Abstract
BackgroundHendra 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.MethodsA 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.ResultsThree 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.ConclusionsHere, 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.
Highlights
Hendra virus (HeV) is an Australian bat-borne zoonotic paramyxovirus that repeatedly spills-over to horses causing fatal disease
Rescue systems have previously been reported for Nipah virus (NiV) [23,24,25], here we report the generation of recombinant HeV in which the green fluorescent protein (GFP) (HeV-GFP) or firefly luciferase gene (HeV-Luc) has been inserted as an additional transcriptional unit between the P and M genes, and we assess their biological characteristics both in vitro and in vivo
Reporter genes were inserted between the P and M genes, including the untranslated regions from the 3’ end of the P gene and the 5’ end of the M gene (Figure 1)
Summary
Hendra virus (HeV) is an Australian bat-borne zoonotic paramyxovirus that repeatedly spills-over to horses causing fatal disease. HeV first emerged in 1994 in Hendra, a suburb of Brisbane, Australia, leading to an outbreak of acute respiratory disease in 21 thoroughbred horses, of which 14 died [1,2,3]. During this outbreak, two horse trainers became infected with the virus, one fatally. Since its emergence in Queensland, Australia in 1994, HeV spillover from flying foxes to horses has regularly recurred: with an increase in disease events occurring over the past two years [5]. Thirty-nine disease events have occurred resulting in the death or euthanasia of seventy-six horses and one dog, with a case fatality of 75% in the horses
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