A simple fluorescent labeling technique to study virus adsorption in Newcastle disease virus infected cells

► This study demonstrates PKH67 and PKH26 labeling of enveloped NDV particles. ► Specificity of labeling was determined by three different techniques. ► Infectivity of the virus particles are not altered significantly by the labeling. ► The technique is fast, simple, convenient and easy to perform. ► The technique can be easily adapted to other enveloped RNA and DNA viruses. The present study demonstrates that the fluorescent general membrane dyes PKH67 and PKH26 are suitable to label Newcastle disease virus, an enveloped virus belonging to the family of paramyxoviridae. Adsorption of the labeled virus particles was tracked, visualized and quantitated using confocal laser scanning microscopy. The specificity of PKH-labeling was determined by colocalization analysis of the PKH signal with NDV-specific immunolabeling, and by using mock-infected controls and infection with detergent-pretreated labeled virus particles. The infectivity of the NDV particles was not affected by the labeling procedure as indicated by the results of a cytotoxicity ATP assay, an apoptosis assay and detection of virus-specific RNA and protein by qPCR and Western blotting, respectively, in cells infected with PKH-labeled and unlabeled virus particles. This technique can be used as an inexpensive, sensitive and rapid alternative method in the analysis of adsorption and internalization of enveloped viruses by the infected cells.