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West Nile Virus (WNV) is now endemic throughout North America, with annual recurrence dependent upon successful overwintering when cold temperatures drive mosquito vectors into inactivity and halt transmission. To investigate whether avian hosts may serve as an overwintering mechanism, groups of eight to ten House Sparrows were experimentally infected with a WN02 genotype of WNV and then held until necropsy at 3, 5, 7, 9, 12, 15, or 18 weeks post-infection (pi) when they were assessed for the presence of persistent infection. Blood was collected from all remaining birds every two weeks pi, and sera tested for WNV RNA and WNV neutralizing antibodies. West Nile virus RNA was present in the sera of some birds up to 7 weeks pi and all birds retained neutralizing antibodies throughout the experiment. The detection of persistently infected birds decreased with time, from 100% (n = 13) positive at 3 weeks post-infection (pi) to 12.5% (n = 8) at 18 weeks pi. Infectious virus was isolated from the spleens of birds necropsied at 3, 5, 7 and 12 weeks pi. The current study confirmed previous reports of infectious WNV persistence in avian hosts, and further characterized the temporal nature of these infections. Although these persistent infections supported the hypothesis that infected birds may serve as an overwintering mechanism, mosquito-infectious recrudescent viremias have yet to be demonstrated thereby providing proof of principle. Author Summary: House Sparrows experimentally infected with West Nile virus [WNV] were necropsied at multiple time points from 3 to 18 weeks post infection (pi). The percent of birds with tissues positive for WNV RNA decreased from 100% at 3 wks to 13% at 18 wks pi; infectious virus was recovered from some birds by tissue co-cultivation and Vero cell passage from 3 to 12 wks pi, even though positive birds retained neutralizing antibody. WNV RNA also was detected in sera at 2 to 7 wks pi. Collectively, these data indicated that House Sparrows frequently developed persistent infections and could serve as an overwintering mechanism for WNV. However, recrudescent viremias suitable to infect mosquitoes have yet to be demonstrated and would seem to require host Immunosuppression. [ABSTRACT FROM AUTHOR]