Recombinant Hendra viruses expressing a reporter gene retain pathogenicity in ferrets
© Marsh et al.; licensee BioMed Central Ltd. 2013
Received: 3 December 2012
Accepted: 21 March 2013
Published: 25 March 2013
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.
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.
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.
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.
Hendra virus (HeV) is a zoonotic paramyxovirus harboured by Australian mainland flying foxes from which it is believed to be transmitted directly to horses. 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 [1–3]. During this outbreak, two horse trainers also became infected with the virus, one fatally. In horses, HeV causes a severe, often fatal, febrile illness associated with respiratory and neurological signs . 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 . 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.
HeV is the prototype species of the genus Henipavirus, within the subfamily Paramyxovirinae[6, 7]. Nipah virus (NiV) is the only other virus officially classified within the Henipavirus genus. NiV was first identified during a major outbreak of acute respiratory disease in pigs in peninsular Malaysia in 1998–99. Over one million pigs were culled in Malaysia to prevent the continued spread of the virus. Over 265 farm and abattoir workers exposed to infected pigs were infected by NiV, resulting in a total of 105 deaths in Malaysia and Singapore [8–10]. Since the outbreak of disease in Malaysia and Singapore, NiV re-emerged in Bangladesh in 2001, with continued re-emergence and human cases almost annually since in Bangladesh and sporadically in India [11, 12]. Differences in transmission have also been observed between the Bangladesh and Malaysian strains of NiV. NiV Bangladesh has been shown to cause direct bat-to-human transmission without the involvement of an intermediary or amplifying host. Human-to-human spread of NiV in Bangladesh has also been documented [12–15]. Because of the broad host range and the high mortality rates associated with these viruses, both HeV and NiV have been classified as a biosafety level 4 (BSL-4) agents.
Recently, we described a novel paramyxovirus, Cedar virus (CedPV), which displayed many of the distinguishing characteristics of the henipaviruses, including similar genome length and organisation, it displayed antigenic cross-reactivity with henipaviruses and used the same host cell molecule (ephrin B2) as a receptor for entry during infection . Interestingly in preliminary animal challenge studies, CedPV did not cause disease in ferrets and guinea pigs, both of which are susceptible to fatal disease by the henipaviruses. In addition, a near full length genome sequence has been described for a bat-borne virus from Ghana, Africa , that shows around 50% sequence identity with the henipaviruses, including CedPV. Henipa-like viruses have also been detected serologically in bats in Thailand , China , Madagascar  and West Africa , with successful virus isolation obtained from Lyle’s flying foxes in Cambodia .
Reverse genetics of negative strand RNA viruses allow for the creation of recombinant infectious and replication-competent viruses with specific mutations or insertions. Often, researchers have inserted the green fluorescent protein (GFP) gene into such viruses allowing for the real-time monitoring of virus replication and spread, either within cell culture or in vivo within an infected host. The expression of GFP allows for the detection of virus infection in tissues without the need for antibody-specific detection methods. The generation of recombinant henipaviruses will be an extremely powerful tool to monitor viral infections both in real-time for imaging and in a high-throughput approach for screening activities. They will also play a pivotal role in our understanding of pathogenesis of henipaviruses at the molecular level through the generation, rescue and testing of specific mutation variants. Rescue systems have previously been reported for NiV [23–25], here we report the generation of recombinant HeV in which the 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.
Results and discussion
Rescue of recombinant viruses
Rescue of recombinant HeV for wild-type, HeV-GFP and HeV-Luc viruses were achieved by co-transfection of human 293T cell in 12.5 cm2 flasks with the full length antigenome plasmid, N, P and L protein expression vector and pCAGGS T7 RNA polymerase. Two days after transfection, Vero cells (5 × 105 cells) were added to the 293T cell monolayers. In successful experiments, CPE was obvious within 3 days following the addition of the Vero cells, with CPE reaching maximal levels around 7 days post transfection. Supernatants were collected from the transfected cell monolayers and clarified by centrifugation and used to infect Vero cell monolayers to prepare virus stocks for further study.
In vitro characterisation
In vivo characterisation
Here we report the first successful rescue of replication-competent recombinant HeV, including both wild-type HeV as well as recombinant viruses encoding either the GFP or luciferase genes by genetic insertion between the P and M genes. The rescue of the HeV-Luc virus resulted in a paramyxovirus with the largest functional reported genome to date. Challenge of ferrets with HeV-GFP demonstrated that the insertion of GFP into the virus genome did not moderate its virulence, with GFP expression being observed in tissues collected from ferrets at euthanasia. The HeV-Luc virus is currently being utilized as a powerful tool for studying host genes important for the replication of HeV (unpublished data).
