Inhibition Of Small Interfering RNA To Influenza Genes Of Hemagglutinin And Neuraminidase And Its Application In Classical Reassortants

siRNAs are non-coding RNAs that can be widely found in organisms.The siRNA-mediated RNAi process can silence target genes efficiently and specifically,which is widely used in antiviral studies.It has been reported that specific siRNAs for M,NP or other segments of influenza could silence its mRNA and reduce virus titers.In our report,multiple siRNAs targeting HA and NA of PR8 was designed,and then its effect on virus titers and genome was detected.What's more,we also attempted to use siRNAs for screening reassortant strains of influenza.In section one,specific siRNAs were designed.Sequencing the genome of PR8 and H3N2,designing siRNAs targeting both HA and NA sequences of PR8 according to the pre-designed rules,and aligning with H3N2 in order to excluded homologous sequences.Eventually,12 siRNAs including three 19 nt common siRNAs and three 25 bp stealth siRNAs respectively specific for both HA and NA were chemical synthesized.In section two,the inhibitory effect and specificity of siRNAs were detected.The transfection reagents,concentrations of siRNAs and the best detecting time were optimized on MDCK and MTY6 cells.Inhibitory effects on PR8 of NA-specific siRNAs transfected in form of single,two and three was detected by NA activity and CCID50 assay.The results showed that NA-105 and NA-199 transfected into cells together has made 86.07% inhibition in replication of PR8,the best combination among all NA-specific siRNAs.And all these siRNAs didn't interfere nonspecifically with H3N2 production.The results of HA specific siRNAs showed that HA-56 and HA-802 co-transfected into cells has made 57.88% inhibition in replication of PR8,the best combination among all HA-specific siRNA.Also,they didn't interfere nonspecifically with H3N2 production.The inhibition rate of HA-specific and NA-specific siRNAs co-transfected into cells indicated approximately 95% inhibition in replication of PR8,which was higher than effects of HA-specific or NA-specific siRNAs transfected respectively,and didn't interfere with H3N2.In section three,reassortants strains between PR8 and H3N2 were prepared using HA-specific and NA-specific siRNAs.Virus mixtures containing PR8 and H3N2 were prepared in the following three ways:(1)H3N2 and PR8 were co-infected into MTY6 cells successively and harvested when 90% cytopathogenic efficiency appeared on MTY6 cells.(2)H3N2 and inactivated PR8 were co-infection chicken embryos and virus mixtures were harvested after 24 h.(3)PR8 and inactivated H3N2 were co-infected into chicken embryos and virus mixtures were harvested after 24 h.MTY6 cells were transfected with NA-105?NA-199 and HA-56?HA-802 respectively to limit growth of PR8 exisiting in virus mixtures.Monoclones were obtained by plaque assay and identified by RT-qPCR and RT-PCR.After turns of screening,reassortants strains were not obtained.At last,high-throughput sequencing methods were performed to identify if the gene mutation appeared in the genome of PR8.After three consecutive generations of PR8 virus suppression using siRNA on MTY6 cells,the high-throughput sequencing was used to detect the genome mutations of influenza virus.