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

Authors: Mecham, James; Brown, Philip

Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 4/4/2007
Publication Date: 7/18/2007
Citation: Mecham, J.O., Brown, P.L. 2007. In situ immune infrared fluorescent staining for detection and quantification of bluetongue virus in insect cell culture. American Society for Virology Meeting. Corvallis, OR. July 14-18, 2007.

Interpretive Summary: Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. of biting midges. In this laboratory, cell lines have been produced from C. sonorensis that have proven to be particularly useful for various studies on BTV and the insect vector. However, since BTV does not produce detectable damage in these cells, indirect methods, such as co-cultivation with susceptible mammalian cells, are necessary for detection and quantifying virus replication in the insect cells. This paper describes the development of a technique to directly visualize and quantify BTV infection in Culicoides cell culture by immune infrared fluorescence staining. The sensitivity of this assay was comparable to plaque assay in mammalian cell culture. This assay will facilitate the use of the Culicoides cell lines in research questions and for isolation and quantification of BTV from biological samples.

Technical Abstract: Bluetongue virus (BTV) is transmitted to sheep, cattle and other ruminants by Culicoides spp. of biting midges. Cell lines developed from C. sonorensis have facilitated studies of virus replication and the role of the insect vector in BTV transmission. However, techniques for directly detecting virus in the insect cells are lacking. We report the application of an In situ immune infrared fluorescent staining technique to visualize and quantify BTV infection in Culicoides cell culture. Insect cell cultures infected with BTV were fixed, permeabilized and reacted with virus-specific monoclonal antibody and infrared labeled secondary antibody. The extent of virus replication in the cells was determined and quantitated by fluorescence measurement using the Odyssey Infrared Imaging System. The sensitivity of virus detection in the insect cell culture using this system was comparable to detection in vertebrate cell culture. The technique was also used to detect and titer BTV from infected Culicoides insects. The results indicate the potential utility of this procedure for both diagnostic work and research application related to BTV infection of the insect vector.