeting Dicer and Argonaute genes, suggesting that the neutralization by RNAi does not play a role in the establishment of an in vitro culture system. The mechanism of the dsRNA stability in Bm5 cells is discussed, as well as the implications for the establishment for an in vitro culture system for BmCPV.
authors.affiliation: Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research "Demokritos", P. Grigoriou & Neapoleos Str, Aghia Paraskevi Attikis, 153 42 Athens, Greece. Electronic address: swevers@bio.demokritos.gr.
authors.forename: Luc
authors.initials: L
authors.lastname: Swevers
authors.affiliation: Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research "Demokritos", P. Grigoriou & Neapoleos Str, Aghia Paraskevi Attikis, 153 42 Athens, Greece.
authors.forename: Anna
authors.initials: A
authors.lastname: Kolliopoulou
authors.affiliation: Guangdong Engineering Research Center of Subtropical Sericulture and Mulberry Resources Protection and Safety, Guangdong Provincial Key Lab of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, People's Republic of China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Biology, University of Crete, Voutes University Campus, 700 13 Heraklion, Crete, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Daskalaki
authors.affiliation: Department of Biology, University of Crete, Voutes University Campus, 700 13 Heraklion, Crete, Greece.
authors.forename: Frederic
authors.initials: F
authors.lastname: Verret
authors.affiliation: Department of Biology, University of Crete, Voutes University Campus, 700 13 Heraklion, Crete, Greece.
authors.forename: Kriton
authors.initials: K
authors.lastname: Kalantidis
authors.affiliation: Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
authors.forename: Guy
authors.initials: G
authors.lastname: Smagghe
authors.affiliation: Guangdong Engineering Research Center of Subtropical Sericulture and Mulberry Resources Protection and Safety, Guangdong Provincial Key Lab of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, People's Republic of China. Electronic address: cyfz@scau.edu.cn.
authors.forename: Jingchen
authors.initials: J
authors.lastname: Sun
mesh_terms: D000818:Animals; D012831:Bombyx / Q000821:virology*; D002478:Cells, Cultured; D020868:Gene Silencing; D008156:Luciferases; D034622:RNA Interference; D012330:RNA, Double-Stranded; D012087:Reoviridae / Q000276:immunology*; D014026:Nicotiana / Q000821:virology; D014162:Transfection
chemical_list: D012330:RNA, Double-Stranded; D008156:Luciferases
keywords: Bm5 cells; BmCPV; Cytoplasmic polyhedrosis virus; RNAi; dsRNA