Generation of VSV pseudotypes using recombinant ΔG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines

Vesicular stomatitis virus (VSV) is a prototypic enveloped animal virus that has been used extensively to study virus entry, replication and assembly due to its broad host range and robust replication properties in a wide variety of mammalian and insect cells. Studies on VSV assembly led to the creation of a recombinant VSV in which the glycoprotein (G) gene was deleted. This recombinant (rVSV-Delta G) has been used to produce VSV pseudotypes containing the envelope glycoproteins of heterologous viruses, including viruses that require high-level biocontainment; however, because the infectivity of rVSV-Delta G pseudotypes is restricted to a single round of replication the analysis can be performed using biosafety level 2 (BSL-2) containment. As such, rVSV-Delta G pseudotypes have facilitated the analysis of virus entry for numerous viral pathogens without the need for specialized containment facilities. The pseudotypes also provide a robust platform to screen libraries for entry inhibitors and to evaluate the neutralizing antibody responses following vaccination. This manuscript describes methods to produce and titer rVSV-Delta G pseudotypes. Procedures to generate rVSV-Delta G stocks and to quantify virus infectivity are also described. These protocols should allow any laboratory knowledgeable in general virological and cell culture techniques to produce successfully replication-restricted rVSV-Delta G pseudotypes for subsequent analysis. (C) 2010 Elsevier B.V. All rights reserved.