Limiting H1N1 Replication Via the dsRNAi Pathway Using Sequence-Specific LdsRNA

The seasonal influenza virus causes a common respiratory illness that can be life threatening to the immunocompromised, the very young, and the elderly. The current strategies for avoiding a serious influenza virus infection are good hygiene practices and the annual influenza vaccine. Unfortunately, last year’s influenza virus vaccine effectiveness ranged between 32-60% for children and adolescents and 46-54% for adults. These low efficacy rates are due to the vaccine often targeting hemagglutinin, a highly variable surface protein. This thesis seeks to create a prophylactic treatment via the RNA interference (RNAi) response that targets the virus’s conserved genes that are integral for replication, to be complementary to vaccines. The RNAi response is an innate immune response triggered by long (>30 bp) double stranded RNA (LdsRNA), which is made by viruses, but not healthy cells. In dsRNAi, viral LdsRNA is recognized and cleaved into small interfering (si)RNAs which are then used to bind complementary viral mRNAs and prevent protein translation, ultimately stopping viral replication. This study aimed to determine the optimal target gene for RNAi-mediated H1N1 replication inhibition. DsRNA was synthesized by in vitro transcription to target two integral H1N1 genes: the nucleoprotein (NP) and part of the RNA polymerase (PB1). MDCK cells were treated with these dsRNAs and infected with a human influenza A virus (IAV), strain H1N1/09/Cal. After 48 hours the media was collected and the viral titre quantified by a TCID50 assay. Both NP and PB1 LdsRNA showed a sequence-specific knockdown in viral titres suggesting that LdsRNA treatments are effective in triggering the RNAi response to protect against the virus. Western blots and RNAi inhibition experiments using a chemical inhibitor (aurintricarboxylic acid; ATA) were performed to support this hypothesis. The western blot results were inconclusive but the ATA antiviral trials show loss of protection with ATA suggesting the RNAi pathway is involved. This research aims to bring us a step closer to an effective RNAi-based prophylactic treatment for influenza virus infections.