H5N1- NS gene 103L, 106I and 103L+ 106I mutations enhance viral replication in mouse lungs and increase IFN-β antagonism in mouse cells. a. Groups of 12 CD-1 mice were infected intransally with 5x103 pfu of the rPR8-H5N1-NS and the three mutants (rPR8-H5N1-NS-L103F, rPR8-H5N1-NS-I106M and rPR8-H5N1-NS-L103F+I106M). Lung homogenates were collected at days 1, 3, 5 and 7 pi (n = 3) and viral titers were assessed by plaque assay. The L103F+I106M mutant generated 100 fold less virus than the others (*p<0.05; by paired two-tailed t-test). b. IFN-β induction in mouse M1 cells. Monolayers of M1 cells in 35 mm dishes were infected at MOI = 2 and incubated for 24 h before collecting supernatants for quantification of IFN-β by ELISA (n = 3 biological replicates with n = 2 technical replicates). The values for rPR8-H5N1-NS-wt and NS1 L103F + I106M were first reported by  and are included here as controls. Values are shown as average +/− standard deviation with significant difference indicated for NS1 L103F + I106M relate to each of the other mutants (*** p<0.001; two-tailed t-test). c. IFN-β induction in mouse lungs. Groups of 12 CD-1 mice were infected intransally with 5x103 pfu of the rPR8-H5N1-NS and the rPR8-H5N1-NS-L103F+I106M. The levels of IFN-β lung homogenates were determined by ELISA at days 1 and 3 (n =3), and 5 (n =6) pi. d. Infectious yield associated with IFN-β induction in mouse M1 cells shown in panel B were quantified for infectious yield by plaque assay. The yield of the L103F+I106M mutant was significantly less than the other 3 viruses (*** p<0.001; two-tailed t-test). e. Infectious yield in monkey Vero cells were quantified for infectious yield by plaque assay. The yield of the L103F+I106M mutant was significantly greater than each of the other 3 viruses (*** p<0.001; two-tailed student’s t-test).