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Hendra virus and Nipah virus are bat-borne paramyxoviruses that are the prototypic members of the genus Henipavirus. The henipaviruses emerged in the 1990s, spilling over from their natural bat hosts and causing serious disease outbreaks in humans and livestock. Hendra virus emerged in Australia and since 1994 there have been 7 human infections with 4 case fatalities. Nipah virus first appeared in Malaysia and subsequent outbreaks have occurred in Bangladesh and India. In total, there have been an estimated 582 human cases of Nipah virus and of these, 54% were fatal. Their broad species tropism and ability to cause fatal respiratory and/or neurologic disease in humans and animals make them important transboundary biological threats. Recent experimental findings in animals have demonstrated that a human monoclonal antibody targeting the viral G glycoprotein is an effective post-exposure treatment against Hendra and Nipah virus infection. In addition, a subunit vaccine based on the G glycoprotein of Hendra virus affords protection against Hendra and Nipah virus challenge. The vaccine has been developed for use in horses in Australia and is the first vaccine against a Biosafety Level-4 (BSL-4) agent to be licensed and commercially deployed. Together, these advances offer viable approaches to address Hendra and Nipah virus infection of livestock and people.

Nipah virus (NiV) and Hendra virus (HeV) are closely related deadly zoonotic paramyxoviruses that have emerged and re-emerged over the last 10 years. In this study, a subunit vaccine formulation containing only recombinant, soluble, attachment glycoprotein from HeV (sG(HeV)) and CpG adjuvant was evaluated as a potential NiV vaccine in the cat model. Different amounts of sG(HeV) were employed and sG-induced immunity was examined. Vaccinated animals demonstrated varying levels of NiV-specific Ig systemically and importantly, all vaccinated cats possessed antigen-specific IgA on the mucosa. Upon oronasal challenge with NiV (50,000TCID50), all vaccinated animals were protected from disease although virus was detected on day 21 post-challenge in one animal. The ability to elicit protective systemic and mucosal immunity in this animal model provides significant progress towards the development of a human subunit vaccine against henipaviruses.

We have previously identified neutralizing human monoclonal antibodies against Nipah virus (NiV) and Hendra virus (HeV) by panning a large nonimmune antibody library against a soluble form of the HeV attachment-envelope glycoprotein G (sGHeV). One of these antibodies, m102, which exhibited the highest level of cross-reactive neutralization of both NiV and HeV G, was affinity maturated by light-chain shuffling combined with random mutagenesis of its heavy-chain variable domain and panning against sGHeV. One of the selected antibody Fab clones, m102.4, had affinity of binding to sGHeV that was equal to or higher than that of the other Fabs; it was converted to IgG1 and tested against infectious NiV and HeV. It exhibited exceptionally potent and cross-reactive inhibitory activity with 50% inhibitory concentrations below 0.04 and 0.6 μg/mL, respectively. The virus-neutralizing activity correlated with the binding affinity of the antibody to sGHeV and sGNiV. m102.4 bound a soluble form of NiV G (sGNiV) better than it bound sGHeV, and it neutralized NiV better than HeV, despite being originally selected against sGHeV. These results suggest that m102.4 has potential as a therapeutic agent for the treatment of diseases caused by henipaviruses. It could be also used for prophylaxis and diagnosis, and as a research reagent

Hendra virus (HeV) and Nipah virus (NiV) are closely related paramyxoviruses that infect and cause disease in a wide range of mammalian hosts. To determine whether host receptor molecules play a role in species-specific and/or virus-specific infection we have cloned and characterized ephrin-B2 and ephrin-B3 ligands from a range of species, including human, horse, pig, cat, dog, bats (Pteropus alecto and Pteropus vampyrus) and mouse. HeV and NiV were both able to infect cells expressing any of the ephrin-B2 and ephrin-B3 molecules. There did not appear to be significant differences in receptor function from different species or receptor usage by HeV and NiV. Soluble ephrin ligands, their receptors and G-specific human monoclonal antibodies differentially blocked henipavirus infections suggesting different receptor affinities, overlapping receptor binding domains of the henipavirus attachment glycoprotein (G) and that the functional domains of the ephrin ligands may be important for henipavirus binding.

Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae , which are distinguished by their ability to cause fatal disease in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) glycoproteins. Previously, we reported on HeV- and NiV-mediated fusion activities and detailed their host-cell tropism characteristics. These studies also suggested that a common cell surface receptor, which could be destroyed by protease, was utilized by both viruses. To further characterize the G glycoprotein and its unknown receptor, soluble forms of HeV G (sG) were constructed by replacing its cytoplasmic tail and transmembrane domains with an immunoglobulin κ leader sequence coupled to either an S-peptide tag (sG S-tag ) or myc-epitope tag (sG myc-tag ) to facilitate purification and detection. Expression of sG was verified in cell lysates and culture supernatants by specific affinity precipitation. Analysis of sG by size exclusion chromatography and sucrose gradient centrifugation demonstrated tetrameric, dimeric, and monomeric species, with the majority of the sG released as a disulfide-linked dimer. Immunofluorescence staining revealed that sG specifically bound to HeV and NiV infection-permissive cells but not to a nonpermissive HeLa cell line clone, suggesting that it binds to virus receptor on host cells. Preincubation of host cells with sG resulted in dose-dependent inhibition of both HeV and NiV cell fusion as well as infection by live virus. Taken together, these data indicate that sG retains important native structural features, and we further demonstrate that administration of sG to rabbits can elicit a potent cross-reactive neutralizing antibody response against infectious HeV and NiV. This HeV sG glycoprotein will be exceedingly useful for structural studies, receptor identification strategies, and vaccine development goals for these important emerging viral agents.

Nipah virus (NiV) and Hendra virus (HeV) are novel paramyxoviruses from pigs and horses, respectively, that are responsible for fatal zoonotic infections of humans. The unique genetic and biological characteristics of these emerging agents has led to their classification as the prototypic members of a new genus within the Paramyxovirinae subfamily called Henipavirus . These viruses are most closely related to members of the genus Morbillivirus and infect cells through a pH-independent membrane fusion event mediated by the actions of their attachment (G) and fusion (F) glycoproteins. Understanding their cell biological features and exploring the functional characteristics of the NiV and HeV glycoproteins will help define important properties of these emerging viruses and may provide new insights into paramyxovirus membrane fusion mechanisms. Using a recombinant vaccinia virus system and a quantitative assay for fusion, we demonstrate NiV glycoprotein function and the same pattern of cellular tropism recently reported for HeV-mediated fusion, suggesting that NiV likely uses the same cellular receptor for infection. Fusion specificity was verified by inhibition with a specific antiserum or peptides derived from the α-helical heptads of NiV or HeV F. Like that of HeV, NiV-mediated fusion also requires both F and G. Finally, interactions between the glycoproteins of the paramyxoviruses have not been well defined, but here we show that the NiV and HeV glycoproteins are capable of highly efficient heterotypic functional activity with each other. However, no heterotypic activity was observed with envelope glycoproteins of the morbilliviruses Measles virus and Canine distemper virus .

Hendra virus (HeV) is a zoonotic emerging virus belonging to the family Paramyxoviridae . HeV causes severe and often fatal respiratory and/or neurologic disease in both animals and humans. Currently, there are no licensed vaccines or antiviral drugs approved for human use. A number of animal models have been developed for studying HeV infection, with the African green monkey (AGM) appearing to most faithfully reproduce the human disease. Here, we assessed the utility of a newly developed recombinant subunit vaccine based on the HeV attachment (G) glycoprotein in the AGM model. Four AGMs were vaccinated with two doses of the HeV vaccine (sG HeV ) containing Alhydrogel, four AGMs received the sG HeV with Alhydrogel and CpG, and four control animals did not receive the sG HeV vaccine. Animals were challenged with a high dose of infectious HeV 21 days after the boost vaccination. None of the eight specifically vaccinated animals showed any evidence of clinical illness and survived the challenge. All four controls became severely ill with symptoms consistent with HeV infection, and three of the four animals succumbed 8 days after exposure. Success of the recombinant subunit vaccine in AGMs provides pivotal data in supporting its further preclinical development for potential human use. IMPORTANCE A Hendra virus attachment (G) glycoprotein subunit vaccine was tested in nonhuman primates to assess its ability to protect them from a lethal infection with Hendra virus. It was found that all vaccinated African green monkeys were completely protected against subsequent Hendra virus infection and disease. The success of this new subunit vaccine in nonhuman primates provides critical data in support of its further development for future human use.

