Fig 4.

A. Replication kinetics of BgV_C-CG (prototype), BgV_C-AT (double mutant NS4B/5), BgV_T-AT (triple mutant NS4A/4B/5) and WNV_KUN in C6/36 cells (28°C—left), BSR cells (37°C—middle) and Vero cells (37°C—right) over five days. The cells were inoculated with each virus at MOI 0.1 in triplicate, incubated 1h 30min at 28°C or 37°C, the inoculum removed and the cells washed before incubating at 28°C or 37°C with fresh medium. The supernatants were harvested at 2, 24, 48, 72, 96 and 120 hours p.i., stored at -80°C until they were titrated on C6/36 cells and the titres determined by fixed cell ELISA. Error bars represent the standard deviation and the dotted lines represent the lower limit of detection. The graph includes only representations of statistical analysis for comparisons to BgV_T-AT for clarity (* = p < 0.05). The results of the comparisons to BgV_C-AT were the same as those to BgV_T-AT. A star can therefore be read as a statistically significant difference to the mutants at that time point. B. BgV variants and WNV_KUN replication in DF-1 cells at 37°C. The cells were inoculated with each virus in triplicate at MOI 1 and incubated at 28°C (C6/36) or 37°C (DF-1). The supernatants were harvested after five days, titrated on C6/36 cells and the titres determined by fixed cell ELISA. The error bars represent the standard deviation and the dotted line represents the lower limit of detection. C. Prototype BgV_C-CG, BgV_C-CG/WNV-prME, and mutant BgV_T-AT, BgV_T-AT/WNV-prME and WNV_KUN replication in vertebrate cells at 37°C. Each cell line was inoculated with each virus in triplicate at MOI 1 as above and incubated at 28°C (C6/36) or 37°C (BSR, DF-1, Vero). The supernatants were harvested, titrated on C6/36 cells and the titres determined by fixed-cell ELISA. Error bars represent the standard deviation and the dotted line represents the lower limit of detection.