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Title: Measurement of Bluetongue Virus Binding to a Mammalian Cell Surface Receptor by an In Situ Immune Fluorescent Staining Technique

item Mecham, James
item MCHOLLAND, LINDA - University Of Wyoming

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/10/2009
Publication Date: 4/1/2010
Citation: Mecham, J.O., Mcholland, L.E. 2010. Measurement of Bluetongue Virus Binding to a Mammalian Cell Surface Receptor by an In Situ Immune Fluorescent Staining Technique. Journal of Virological Methods.

Interpretive Summary: Bluetongue virus is an orbivirus that infects both insect and mammalian hosts. The nature of the virus-cell interactions in initiating infection is incompletely understood. In the mammalian host, limited data suggests that the first step in BTV infection involves specific binding of viral proteins to cell surface glycoprotein receptors. Additional work is needed to identify and characterize the specific cell surface receptors targeted by BTV. This can be investigated by determining the effects of chemical and enzymatic treatments, or competition by well defined molecules on virus-cell interactions. This requires accurate measurement of virus binding to susceptible cells, and routinely uses radioactively labeled virus. Since there is widespread interest in developing non-isotopic research tools, a novel immune fluorescent assay was developed to accurately measure BTV binding to susceptible cells. This technique will facilitate studies to characterize and identify receptors for BTV and other viruses. Such information will aid in understanding cell tropism and pathogenesis of BTV infections.

Technical Abstract: A quantifiable in situ immune fluorescent assay (IFA) was developed to measure bluetongue virus (BTV) binding to mammalian cells. The utility of the assay was demonstrated with both Chinese hamster ovary (CHO) and bovine pulmonary artery endothelial (CPAE) cells. Since heparin sulfate (HS) has been shown to function as a receptor for a number of viruses, its role as a receptor for BTV was evaluated with the in situ IFA. Binding of BTV to both CHO and CPAE cells was inhibited in a dose dependent manner by HS. In addition, HS deficient CHO cells showed greatly diminished binding of BTV when compared to the parental cell line. The IFA protocol will find application, as a non-isotopic, quantifiable technique, to study virus-cell receptor interactions. Information gained from such studies will expand our understanding of the early steps in virus replication.