FIG 2 .

HEF is a key determinant of the exceptional acid and temperature stability of IDV. (A) Schematic representation of A/WSN/33, D/OK/11, and chimeric A/D-HEF viruses used in this study. Specifically, we generated a D/OK/11 HEF expression plasmid in the context of pHW2000-derived dual-promoter reverse genetic system (RGS) expression construct of the A/WSN/33 neuraminidase (NA) segment. The complete HEF cDNA from D/OK/11 is flanked by 183 nucleotides of the 3′ NA viral RNA (vRNA) and 157 nucleotides of the 5′ NA vRNA, and initiation codons in the 3′ NA vRNA are mutated to express HEF protein only. Chimeric A/D-HEF virus was generated through cotransfection of 293T and MDCK cells with the chimeric HEF plasmid together with A/WSN/33-derived PA, PB1, PB2, NP (nucleoprotein), M (matrix), and NS (nonstructural) RGS plasmids. (B) A/WSN/33, IDV D/OK/11, and chimeric A/D-HEF were treated in solution at 53°C for different times, followed by incubation in 4°C for 30 min prior to infection experiments. (C) A/WSN/33, D/OK/11, and chimeric A/D-HEF were treated in solution over a range of pH values from pH 3.0 to 7.0 for 30 min, followed by neutralization and incubation for another 30 min in 4°C prior to the infectivity experiment. The data presented in this figure are representative of three independent experiments, with each assay sample tested in duplicate. The error bars represent standard deviations and indicate the variations among experiments.