Cells and virus
293T cells (ATCC CRL-11268) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (Invitrogen), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, 4 mM L-glutamine. African green monkey (Vero – ATCC CCL-81) cells were maintained in EMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. HeV (Hendra virus/horse/1994/Hendra isolate, GenBank Accession AF017149) was isolated in Vero cells from the lung of a horse infected in the Brisbane outbreak in 1994 and was passaged and triple plaque purified in Vero cells. All work with live HeV was carried out within the BSL4 facilities at the Australian Animal Health Laboratory, Geelong Australia.
Generation of plasmids and rescue of recombinant HeVs
Vero cells were infected with HeV and when maximal CPE was observed cell supernatants were collected and virus concentrated by ultracentrifugation (280,000 g for 45 minutes). Pellet was then resuspended in buffer RLT (Qiagen) and removed from the BSL4 laboratory. RNA was extracted using a Qiagen RNEasy kit and reverse transcribed to cDNA. Using a panel of PCR primers, large (3–5 kb) fragments were generated by PCR and cloned into pOLTV5  between a T7 RNA promoter and a hepatitis delta virus ribozyme. Full open reading frames for HeV N, P and L genes, the essential components needed for viral RNA transcription and replication, were cloned into pTM-I, a T7 driven mammalian expression plasmid. In addition, the T7 RNA polymerase gene was PCR amplified from BSR/T7 cells and cloned into pCAGGS. All plasmid inserts were sequenced to obtain at least 2-fold sequence coverage to ensure no mutations relative to the reference sequences.
To generate recombinant viruses, 1 × 106 293T cells were transfected with pTM-I HeV N (1.25 μg), pTM-I HeV P (0.8 μg), pTM-I HeV L (0.4 μg), full-length HeV genome plasmid (3.5 μg) and pCAGGS T7 (2 μg) using 7 μL Lipofectamine 2000 (Invitrogen). Media was changed on cells at 6 hours post transfection and then 5 × 105 Vero cells were added to transfected 293T cells after 2 days. Cells were observed for 7 days for the generation of syncytia, at which time, supernatant was passaged to fresh Vero cells (75 cm2 flasks) to amplify recombinant virus. When maximal CPE was obtained, supernatants were clarified by low speed centrifugation (1000 g), aliquoted, stored at −80°C and the 50% tissue culture infective dose (TCID50)/mL calculated using the Reed and Muench method .
Procedures involving live animals were approved by the Commonwealth Scientific and Industrial Research Organisation, Australian Animal Health Laboratory and Animal Ethics Committee. Four male ferrets, aged 12–18 months, were exposed oronasally to 5000 TCID50 of HeV-GFP. This challenge dose was selected as the wild-type isolate has been shown to be lethal in ferrets at this dose (unpublished observations). Ferrets were monitored daily for indications of disease including fever and alertness. Euthanasia was performed at predetermined humane endpoints as previously described . Shedding samples, blood and urine were collected on days 3 and 6 post-exposure as well as immediately prior to euthanasia and various tissues collected at post mortem examination.
Imaging of tissues
Samples of brain, lung, spleen and kidney tissues from HeV-GFP infected ferrets were dissected into approximately 1 cm cubes and fixed for 48 hr in 4% paraformaldehyde in phosphate buffered saline (PBS). They were stored at 4°C in PBS. Sections were cut at 70 μm on a Leica VT1000 vibrating microtome (Leica Microsystems). Sections were blocked in PBS containing 0.5% bovine serum albumin (PBS/BSA) overnight and then incubated in rabbit anti-HeV N protein diluted 1:1000 in PBS/BSA. Following 3 × 10 min washes in PBS, the bound antibody was detected with goat anti-rabbit IgG conjugated to Alexa 568 (Life Technologies) diluted 1:200 in PBS/BSA, followed by 3 × 10 min washes in PBS. Nuclei were labelled by incubation of sections in a 1:1000 dilution of DAPI (Sigma, Sydney) for 30 min and following 2 × H2O washes, the sections were mounted in Vectashield (Vector laboratories). Coverslips were sealed with nail varnish and the sections imaged with a Leica SP5 confocal microscope.
We thank Leanne Dickson and Dianne Green for providing technical assistance and Jackie Pallister and Gary Crameri for critical review of the manuscript. This work was supported in part by a CSIRO Office of the Chief Executive Postdoctoral fellowship (GAM), a CEO Science Leader Award (LFW) from the CSIRO Office of the Chief Executive and NIH grant AI054715 (CCB). The authors acknowledge the support of the Australian Microscopy and Microanalysis Facility (AMMRF) for the AAHL Biosecurity Microscopy Laboratory.
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