Nipah virus (NiV) (Genus Henipavirus) is a recently emerged zoonotic virus that causes severe disease in humans and has been found in bats of the genus Pteropus. Whilst NiV has not been detected in Australia, evidence for NiV-infection has been found in pteropid bats in some of Australia's closest neighbours. The aim of this study was to determine the occurrence of henipaviruses in fruit bat (Family Pteropodidae) populations to the north of Australia. In particular we tested the hypothesis that Nipah virus is restricted to west of Wallace's Line. Fruit bats from Australia, Papua New Guinea, East Timor and Indonesia were tested for the presence of antibodies to Hendra virus (HeV) and Nipah virus, and tested for the presence of HeV, NiV or henipavirus RNA by PCR. Evidence was found for the presence of Nipah virus in both Pteropus vampyrus and Rousettus amplexicaudatus populations from East Timor. Serology and PCR also suggested the presence of a henipavirus that was neither HeV nor NiV in Pteropus alecto and Acerodon celebensis. The results demonstrate the presence of NiV in the fruit bat populations on the eastern side of Wallace's Line and within 500 km of Australia. They indicate the presence of non-NiV, non-HeV henipaviruses in fruit bat populations of Sulawesi and Sumba and possibly in Papua New Guinea. It appears that NiV is present where P. vampyrus occurs, such as in the fruit bat populations of Timor, but where this bat species is absent other henipaviruses may be present, as on Sulawesi and Sumba. Evidence was obtained for the presence henipaviruses in the non-Pteropid species R. amplexicaudatus and in A. celebensis. The findings of this work fill some gaps in knowledge in geographical and species distribution of henipaviruses in Australasia which will contribute to planning of risk management and surveillance activities.

In the 1990s, Hendra virus and Nipah virus (NiV), two closely related and previously unrecognized paramyxoviruses that cause severe disease and death in humans and a variety of animals, were discovered in Australia and Malaysia, respectively. Outbreaks of disease have occurred nearly every year since NiV was first discovered, with case fatality ranging from 10 to 100%. In the African green monkey (AGM), NiV causes a severe lethal respiratory and/or neurological disease that essentially mirrors fatal human disease. Thus, the AGM represents a reliable disease model for vaccine and therapeutic efficacy testing. We show that vaccination of AGMs with a recombinant subunit vaccine based on the henipavirus attachment G glycoprotein affords complete protection against subsequent NiV infection with no evidence of clinical disease, virus replication, or pathology observed in any challenged subjects. Success of the recombinant subunit vaccine in nonhuman primates provides crucial data in supporting its further preclinical development for potential human use.

Hendra virus and Nipah virus are recently discovered and closely related emerging viruses that now comprise the genus henipavirus within the sub-family Paramyxoviridae and are distinguished by their broad species tropism and in addition to bats can infect and cause fatal disease in a wide variety of mammalian hosts including humans. The high mortality associated with human and animal henipavirus infections has highlighted the importance and necessity of developing effective immunization strategies. The development of suitable animal models of henipavirus infection and pathogenesis has been critical for testing the efficacy of potential therapeutic approaches. Several henipavirus challenge models have been used and recent successes in both active and passive immunization strategies against henipaviruses have been reported which have all targeted the viral envelope glycoproteins.

Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.

