Depending on the stringency of siRNA-target base pairing, siRNA treatment may cause selection of siRNA-resistant viruses, and this has been shown with HIV and HCV [17, 27, 28] , and therefore abrogated the further medication or treatments. Using lentiviral delivery system, we established stable cell lines persistently expressing shRNA, which provided a more convenient experimental approach to study long term inhibition effect of siRNAs and screen for siRNA resistant virus mutants in quasispecies in vitro. Our results showed that H5N1 virus cultured in shM2-MDCK were equally susceptible to siM2 as the original virus even after 40 passages. Moreover, sequencing of siM2 targeted region in 10 such independent plaque purified virus isolates revealed sequence identical to the parental one. The current data have shown no insertion, deletion and nucleotide substitution in the siRNA target sequence, therefore demonstrated siM2 possessed good long term inhibition effect for influenza virus replication without the problem of siRNA resistant mutants.