Figure 4: Growth kinetics of H7N9 viruses carrying different PB2 variants in mammalian and avian cells.

Reverse genetic versions of H7N9 viruses containing avian-PB2, avian-PB2-526R, PB2-526R/627K, PB2-627K, PB2-526R/701N or PB2-701N, along with the remaining seven gene segments of A/Zhejiang/DTID-ZJU01/2013, were used to infect (a) MDCK cells at an MOI of 0.001 and (b) HEK293T cells and (c) A549 cells at an MOI of 0.01. The cells were cultured at 37 °C and supernatants collected at 24, 48 and 72 h post infection and subjected to plaque assays in MDCK cells to determine virus titres. (d) A subset of reverse genetic versions of H7N9 viruses containing avian-PB2, avian-PB2-526R, PB2-526R/627K or PB2-627K or rescued reverse genetic A/Chicken/Zhejiang/DTID-ZJU01/2013 (Avian RG), were used to infect DF-1 cells at an MOI of 0.01 and cells were then cultured at 39 °C. Supernatants were collected at 24, 48 and 72 h post infection and subjected to plaque assays in MDCK cells to determine virus titres. The values displayed are the log₁₀ means±s.d. from three separate experiments. Statistical significance was analysed by one-way analysis of variance, corrected by the Bonferroni post-test: ***P<0.001 and **P<0.01. PFU, plaque-forming units; h.p.i., hours post infection.