The effect of cold or isolation stress on neuroinvasiveness and virulence was investigated in mice inoculated with an attenuated WNV (WN-25) strain. The WN-25 variant differed from the parent strain by its inability to kill mice after I.P. injection though it was able to immunize even after injection with low doses of virus. Exposure of inoculated mice for 5 minutes a day to cold water (1 +/- 0.5 degrees C) for 8 days resulted in 60% mortality, while in nonstressed infected mice no death was observed. Cold or isolation stress increased the virus level in the brain to 8.9 and 7.4 log 10 PFU as compared to no virus in the infected control. Moreover, it was found that virus level in the spleen of stressed mice reached 3.4 and 3.7 log 10 PFU respectively, while in non-stressed mice no virus was detected. The virus which was isolated from the brain of moribund stressed mice was extremely virulent: I.P. inoculation of as little as 10 PFU caused death to normal non-stressed mice. We suggest that cold or isolation stress conditions in mice inoculated with an attenuated strain induce a selection process. The virus which was isolated from the brain of stressed mice changes its virulence and kills like wild type WNV.