Inhibitory effect of small interfering RNA on dengue virus replication in mosquito cells.
BACKGROUND
Dengue viruses (DENs) are the wildest transmitted mosquito-borne pathogens throughout tropical and sub-tropical regions worldwide. Infection with DENs can cause severe flu-like illness and potentially fatal hemorrhagic fever. Although RNA interference triggered by long-length dsRNA was considered a potent antiviral pathway in the mosquito, only limited studies of the value of small interfering RNA (siRNA) have been conducted.


RESULTS
A 21 nt siRNA targeting the membrane glycoprotein precursor gene of DEN-1 was synthesized and transfected into mosquito C6/36 cells followed by challenge with DEN. The stability of the siRNA in cells was monitored by flow cytometry. The antiviral effect of siRNA was evaluated by measurement of cell survival rate using the MTT method and viral RNA was quantitated with real-time RT-PCR. The presence of cells containing siRNA at 0.25, 1, 3, 5, 7 days after transfection were 66.0%, 52.1%, 32.0%, 13.5% and 8.9%, respectively. After 7 days incubation with DEN, there was reduced cytopathic effect, increased cell survival rate (76.9 ± 4.5% vs 23.6 ± 14.6%) and reduced viral RNA copies (Ct value 19.91 ± 0.63 vs 14.56 ± 0.39) detected in transfected C6/36 cells.


CONCLUSIONS
Our data showed that synthetic siRNA against the DEN-1 membrane glycoprotein precursor gene effectively inhibited DEN-1 viral RNA replication and increased C6/36 cell survival rate. siRNA may offer a potential new strategy for prevention and treatment of DEN infection.
Guangzhou Center for Disease Control and Prevention, 23 Zhongshan 3rd Road, Guangzhou, Guangdong, China.
Xinwei
X
Wu
Hua
H
Hong
Jinya
J
Yue
Yejian
Y
Wu
Xiangzhong
X
Li
Liyun
L
Jiang
Lei
L
Li
Qiaoyan
Q
Li
Guoquan
G
Gao
Xia
X
Yang
D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D009033:Culicidae; D003716:Dengue Virus / Q000502:physiology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D014779:Virus Replication
D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D012367:RNA, Viral; D013778:Tetrazolium Salts; D013844:Thiazoles; D014764:Viral Proteins; C022616:thiazolyl blue