Fig 4. The effect of the SNP A94G in WT and DelVG.

(A) A circle-packing diagram visualizes the mutation frequencies in both the WT virus and the major two dominating DelVGs in both lineages. Each circle represents a distinct SNP, with its size proportional to the mutation frequency. The most frequent mutations are listed below the figure and highlighted as light green (WT) and orange (DelVG). (B) The impact of the A94G mutation on the WT virus was illustrated through agarose gel, qPCR & Sanger sequencing using PB2-specific primers. (C) Left panel, the minigenome replicon assay is employed to measure the polymerase activity in the presence of DI250 or DI250:A94G. pUC19 was used as a negative control. Right panel, competition assay was performed between WT virus and clonal populations of either DI250 or DI250:A94G, using a 1:10 WT:DIP input ratio normalized by NP copy number. Progeny from these infections were subsequently quantified by plaque assay. (D) 293 cells were transfected with 500ng plasmids that express both the negative and positive strands of DI250 or DI250:A94G. Following the transfection, the virus was introduced 24 hours later. After another 24 hours post-infection, 50μl of the viral progeny was used to initiate infection in fresh MDCK cells, a process repeated 14 times. The proportion of DelVG to NP segment was determined using qPCR, shown in the top panel, while the TCID50 for each passage was assessed and displayed in the bottom panel. The experiment was carried out in duplicate. Statistical significance was determined by Welch’s t-test (*, p = 0.01; **, p = 0.001).