Virus rescue rZH501-M847-G, a recombinant ZH501 carrying G at nucleotide 847 of anti-viral-sense M, was recovered by cotransfection of pProT7-wS(+), pProT7-wM(+)-M847-G, pProT7-wL(+), pT7-IRES-N, pT7-IRES-L and pCAGGS-G into BHK/ T7-9 cells by TransIT-LT1 (Mirus, Madison, WI), as described previously [45] . rZH501-M847-A was recovered by co-transfec- Figure 9 . The virulence of rZH501-M847-A-type virus obtained from the brain of rZH501-M847-G mouse. Five 5-week-old female CD-1 mice were inoculated i.p. with 10 1 , 10 2 , 10 3 , 10 4 or 10 5 PFU of the brain homogenates from the 8b mouse (Fig. 8A ). Control mice were inoculated with HBSS. Mortality was recorded daily for 28 days p.i. No mice died after 9 days p.i. doi:10.1371/journal.pone.0009986.g009 tion of pProT7-wS(+), pProT7-wM(+)-M847-A, pProT7-wL(+), pT7-IRES-N, and pT7-IRES-L into BHK/T7-9 cells. The culture medium was replaced with fresh medium at 24 h post transfection. At 5 days post transfection, the culture supernatants were collected, clarified and then inoculated into VeroE6 cells. The supernatant of infected VeroE6 cells at 3 days p.i. was collected and used for titration of virus infectivity by plaque assay and for the subsequent experiments. Sequence analysis of M segment RNA confirmed the presence of the expected nucleotide at M847 in rZH501-M847-G and rZH501-M847-A.