Influenza (flu) pandemics have posed a great threat to human health in the last century. However, current vaccination strategies and antiviral drugs provide limited protection. RNA interference (RNAi) is an effective means of suppressing influenza virus replication. PB1 is the critical protein subunit of the influenza virus RNA polymerase. The gene encoding this protein, PB1 , is highly conserved among different subtypes of IAV and was therefore chosen as the target in this study. The oligonucleotide, PB1 -shRNA, contains a 21-bp siRNA corresponding to nucleotides 1,632 to 1,652 of PB1 linear vRNA with BamHI or EcoRI restriction enzyme sites incorporated at the ends. The PB1 -shRNA oligonucleotide was directionally cloned into the RNAi-ready pSIREN-shuttle vector. The correct structure of the resulting pSIREN/ PB1 plasmid was confirmed by restriction endonuclease digestion. Madin-Darby canine kidney (MDCK) cells were transfected with pSIREN/ PB1 and subsequently infected with IAV at an MOI of 0.1 (A/PR/8/34, H1N1). The virus titer in cell culture supernatants was determined 48 hours later, and it was found that virus growth was inhibited by more than 50-fold relative to controls. Furthermore, embryonated eggs and mice were inoculated with liposome-encapsulated pSIREN/ PB1 and then challenged with the A/PR/8/34 virus. The results showed at least a 100-fold inhibition in virus replication in egg allantoic fluid and a survival rate of between 50% and 100% in experimental mice. This study demonstrates that PB1 -shRNA expressed by the recombinant plasmid pSIREN/ PB1 inhibits influenza A virus replication both in vitro and in vivo. These observations provide a foundation for the development of a new and efficient treatment of influenza infections.
Archives of Virology – Springer Journals
Published: Nov 1, 2011
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