Studying the Binding and Entry of the Intracellular and Extracellular Enveloped Forms of Vaccinia Virus

This chapter describes the methods for the study of binding and entry of the two different forms of vaccinia virus (VV)—the intracellular mature virus (IMV) and extracellular enveloped virus (EEV)—using immunofluorescent staining and confocal microscopy. After binding to or penetration of the cells, IMV, EEV, and virus cores are distinguished by different antibodies. Bound virus or penetrated cores are visualized and recorded by confocal microscopy and can be accurately counted. Although specific antibodies to IMV, EEV, and virus cores are required, this method is highly quantitative and also allows the recognition of virus aggregates, which would not be possible using other techniques, such as flow cytometry and radiolabeling of virus particles. Furthermore, this method bypasses the need for EEV purification that may damage the EEV membrane and release an IMV particle.