In silico designing of siRNA targeting PB 1 gene of Influenza A virus and in vitro validation
Bhawana Jain, Amita Jain, Om Prakash, Ajay Kr Singh, Tanushree Dangi, Mastan Singh, Kaleshwar P Singh.
Abstract
Genomic variability makes Influenza A virus (IAV) the least susceptible to existing vaccines or anti-influenza drugs. siRNA targeting viral gene silent the gene by cleaving mRNA.
Objective: To develop siRNA targeting polymerase basic 1 (PB1) gene and to validate its efficiency in vitro.
Materials and Methods: siRNA was designed rationally, targeting the most conserved region of PB1 gene of IAV strains. To choose the most efficient siRNA, three levels screening method was developed. One was selected due to its unique position in conserved region. siRNA efficacy was confirmed in vitro on Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/2011(H3N2)] and Influenza A/pdmH1N1 [A/India/LKO2151/2012(H1N1)].
Result and Conclusion: Total 147 strains worldwide and 43 Indian strains, when aligned, showed seven sets of conserved regions (> 30 bp stretch and < 5% mismatches). The longest ORF was targeted by the selected siRNA, which showed 57% inhibition in replication of Influenza A/pdmH1N1 and 60.6% inhibition in replication of Influenza A/H3N2 at 72 hpi and 48 hpi respectively on MDCK cell line. This study shows that siRNA targeting PB1 may be moderately effective in controlling IAV replication so can be used as anti-IAV therapeutic agent.
Key words: Influenza A virus, siRNA designing, multiple sequence alignment, MDCK cell line, in vitro validation, PB1 gene
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