Inhibition of West Nile virus replication in cells stably transfected with vector-based shRNA expression system.
In this study, the efficacies of short hairpin RNAs (shRNAs) targeting different regions of West Nile virus (WNV) strain Sarafend genome were investigated. Short hairpin RNAs targeting Capsid, NS2B and NS4B genes were cloned into pSilencer 3.1-H1 neo and designated as pshCapsid, pshNS2B and pshNS4B, respectively. Vero cells that were positively transfected were selected for creating stable cell lines expressing shRNAs constitutively. These cells were subjected to West Nile virus at multiplicity of infection (M.O.I.) of 10. The cells stably transfected with pshCapsid gave the best silencing effect among the three stable cell lines (transfected with pshCapsid, pshNS2B and pshNS4B) at both 12- and 24 h p.i. When compared to the non-transfected WNV-infected cells, pshCapsid stably transfected cells showed more than 4 log(10) unit reduction in viral transcripts and greater than 3 log(10) unit reduction in virus production. Cells stably transfected with pshNS2B did not exhibit as strong an inhibition when compared to the pshCapsid stably transfected cells having only 2 log(10) unit reduction in virus titre. The pshNS4B-stably transfected cells did not suppress WNV replication. Hence, from this study, pshCapsid has the potential to be developed into effective antiviral strategy for WNV infection.
Flavivirology Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, 5 Science Drive 2, National University of Singapore, 117597 Singapore, Singapore.
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D000818:Animals; D002522:Chlorocebus aethiops; D005822:Genetic Vectors; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*; D014902:West Nile virus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D034741:RNA, Small Interfering; D012367:RNA, Viral; D014764:Viral Proteins