FIG 5.

Amino acid 52N in H7N9 NP contributes to reduced sensitivity to murine Mx1. (A) Direct comparison of the Mx1 sensitivity of SH/1-NP(wt), SH/1-NP(N52Y), and VN/04 in the VN/04 polymerase reconstitution system and increasing amounts of Mx1 as described in the legend of Fig. 4C. Expression of Mx1 and viral PB2 was monitored by Western blotting. (B) Introduction of SH/1-NP(N52Y) or VN/04-NP does not alter virus growth of SH/1 in MDCKII cells. Cells were infected at an MOI of 0.01 with the indicated viruses in the presence of 0.5 μg/ml trypsin. At the indicated time points postinfection (p.i.), virus titers were determined by plaque assay. Error bars indicate the standard deviations of one experiment performed in duplicates. (C and D) Lung titers of H7N9 SH/1 viruses encoding an N52Y exchange in NP or the NP of VN/04. C57BL/6 (C) and B6.A2G-Mx1 (D) mice (n = 3) were infected intranasally with 100 PFU. At 3 and 6 days postinfection, virus titers were determined in lung homogenates by plaque assay. (E and F) Lung titers of H7N9 in IFN-pretreated animals. C57BL/6 (E) and B6.A2G-Mx1 (F) mice (n = 3) were intranasally pretreated with 20,000 units of IFN-α or PBS (ctrl). After 24 h the animals were infected intranasally with 200 PFU of the indicated viruses for 2 days, and virus titers were determined in lung homogenates by plaque assay. Student's t test was performed to determine the P values. *, P < 0.05; **P < 0.01; ***, P < 0.001; ns, not significant. D, day.