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.

Figure 1

Schematic representation of the genomes of each of the recombinant viruses. Full gene cassettes including the 5’UTR from the M gene, the reporter gene and the 3’ UTR of the P gene were inserted for expression of the GFP or luciferase.

Figure 2

Kinetics of recombinant virus growth in Vero cells. Vero cells were infected at an MOI of 0.01 and samples collected for titration at 8, 24, 48 and 72 hours post infection. Results are expressed as the average of 3 independent infections, with error bars representing standard deviation.

Figure 3

Expression of GFP in Vero cells infected with HeV-GFP recombinant virus. Vero cells were infected at an MOI of 0.05 and imaged using a AMG EVOS FL fluorescent inverted microscope with a monochrome camera at 24 and 48 hr post infection.

Figure 4

Expression of luciferase by the recombinant HeV-Luc. Vero, HeLa, HEp2 and U373 human cells were infected with various amounts of HeV-Luc. At 24 hours post infection, luciferase activity in cells was measured by lysing cells using BrightGlo reagent (Promega) and reading on a Synergy H4 microplate reader (Biotek Instruments Inc). Results represent the average of 4 independent infections with error bars representing standard deviation. All values have been normalised to uninfected cells (relative light units of 1).

Figure 5

Necrotising bronchoalveolitis with epithelial syncytia in lung of ferret infected with HeV-GFP virus. (a) H and E and (b) immunohistochemical staining of HeV N protein showing presence of antigen in red. Arrow indicates syncytia.

Figure 6

Relative abundance of HeV-GFP RNA in different tissues of the ferret. Various tissues were collected at post mortem examination and analysed for HeV-GFP viral load by real-time PCR. Values are expressed relative to ribosomal 18S copies.

Figure 7

Expression of GFP in tissues sectioned from HeV-GFP challenged ferrets. Ferrets were challenged with 5,000 TCID₅₀ of HeV-GFP and succumbed to disease on days 7 and 8. At necropsy, kidney (a,b,c) and lung (d,e,f) samples were collected, fixed with paraformaldehye, sectioned and imaged by confocal microscopy. To assess sensitive of GFP expression (green; a and d) to traditional staining techniques, HeV nucleocapsid protein (red; b and e) was co-stained using rabbit anti- HeV N sera detected with anti-rabbit conjugated Alexafluor 568. Composite images (c and f) show both GFP and HeV N labelling. Scale bars: c =25 μm, f = 40 μm.