Hendra virus (HeV) is a recently emerged zoonotic paramyxovirus that can cause a severe and often fatal disease in horses and humans. HeV is categorized as a biosafety level 4 agent, which has made the development of animal models and testing of potential therapeutics and vaccines challenging. Infection of African green monkeys (AGMs) with HeV was recently demonstrated, and disease mirrored fatal HeV infection in humans, manifesting as a multisystemic vasculitis with widespread virus replication in vascular tissues and severe pathologic manifestations in the lung, spleen, and brain. Here, we demonstrate that m102.4, a potent HeV-neutralizing human monoclonal antibody (hmAb), can protect AGMs from disease after infection with HeV. Fourteen AGMs were challenged intratracheally with a lethal dose of HeV, and 12 subjects were infused twice with a 100-mg dose of m102.4 beginning at either 10, 24, or 72 hours after infection and again about 48 hours later. The presence of viral RNA, infectious virus, and HeV-specific immune responses demonstrated that all subjects were infected after challenge. All 12 AGMs that received m102.4 survived infection, whereas the untreated control subjects succumbed to disease on day 8 after infection. Animals in the 72-hour treatment group exhibited neurological signs of disease, but all animals started to recover by day 16 after infection. These results represent successful post-exposure in vivo efficacy by an investigational drug against HeV and highlight the potential impact a hmAb can have on human disease.

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are emerging zoonotic paramyxoviruses that can cause severe and often lethal neurologic and/or respiratory disease in a wide variety of mammalian hosts, including humans. There are presently no licensed vaccines or treatment options approved for human or veterinarian use. Guinea pigs, hamsters, cats, and ferrets, have been evaluated as animal models of human HeV infection, but studies in nonhuman primates (NHP) have not been reported, and the development and approval of any vaccine or antiviral for human use will likely require efficacy studies in an NHP model. Here, we examined the pathogenesis of HeV in the African green monkey (AGM) following intratracheal inoculation. Exposure of AGMs to HeV produced a uniformly lethal infection, and the observed clinical signs and pathology were highly consistent with HeV-mediated disease seen in humans. Ribavirin has been used to treat patients infected with either HeV or NiV; however, its utility in improving outcome remains, at best, uncertain. We examined the antiviral effect of ribavirin in a cohort of nine AGMs before or after exposure to HeV. Ribavirin treatment delayed disease onset by 1 to 2 days, with no significant benefit for disease progression and outcome. Together our findings introduce a new disease model of acute HeV infection suitable for testing antiviral strategies and also demonstrate that, while ribavirin may have some antiviral activity against the henipaviruses, its use as an effective standalone therapy for HeV infection is questionable.

Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID50 within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody., Author Summary Nipah virus and Hendra virus are closely related and highly pathogenic zoonoses whose primary natural reservoirs are several species of Pteropus fruit bats. Both Nipah and Hendra viruses can cause severe and often fatal disease in a variety of mammalian hosts, including humans. The henipaviruses are categorized as biosafety level 4 (BSL-4) agents, which has limited the development of animal models and the testing of potential therapeutics and vaccine countermeasures. We show here a new ferret model of Nipah virus pathogenesis in which the underlying pathology closely mirrors the illness seen in Nipah virus-infected humans, including both respiratory and neurological disease. We also show that m102.4, a cross-reactive neutralizing human monoclonal antibody that targets the viral attachment glycoprotein, completely protected ferrets from disease when given ten hours after a lethal Nipah virus challenge. This study is the first successful and viable post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.

