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Open Access Highly Accessed Research

Recombinant Hendra viruses expressing a reporter gene retain pathogenicity in ferrets

Glenn A Marsh1*, Elena R Virtue14, Ina Smith1, Shawn Todd1, Rachel Arkinstall1, Leah Frazer1, Paul Monaghan1, Greg A Smith1, Christopher C Broder2, Deborah Middleton1 and Lin-Fa Wang13

Author Affiliations

1 CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, Geelong, VIC 3220, Australia

2 Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD 20814, USA

3 Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore 169857, Singapore

4 Current address - Monash Institute of Medical Research, Clayton, VIC 3168, Australia

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Virology Journal 2013, 10:95  doi:10.1186/1743-422X-10-95

Published: 25 March 2013

Abstract

Background

Hendra virus (HeV) is an Australian bat-borne zoonotic paramyxovirus that repeatedly spills-over to horses causing fatal disease. Human cases have all been associated with close contact with infected horses.

Methods

A full-length antigenome clone of HeV was assembled, a reporter gene (GFP or luciferase) inserted between the P and M genes and transfected to 293T cells to generate infectious reporter gene-encoding recombinant viruses. These viruses were then assessed in vitro for expression of the reporter genes. The GFP expressing recombinant HeV was used to challenge ferrets to assess the virulence and tissue distribution by monitoring GFP expression in infected cells.

Results

Three recombinant HeV constructs were successfully cloned and rescued; a wild-type virus, a GFP-expressing virus and a firefly luciferase-expressing virus. In vitro characterisation demonstrated expression of the reporter genes, with levels proportional to the initial inoculum levels. Challenge of ferrets with the GFP virus demonstrated maintenance of the fatal phenotype with disease progressing to death consistent with that observed previously with the parental wild-type isolate of HeV. GFP expression could be observed in infected tissues collected from animals at euthanasia.

Conclusions

Here, we report on the first successful rescue of recombinant HeV, including wild-type virus and viruses expressing two different reporter genes encoded as an additional gene cassette inserted between the P and M genes. We further demonstrate that the GFP virus retained the ability to cause fatal disease in a well-characterized ferret model of henipavirus infection despite the genome being an extra 1290 nucleotides in length.

Keywords:
Henipavirus; Hendra virus; Reverse genetics; Rescue system; Pathogenesis; Paramyxovirus; Ferret; Zoonotic disease