In situ immune infrared fluorescent staining for detection and quantitation of bluetongue virus in Culicoides insect cell culture

Bluetongue virus (BTV) is transmitted to sheep, cattle and other ruminants by Culicoides spp. of biting midges. Cell lines have been developed from Culicoides sonorensis; however, techniques to detect and quantitate viable virus directly in these insect cells are lacking. In situ immune infrared fluorescent staining techniques were developed to visualize and quantitate BTV infection in Culicoides cell culture by both an endpoint titration and an agarose overlay fluorescent focus assay. Insect cell cultures infected with BTV were fixed, permeabilized and reacted with virus-specific monoclonal antibody and fluorescent-labeled secondary antibody. Virus replication in the infected cells was visualized and quantitated by measuring fluorescence with an infrared imager. The sensitivity of virus detection in insect cell culture using these techniques was comparable to or better than detection by standard techniques in vertebrate cell culture.