Hendra virus (HeV) is a member of the broadly tropic and highly pathogenic paramyxovirus genus Henipavirus . HeV is enveloped and infects cells by using membrane-anchored attachment (G) and fusion (F) glycoproteins. G possesses an N-terminal cytoplasmic tail, an external membrane-proximal stalk domain, and a C-terminal globular head that binds the recently identified receptors ephrinB2 and ephrinB3. Receptor binding is presumed to induce conformational changes in G that subsequently trigger F-mediated fusion. The stalk domains of other attachment glycoproteins appear important for oligomerization and F interaction and specificity. However, this region of G has not been functionally characterized. Here we performed a mutagenesis analysis of the HeV G stalk, targeting a series of isoleucine residues within a hydrophobic α-helical domain that is well conserved across several attachment glycoproteins. Nine of 12 individual HeV G alanine substitution mutants possessed a complete defect in fusion-promotion activity yet were cell surface expressed and recognized by a panel of conformation-dependent monoclonal antibodies (MAbs) and maintained their oligomeric structure. Interestingly, these G mutations also resulted in the appearance of an additional electrophoretic species corresponding to a slightly altered glycosylated form. Analysis revealed that these G mutants appeared to adopt a receptor-bound conformation in the absence of receptor, as measured with a panel of MAbs that preferentially recognize G in a receptor-bound state. Further, this phenotype also correlated with an inability to associate with F and in triggering fusion even after receptor engagement. Together, these data suggest the stalk domain of G plays an important role in the conformational stability and receptor binding-triggered changes leading to productive fusion, such as the dissociation of G and F.

Hendra virus (HeV) is an emerging paramyxovirus capable of infecting and causing disease in a variety of mammalian species, including humans. The virus infects its host cells through the coordinated functions of its fusion (F) and attachment (G) glycoproteins, the latter of which is responsible for binding the virus receptors ephrinB2 and ephrinB3. In order to identify the receptor binding site, a panel of G glycoprotein constructs containing mutations was generated using an alanine-scanning mutagenesis strategy. Based on a predicted G structure, charged amino acids residing in regions that could be homologous to those in the measles virus H attachment glycoprotein known to be involved in its protein receptor interaction were targeted. Using a coprecipitation-based assay, seven single-amino-acid substitutions in HeV G were identified as having significantly impaired binding to both the ephrinB2 and ephrinB3 viral receptors: D257A, D260A, G439A, K443A, G449A, K465A, and D468A. The impairment of receptor interaction conferred a concomitant diminution in their abilities to promote membrane fusion when coexpressed with F. The G glycoprotein mutants were also recognized by three or more conformation-dependent monoclonal antibodies of a panel of five, were expressed on the cell surface, and retained their abilities to bind and coprecipitate F. Interestingly, some of these mutant G glycoproteins coprecipitated with F more efficiently than wild-type G. Taken together, these data provide strong biochemical and functional evidence that some of these residues could be part of a conformation-dependent, discontinuous, and overlapping ephrinB2 and -B3 binding domain within the HeV G glycoprotein.

Hendra virus (HeV) and Nipah virus (NiV) are related emerging paramyxoviruses classified in the genus Henipavirus. Both cause fatal disease in animals and humans and are classified as biosafety level 4 pathogens. Here we detail two new multiplexed microsphere assays, one for antibody detection and differentiation and another designed as a surrogate for virus neutralization. Both assays utilize recombinant soluble attachment glycoproteins (sG) whereas the latter incorporates the cellular receptor, recombinant ephrin-B2. Spectrally distinct sG(HeV)- and sG(NiV)-coupled microspheres preferentially bound antibodies from HeV- and NiV-seropositive animals, demonstrating a simple procedure to differentiate antibodies to these closely related viruses. Soluble ephrin-B2 bound sG-coupled microspheres in a dose-dependent fashion. Specificity of binding was further evaluated with henipavirus G-specific sera and MAbs. Sera from henipavirus-seropositive animals differentially blocked ephrin-B2 binding, suggesting that detection and differentiation of HeV and NiV neutralizing antibodies can be done simultaneously in the absence of live virus.

