Fig. 5

Replication kinetics of H4N1 AIVs in MDCK cells across different concentrations of TPCK-trypsin (a, c). MDCK cells were inoculated in triplicate with a multiplicity of infection (MOI) of 0.0001. Post-inoculation, the cells were incubated at 37 °C, and culture supernatants were collected at 12, 24, 36, 48, 60, and 72 h post-inoculation (hpi) for viral load measurement using digital PCR (dPCR). Cytopathic effects (CPE) were monitored using an inverted phase contrast microscope (Olympus) at ×100 magnification (b, d). (a) The growth curve indicates that at a TPCK-trypsin concentration of 2 µg/mL, the average viral load of CS01, CS01-A4, and CS01-A7 rapidly increased until 36 hpi, followed by a gradual decline. Notably, the viral load of CS01-A7 was significantly higher than that of CS01 and CS01-A4 prior to 36 hpi. (b) By 24 hpi, pronounced CPE was observed in CS01-A7; however, significant CPE in CS01 and CS01-A4 was evident at 48 hpi. (c) At a TPCK-trypsin concentration of 1 µg/mL, the growth curve shows that the average viral loads of CS01, CS01-A4, and CS01-A7 increased over time, except at 72 hpi. Moreover, except for 12 hpi, the viral load of CS01-A7 remained higher than that of the other viruses. Each data point represents the mean of three independent replicate experiments, and the CPE images are representative selections. Statistical significance between viral loads at each hpi was analyzed by two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (Supplementary Table S1)