A functional henipavirus envelope glycoprotein pseudotyped lentivirus assay system.

Dimple Khetawat,Christopher C Broder

doi:10.1186/1743-422x-7-312

Dimple Khetawat, Christopher C Broder

Open Access

https://doi.org/10.1186/1743-422x-7-312

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Abstract

BackgroundHendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins.ResultsFunctional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F.ConclusionsTogether, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.

Highlights

Hendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus
Henipavirus F and G envelope glycoprotein pseudotyped lentivirus particles It is often desirable to study the functions of viral envelope glycoproteins that are involved in attachment, membrane fusion and entry in the context of a viral particle
Pseudotyped virus particles generated with the NiV F and G glycoproteins were able to infect and produce luciferase reporter gene activity at various levels on all permissive receptor expressing cells (Figure 1A) while no signal was observed with the receptor negative HeLa-USU or with control virus particles generated by transfection with empty vector

Summary

Introduction

Hendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Commonly known as flying foxes in the family Pteropodidae, are the principal natural reservoirs for both NiV and HeV (reviewed in [2]) recent evidence of henipavirus infection in a wider range of both frugivorous and insectivorous bats has been reported [5,6] Since their identification, both HeV and NiV have caused repeated spillover events. Among these spillover events of NiV the human mortality rate has been higher (~75%) along with evidence of person-to-person transmission [12,13] and direct transmission of virus from flying foxes to humans via contaminated food [14]

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