Fig 1.

Comparison of plaque size and growth kinetics of WNV NS1 RQ10NK revertants. (A) WNV NS1 RQ10NK (left) was serially passaged in BHK21-15 cells. Within a few passages emerged a more rapidly growing small-plaque variant (WNV NS1 NK10YK, second left) and a large-plaque variant (WNV NS1 NK10KK, second right), which had a plaque size similar to that of the wild-type virus (WNV NS1 RQ, right). Sequences were generated after plaque purification and were representative of multiple isolates from each group. (B) Single-step growth kinetics of WNV NS1 RQ10NK and revertant viruses. WNV NS1 RQ10NK, WNV NS1 NK10YK, WNV NS1 NK10KK, and WNV NS1 RQ (WT) were added to BHK21-15 cells at an MOI of 1. At each of the indicated time points, virus in the supernatant was titrated by focus-forming assay. The data are the representative results of three independent experiments performed in triplicate. The growth of WNV NS1 NK10YK, WNV NS1 NK10KK, and WNV NS1 RQ (WT) was enhanced compared to that of WNV NS1 RQ10NK at 12, 24, 36, and 48 h (P < 0.05). Note that the growth curves for WNV NS1 NK10KK and WNV NS1 RQ (WT) are quite similar and difficult to distinguish on this graph. (C) Focus size comparison of WNV NS1 RQ10NK, WNV NS1 NK10YK, WNV NS1 NK10KK, and WNV NS1 RQ (WT). The data were obtained from several independent wells and were analyzed using a Biospot counter (Cellular Technology). Asterisks indicate statistical significance (***, P < 0.0001); n.s., not statistically significant. As described in Results, the WNV NS1 NK10YK revertant virus also contained a second site mutation in NS4B (F86C).