title: Recombinant Hendra viruses expressing a reporter gene retain pathogenicity in ferrets.
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.
authors.affiliation: CSIRO Animal, Food and Health Sciences, Australian Animal Health Laboratory, Geelong, VIC 3220, Australia. glenn.marsh@csiro.au
authors.forename: Glenn A
authors.initials: GA
authors.lastname: Marsh
authors.forename: Elena R
authors.initials: ER
authors.lastname: Virtue
authors.forename: Ina
authors.initials: I
authors.lastname: Smith
authors.forename: Shawn
authors.initials: S
authors.lastname: Todd
authors.forename: Rachel
authors.initials: R
authors.lastname: Arkinstall
authors.forename: Leah
authors.initials: L
authors.lastname: Frazer
authors.forename: Paul
authors.initials: P
authors.lastname: Monaghan
authors.forename: Greg A
authors.initials: GA
authors.lastname: Smith
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Broder
authors.forename: Deborah
authors.initials: D
authors.lastname: Middleton
authors.forename: Lin-Fa
authors.initials: LF
authors.lastname: Wang
mesh_terms: D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D005289:Ferrets; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D045404:Hendra Virus / Q000235:genetics* / Q000472:pathogenicity*; D045464:Henipavirus Infections / Q000821:virology*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008297:Male; D013194:Staining and Labeling / Q000379:methods; D016019:Survival Analysis; D014774:Virulence
chemical_list: D049452:Green Fluorescent Proteins; D008156:Luciferases