FIG. 4.

Temperature sensitivity of plaque formation by K.ts recombinant viruses. (a) Several isolates of each group of KOS recombinant viruses were analyzed for plaque formation in Vero cells at 39°C versus 33°C. Each of the K.ts isolates exhibited a reduction of approximately 5 logs in plaque formation. (b) Complementation of plaque formation in HS30 cells. Plaque formation in Vero or HS30 cells was quantitated at 39°C and the ratio determined. tsB7 plaque formation was of the order of 3 log units higher in HS30 cells, a result also obtained with each of the K.ts isolates. Comparatively minor differences between Vero and HS30 cells were observed for the other strains. (c) Plaque size for K.HF-B and K.ts-2 viruses at 33°C. The K.ts-2 plaque size was approximately one-third of the area of the corresponding isolates containing the HFEM UL36 gene. Plaque size was estimated using Image Pro Plus software from approximately 50 plaques for each virus, with representative images shown by crystal violet staining. Error bars indicate standard deviations. (d) Temperature sensitivity of gene expression. Vero cells were infected (MOI, 5) with K.ts-2, K.HF-B, K.R-3, or wt KOS at 33°C or 39°C, and extracts collected at 24 h postinfection and analyzed for the accumulation of viral proteins VP5 and ICP8. Actin levels were measured as a loading control.