Dengue virus growth, purification, and fluorescent labeling

Abstract

The early events of the dengue virus life cycle involve virus binding, internalization, traffi cking, and fusion. Fluorescently labeled viruses can be used to visualize these early processes. As dengue virus has 180 identical copies of the envelope protein attached to the membrane surface and is surrounded by a lipid membrane, amine-reactive (Alexa Fluor) or lipophilic (DiD) dyes can be used for virus labeling. These dyes are highly photostable and are ideal for studies involving cellular uptake and endosomal transport. To improve virus labeling effi ciency and minimize the nonspecifi c labeling of nonviral proteins, virus concentration and purifi cation precede fl uorescent labeling of dengue viruses. Besides using these viruses for single-particle tracking, DiD-labeled viruses can also be used to distinguish serotype-specifi c from cross-neutralizing antibodies. Here the details of virus concentration, purifi cation, virus labeling, applications, and hints of troubleshooting are described.