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. 2018 May 18;10(5):272. doi: 10.3390/v10050272

Figure 5.

Figure 5

PA and PB1 substitutions increase CA/07 replication in mouse cells. Mouse embryonic fibroblasts were infected with parental CA/07 or recombinant CA/07-PA,PB1-MA viruses at a multiplicity of infection (MOI) of 0.1. (A) Cells were fixed at 18 h post infection (hpi) and analysed by immunofluorescence microscopy staining with anti-M1 antibody (red); nuclei were labelled with Hoechst dye (blue). (B) Cell lysates harvested at 18 hpi were analysed by Western blotting with antibodies specific for viral M1, NP, HA and cellular actin. (C) Total RNA isolated at 1, 6 and 18 hpi was analysed by reverse transcription quantitative PCR (RT-qPCR) to measure levels of influenza A virus (IAV) NP and NS genome segments. Values were normalized to 18S rRNA levels and expressed as the ratio of CA/07-PA,PB1-MA to parental CA/07. (D) Virion production was measured at 12, 18 and 24 hpi using the plaque assay in MDCK cells. In (C,D), error bars represent standard deviations (n = 3). * p-value < 0.05, paired Student’s t test.