Background The recent emergence of four new members of the paramyxovirus family has heightened the awareness of and re-energized research on new and emerging diseases. In particular, the high mortality and person to person transmission associated with the most recent Nipah virus outbreaks, as well as the very recent re-emergence of Hendra virus, has confirmed the importance of developing effective therapeutic interventions. We have previously shown that peptides corresponding to the C-terminal heptad repeat (HR-2) of the fusion envelope glycoprotein of Hendra virus and Nipah virus were potent inhibitors of both Hendra virus and Nipah virus-mediated membrane fusion using recombinant expression systems. In the current study, we have developed shorter, second generation HR-2 peptides which include a capped peptide via amidation and acetylation and two poly(ethylene glycol)-linked (PEGylated) peptides, one with the PEG moity at the C-terminus and the other at the N-terminus. Here, we have evaluated these peptides as well as the corresponding scrambled peptide controls in Nipah virus and Hendra virus-mediated membrane fusion and against infection by live virus in vitro. Results Unlike their predecessors, the second generation HR-2 peptides exhibited high solubility and improved synthesis yields. Importantly, both Nipah virus and Hendra virus-mediated fusion as well as live virus infection were potently inhibited by both capped and PEGylated peptides with IC50 concentrations similar to the original HR-2 peptides, whereas the scrambled modified peptides had no inhibitory effect. These data also indicate that these chemical modifications did not alter the functional properties of the peptides as inhibitors. Conclusion Nipah virus and Hendra virus infection in vitro can be potently blocked by specific HR-2 peptides. The improved synthesis and solubility characteristics of the second generation HR-2 peptides will facilitate peptide synthesis for pre-clinical trial application in an animal model of Henipavirus infection. The applied chemical modifications are also predicted to increase the serum half-life in vivo and should increase the chance of success in the development of an effective antiviral therapy.

Hendra virus (HeV) and Nipah virus (NiV) belong to the genus Henipavirus of the family Paramyxoviridae and are unique in that they exhibit a broad species tropism and cause fatal disease in both animals and humans. They infect cells through a pH-independent membrane fusion process mediated by their fusion and attachment glycoproteins. Previously, we demonstrated identical cell fusion tropisms for HeV and NiV and the protease-sensitive nature of their unknown cell receptor and identified a human cell line (HeLa-USU) that was nonpermissive for fusion and virus infection. Here, a microarray analysis was performed on the HeLa-USU cells, permissive HeLa-CCL2 cells, and two other permissive human cell lines. From this analysis, we identified a list of genes encoding known and predicted plasma membrane surface-expressed proteins that were highly expressed in all permissive cells and absent from the HeLa-USU cells and rank-ordered them based on their relative levels. Available expression vectors containing the first 10 genes were obtained and individually transfected into HeLa-USU cells. One clone, encoding human ephrin-B2 (EFNB2), was found capable of rendering HeLa-USU cells permissive for HeV- and NiV-mediated cell fusion as well as infection by live virus. A soluble recombinant EFNB2 could potently block fusion and infection and bind soluble recombinant HeV and NiV attachment glycoproteins with high affinity. Together, these data indicate that EFNB2 serves as a functional receptor for both HeV and NiV. The highly conserved nature of EFNB2 in humans and animals is consistent with the broad tropism exhibited by these emerging zoonotic viruses.

Hendra virus (HeV) is an emerging paramyxovirus first isolated from cases of severe respiratory disease that fatally affected both horses and humans. Understanding the mechanisms of host cell infection and cross-species transmission is an important step in addressing the risk posed by such emerging pathogens. We have initiated studies to characterize the biological properties of the HeV envelope glycoproteins. Recombinant vaccinia viruses encoding the HeV F and G open reading frames were generated and glycoprotein expression was verified by metabolic labeling and detection using specific antisera. Glycoprotein function and cellular tropism were examined with a quantitative assay for HeV-mediated membrane fusion. Fusion specificity was verified through specific inhibition by anti-HeV antiserum and a peptide corresponding to one of the α-helical heptad repeats of F. HeV requires both F and G to mediate fusion. Permissive target cells have been identified, including cell lines derived from cat, bat, horse, human, monkey, mouse, and rabbit. Fusion negative cell types have also been identified. Protease treatments of the target cells abolished fusion activity, suggesting that the virus is employing a cell-surface protein as its receptor.