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Journal of Virology, December 2002, p. 12925-12933, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.12925-12933.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

RNA Silencing of Dengue Virus Type 2 Replication in Transformed C6/36 Mosquito Cells Transcribing an Inverted-Repeat RNA Derived from the Virus Genome

Zach N. Adelman,{dagger} Irma Sanchez-Vargas, Emily A. Travanty, Jon O. Carlson, Barry J. Beaty, Carol D. Blair, and Ken E. Olson*

Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523

Received 28 May 2002/ Accepted 10 September 2002

Double-stranded RNA (dsRNA) initiates cellular posttranscriptional responses that are collectively called RNA silencing in a number of different organisms, including plants, nematodes, and fruit flies. In plants, RNA silencing has been associated with protection from virus infection. In this study, we demonstrate that dsRNA-mediated interference also can act as a viral defense mechanism in mosquito cells. C6/36 (Aedes albopictus) cells were stably transformed with a plasmid designed to transcribe an inverted-repeat RNA (irRNA) derived from the genome of dengue virus type 2 (DEN-2) capable of forming dsRNA. Clonal cell lines were selected with an antibiotic resistance marker and challenged with DEN-2. The cell lines were classified as either susceptible or resistant to virus replication, based on the percentage of cells expressing DEN-2 envelope (E) antigen 7 days after challenge. Eight out of 18 (44%) cell lines designed to express irRNA were resistant to DEN-2 challenge, with more than 95% of the cells showing no DEN-2 antigen accumulation. One of the DEN-2-resistant cell lines, FB 9.1, was further characterized. DEN-2 genome RNA failed to accumulate in FB 9.1 cells after challenge. Northern blot hybridization detected transcripts containing transgene sequences of both sense and antisense polarity, suggesting that DEN-2-specific dsRNA was present in the cells. In addition, a class of small RNAs 21 to 25 nucleotides in length was detected that specifically hybridized to labeled sense or antisense DEN-2 RNA derived from the target region of the genome. These observations were consistent with RNA silencing as the mechanism of resistance to DEN-2 in transformed mosquito cells.

* Corresponding author. Mailing address: Arthropod-Borne and Infectious Diseases Laboratory, Foothills Research Campus, Colorado State University, Fort Collins, CO 80523. Phone: (970) 491-8604. Fax: (970) 491-8323. E-mail: kolson{at}lamar.colostate.edu.

{dagger} Present address: Molecular Biology and Biochemistry, University of California—Irvine, Irvine, CA 92697-3900.

Journal of Virology, December 2002, p. 12925-12933, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.12925-12933.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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