Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green f luorescent protein gene instead of the receptor-binding G protein gene (VSV⌬G*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSV⌬G*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSV⌬G* complemented with VSV G protein (VSV⌬G*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSV⌬G*-ResGP but not to VSV⌬G*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and͞or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV) MATERIALS AND METHODSPlasmid Construction. The plasmid pVSVFL(+) expressing the 11,161-nt positive-strand (antigenomic) VSV RNA sequence was constructed from four DNA fragments cloned into pBluescript SK(+) (Stratagene). The starting plasmid for the construction, pVSVFL(-), expressed the complete negativesense VSV genomic RNA (Indiana serotype) from a T7 promoter. This plasmid was generated in a nine-step cloning procedure that involved joining the five original cDNA clones of the VSV mRNAs (15-17) with gene junction fragments and terminal fragments. These fragments were generated by reverse transcription and PCR

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-ΔG) 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-ΔG pseudotypes is restricted to a single round of replication the analysis can be performed using biosafety level 2 (BSL-2) containment. As such, rVSV-ΔG 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-ΔG pseudotypes. Procedures to generate rVSV-ΔG 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-ΔG pseudotypes for subsequent analysis.