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. 2018 Jul 17;92(15):e00295-18. doi: 10.1128/JVI.00295-18

FIG 5.

FIG 5

The L231A mutation strongly impacts infectivity in MeWo and MRC-5 cells. VZV-ORF9-V5 (WT) and mutant strain infectivity was assessed in MeWo cells (A and B) or MRC-5 cells (C to E). (A) Infection focus measurement at 48 hpi. MeWo cells were infected for 48 h, and the size of the infection foci was determined using CellProfiler software and expressed as the number of pixels present in each infection focus. The box plot depicts the 1st and 3rd quantiles (the lower and upper limits of the boxes, respectively) and the median (heavy black lines). Error bars represent minimum and maximum values. (B) Infectivity of infected cells. A known number of infected cells was used to infect MeWo cells seeded in a 24-well plate, and the number of infectious foci was determined in each well 3 days later. The graph shows the mean ratio of infectious cells/infected cells; error bars represent the standard deviation (SD). (C) Growth curves in MRC-5 cells. The graph shows the results of one representative experiment out of four; error bars represent the standard error of the mean (SEM). (D) The amount of the VZV genome over time was quantified by qPCR on DNA extracts. Primers in the human p21 promoter were used for normalization. Serial dilutions of a BAC-VZV of a known concentration were used to build a standard curve. Results are expressed as the absolute number of VZV genomes per nanogram of total DNA. Means from three independent experiments are depicted; error bars represent the SD. (E) In parallel, qRT-PCR was performed on RNA extracts to quantify the expression of IE (orf4), E (orf47), and L (orf40) genes. The expression of 18S rRNA was used to calculate the change in the threshold cycle number, and for each gene, relative expression levels were calculated, with the expression level for the WT at 24 hpi being used as a control. Means from three independent experiments are depicted; error bars represent the SD. (A, B, D, and E) A two-tailed t test was used to compare, at each time point, each mutant to the WT strain. *, P < 0.05; **, P < 0.01.