RNA interference mediated inhibition of influenza virus NP and PA gene expressions and multiplication in MDCK cells

    LI Yaoª²Chen, ZHAO Zhiª²Gang, LI Kangª²Sheng

    Department of Microbiology and Immunology, Department of Urology, Second Affiliated Hospital, Medical College, Shantou University, Shantou 510041, China

    ¡¾Abstract¡¿ AIM: To apply the small interfering RNAs targeting NP or/and PA gene of influenza virus to inhibit the expression of NP or/and PA and multiplication of influenza virus in Madinª²Darby canine kidney (MDCK) cells. METHODS: The recombinant plasmids, pEGFP/NP, pGenesil/PA and pEGFP/NP+ PA were constructed and transfected into MDCK cells. The transfected MDCK cells were infected with subtype H5N1 strain. The transfection efficiency was determined using fluorescence microscopy. The expression levels of NP gene were determined by Western blot and the transcriptions of NP or/and PA gene were detected by semiª²quantitative RTª²PCR. The culture supernatants were assayed for hemagglutination activity (HA) at different time points after infection in order to determine the effect of the siRNA on the multiplication of infectious virus. RESULTS: The results from fluorescence microscopy suggested that the transfection efficiency was about 65.0%. The introductions of plasmids pEGFP/ NP and pEGFP/NP+PA showed efficient in specifically inhibiting the expression of NP according to the results of Western blot, with inhibitory rates of 64.5% and 69.6%. Semiª²quantitative RTª²PCR showed that the transcription of NP gene was reduced by nearly 66.0% in infected MDCK cells after transfected by pEGFP/NP, and PA gene was reduced by nearly 63.0% in infected MDCK cells after transfected by pGenesil/PA. The transcriptions of NP and PA genes were reduced by nearly 71.4% and 69.3% synchronously in infected MDCK cells transfected by pEGFP/NP+PA. In contrast, the control plasmid did exhibit no inhibitory effect on the protein expressions and the transcription of NP or/and PA gene. The inhibitory rates of influenza virus in MDCK cells transfected with pEGFP/ NP, pGenesil/PA and pEGFP/NP+PA were 87.0%, 75.0% and 96.9% respectively according to the HA results, which demonstrated that the inhibitory ability of pEGFP/NP+PA was the strongest among the various groups. CONCLUSION: The small interfering RNAs targeting NP or/and PA gene shows a dramatic inhibition on protein expression, RNA transcription and multiplication of influenza virus in MDCK cells. ......