Background
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 [
4]. 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.
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 [
16]. 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 [
17], that shows around 50% sequence identity with the henipaviruses, including CedPV. Henipa-like viruses have also been detected serologically in bats in Thailand [
18], China [
19], Madagascar [
20] and West Africa [
21], with successful virus isolation obtained from Lyle’s flying foxes in Cambodia [
22].
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.
Conclusions
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).
Methods
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 [
27] 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 × 10
6 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 × 10
5 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 cm
2 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 (TCID
50)/mL calculated using the Reed and Muench method [
28].
Animal studies
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 TCID
50 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 [
29]. 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.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
GAM conceived the study, carried out the molecular work, in vitro characterisation, processed animal tissues, analysed samples and drafted the manuscript. ERV participated in the molecular work. IS provided clones and participated in the molecular work. ST participated in the molecular work. RA carried out the animal infection trials. LF assisted with the animal work. PM performed the confocal imaging work. GAS provided clones and participated in the molecular work. CCB conceived the study, provided funds and edited the manuscript. DM designed the animal study, provided veterinary pathological assistance and drafted the manuscript. LFW conceived the study and drafted the manuscript. All authors read and approved the final manuscript.