FIG. 1.

Growth characteristics of viruses differing in the NES and C terminus of NS1. Recombinants of strain 984 have been analyzed that contain HPAI NES and the HPAI C terminus (H/H), HPAI NES and the LPAI C terminus (H/L), LPAI NES and the HPAI C terminus (L/H), and LPAI NES and the LPAI C terminus (L/L). (A) Virus replication in CF cultures. Cells were inoculated at an MOI of 0.0005 PFU per cell. Growth curves show the averages of two independent experiments. (B) Spread of infection in CF monolayers. After infection, cells were covered with an Avicel overlay for 24 h to avoid virus spread by diffusion. Infected cells were stained with rabbit anti-FPV (H7N1) serum, HRP-conjugated donkey anti-rabbit serum, and TrueBlue peroxidase substrate. Plaque sizes of 100 were measured by using ImageJ software in two independent experiments. Pictures display a section of infected and stained cells. The sizes of yellow-edged plaques were measured for quantitative analysis. (C) Infectivity of the mutant viruses was measured by determining the EID₅₀ using 11-day-old embryonated chicken eggs. Eggs were infected with dilutions of 0.01 to 100 PFU for determination of the EID₅₀.