The screening of potential siRNA resistant mutants were performed in our established stable shRNA-expressing cell lines according to previously described protocols [42] with some modification. Briefly, the shM2-MDCK cells in a T25 cm 2 flask were infected with H5N1 virus. After cultured for 2 days, the supernatants were harvested. Part of the supernatants was inoculated to shM2-MDCK for next passage of the virus culture, another part was subjected for plaque assay to determine if potential siRNA-resistant virus appeared. Every 10 passages, ten bigger size of plaques in the plaque assay were picked for sequencing to detect any mutation in the siRNA targeting region using a pair of primers: forward, 59-AAG GCA GAT GGT GCA GGC AAT-39 and reverse, 59-TAC TCC AGC TCT ATG CTG ACA-39.