title: Visualizing dengue virus through Alexa Fluor labeling.
abstract: The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development of methods to probe this interaction would be useful. Recent advancements in fluorophores development and imaging technology can be exploited to improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually. The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, which contains a single positive strand RNA genome. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer's protocol for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block productive infection of virus and also induce cell cytotoxicity. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine, making them ideal for studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue virus by virus-specific antibody and its putative receptors in host cells. This method could have useful applications in virological studies.
authors.affiliation: Defence Medical and Environmental Research Institute, DSO National Laboratories.
authors.forename: Summer
authors.initials: S
authors.lastname: Zhang
authors.forename: Hwee Cheng
authors.initials: HC
authors.lastname: Tan
authors.forename: Eng Eong
authors.initials: EE
authors.lastname: Ooi
mesh_terms: D003716:Dengue Virus / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D009930:Organic Chemicals / Q000737:chemistry; D014773:Virology / Q000379:methods*
chemical_list: C417664:Alexa594; D005456:Fluorescent Dyes; D009930:Organic Chemicals