Treatment of mice with cyclophosphamide resulted in recrudescence of WNV.

− negative, + positive.
1Skin at the inoculation site consisted of the left rear footpad.
2Lymph nodes consisted of both popliteal and both inguinal lymph nodes.
Adult, female B6 mice were inoculated s.c. in the left rear footpad with diluent alone (mock) or with 103 PFU of WNV. Mice were treated with cyclophosphamide or PBS on days 28 and 33. Mice were sacrificed on day 45 p.i., and WNV infection was confirmed in all WNV-inoculated mice by seroconversion. Six tissues (skin–inoculation site, spinal cord, brain, lymph nodes, spleen, and kidney) per mouse were harvested for virus isolation and RT-PCR for WNV. Virus isolation was performed by co-culturing the homogenized tissues on Vero cell monolayers. All samples were passed two more times onto fresh monolayers, and tissue culture supernatants from the third passage were tested for the presence of WNV by RT-PCR. Mouse #12 and mouse #25 were sick during acute phase of disease (7 to 14 days p.i.). All other mice did not show any clinical disease.