Fig. 3: The biological characteristics of influenza A(H9N2) in vivo and in vitro.

Growth curves after inoculation of each virus at a multiplicity of infection (MOI) of 0.001 into (a) MDCK cells and at an MOI of 0.001 into (b) CEF cells. Each point on the curve is the mean ± standard deviation from three independent experiments. The independent-samples t test was used for analysis. c Thermo stability of the influenza A(H9N2) viruses. The influenza A(H9N2) viruses were incubated for 1, 2, 3, 4, and 5 h at 50 °C. The titers of heat-treated recombination viruses were determined by TCID₅₀ assay in MDCK cells. Western blots of CEF (d) and A549 (e) cell cultures inoculated with five influenza A(H9N2) virus at an MOI of 0.01 in the presence of 1 μg/ml trypsin for 24 h. f Syncytia formation induced by five influenza A(H9N2) viruses in Vero cells. Cultures of Vero cells were inoculated with H9N2 viruses at MOI of 1 for 16 h, followed by incubation with 10 μg/ml of TPCK-trypsin for 15 min to cleave expressed HA0. To induce syncytia formation, Vero cells were incubated for 5 min with fusion buffers of pH values ranging from 5.0 to 5.8, with 0.2 unit increments, and were visualized 3 h later. Representative images at pH values of 5.0 to 5.8 derived from three replicates are shown. Five groups of ten 5-week-old SPF chickens were infected intranasally with 10⁶ EID₅₀/200 μL of each virus. g Viral titers in the lungs of three inoculated chickens. h Viral titers in the duodenum of three inoculated chickens. i Viral titers in the kidney of three inoculated chickens. j Viral titers in the brain of three inoculated chickens. k Antibody titers of inoculated and exposed chickens at 14 dpi. An HI titer ≥1:16 was considered seroconversion. Five groups of three female BALB/c mice were intranasally inoculated with 10⁶ EID₅₀/50 μL of the H9N2 viruses. Three mice from each group were euthanized at 4 dpi. Viral titers in the mouse lungs (l) were shown. The dashed lines represent the detection limit. The independent-samples t test was used for analysis.