Recuperative potential of Indian medicinal plant compounds- a tool to encumber henipaviruses: an in -silico study.

Henipaviruses, highly fatal zoonotic viruses with mortality rates up to 100%, pose a significant threat to humans. Despite sporadic cases, including infections from Cedar, Langya, and Nipah Viruses, there are no established drugs or vaccines for treatment. This lack of specific medication led us to explore 57 non-toxic compounds from Indian Medicinal Plants, selected from 232 compounds, aiming to combat these viruses. Through in silico ADMET analyses, Three compounds-andrographolide, pterygospermin and Salidroside-stood out for their exceptional non-toxic properties. These compounds underwent in silico target prediction, molecular docking and dynamics with Cedar, Langya, and Nipah Virus proteins from the Protein Data Bank. Among them, Andrographolide displayed the most promising negative free energy scores and stability in Cedar Virus-Attachment G-Protein binding pockets. Pterygospermin and Salidroside showed efficacy against Langya and Nipah Virus target proteins throughout the simulation. These compounds not only exhibited antiviral properties but also demonstrated immunomodulatory, anti-inflammatory, and hepatoprotective effects by our in-silico studies. Their potential as treatments or preventive measures against henipaviral infections makes them promising candidates for further research and development. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00236-x.

Foals of mares vaccinated for Hendra virus have a suboptimal response to HeV vaccination.

Hendra virus (HeV) is lethal to horses and a zoonotic threat to humans in Australia, causing severe neurological and/or respiratory disease with high mortality. An equine vaccine has been available since 2012. Foals acquire antibodies from their dams by ingesting colostrum after parturition, therefore it is assumed that foals of mares vaccinated against HeV will have passive HeV antibodies circulating during the first several months of life until they are actively vaccinated. However, no studies have yet examined passive or active immunity against HeV in foals. Here, we investigated anti-HeV antibody levels in vaccinated mares and their foals. Testing for HeV neutralising antibodies is cumbersome due to the requirement for Biosafety level 4 (BSL-4) containment to conduct virus neutralisation tests (VNT). For this study, a subset of samples was tested for HeV G-specific antibodies by both an authentic VNT with infectious HeV and a microsphere-based immunoassay (MIA), revealing a strong correlation. An indicative neutralising level was then applied to the results of a larger sample set tested using the MIA. Mares had high levels of HeV-specific neutralising antibodies at the time of parturition. Foals acquired high levels of maternal antibodies which then waned to below predictive protective levels in most foals by 6 months old when vaccination commenced. Foals showed a suboptimal response to vaccination, suggesting maternal antibodies may interfere with active vaccination. The correlation analysis between the authentic HeV VNT and HeV MIA will enable further high throughput serological studies to inform optimal vaccination protocols for both broodmares and foals.

A Comparative Assessment of the Pathogenic Potential of Newly Discovered Henipaviruses.

Recent advances in high-throughput sequencing technologies have led to the discovery of a plethora of previously unknown viruses in animal samples. Some of these newly detected viruses are closely related to human pathogens. A prime example are the henipaviruses. Both Nipah (NiV) and Hendra virus (HeV) cause severe disease in humans. Henipaviruses are of zoonotic origin, and animal hosts, including intermediate hosts, play a critical role in viral transmission to humans. The natural reservoir hosts of NiV and HeV seem to be restricted to a few fruit bat species of the Pteropus genus in distinct geographic areas. However, the recent discovery of novel henipa- and henipa-like viruses suggests that these viruses are far more widespread than was originally thought. To date, these new viruses have been found in a wide range of animal hosts, including bats, shrews, and rodents in Asia, Africa, Europe, and South America. Since these viruses are closely related to human pathogens, it is important to learn whether they pose a threat to human health. In this article, we summarize what is known about the newly discovered henipaviruses, highlight differences to NiV and HeV, and discuss their pathogenic potential.

An in vivo BSL-2 model for henipavirus infection based on bioluminescence imaging of recombinant Cedar virus replication in mice.

Henipaviruses are enveloped single-stranded, negative-sense RNA viruses of the paramyxovirus family. Two henipaviruses, Nipah virus and Hendra virus, cause a systemic respiratory and/or neurological disease in humans and ten additional species of mammals, with a high fatality rate. Because of their highly pathogenic nature, Nipah virus and Hendra virus are categorized as BSL-4 pathogens, which limits the number and scope of translational research studies on these important human pathogens. To begin to address this limitation, we are developing a BSL-2 model of authentic henipavirus infection in mice, using the non-pathogenic henipavirus, Cedar virus. Notably, wild-type mice are highly resistant to Hendra virus and Nipah virus infection. However, previous work has shown that mice lacking expression of the type I interferon receptor (IFNAR-KO mice) are susceptible to both viruses. Here, we show that luciferase-expressing recombinant Cedar virus (rCedV-luc) is also able to replicate and establish a transient infection in IFNAR-KO mice, but not in wild-type mice. Using longitudinal bioluminescence imaging (BLI) of luciferase expression, we detected rCedV-luc replication as early as 10 h post-infection. Viral replication peaks between days 1 and 3 post-infection, and declines to levels undetectable by BLI by 7 days post-infection. Immunohistochemistry is consistent with viral infection and replication in endothelial cells and other non-immune cell types within tissue parenchyma. Serology analyses demonstrate significant IgG responses to the Cedar virus surface glycoprotein with potent neutralizing activity in IFNAR-KO mice, whereas antibody responses in wild-type animals were non-significant. Overall, these data suggest that rCedV-luc infection of IFNAR-KO mice represents a viable platform for the study of in vivo henipavirus replication, anti-henipavirus host responses and henipavirus-directed therapeutics.

Enzyme-Linked Immunosorbent Assay Using Henipavirus-Receptor EphrinB2 and Monoclonal Antibodies for Detecting Nipah and Hendra Viruses.

The Nipah virus (NiV) and the Hendra virus (HeV) are highly pathogenic zoonotic diseases that can cause fatal infections in humans and animals. Early detection is critical for the control of NiV and HeV infections. We present the development of two antigen-detection ELISAs (AgELISAs) using the henipavirus-receptor EphrinB2 and monoclonal antibodies (mAbs) to detect NiV and HeV. The NiV AgELISA detected only NiV, whereas the NiV/HeV AgELISA detected both NiV and HeV. The diagnostic specificities of the NiV AgELISA and the NiV/HeV AgELISA were 100% and 97.8%, respectively. Both assays were specific for henipaviruses and showed no cross-reactivity with other viruses. The AgELISAs detected NiV antigen in experimental pig nasal wash samples taken at 4 days post-infection. With the combination of both AgELISAs, NiV can be differentiated from HeV. Complementing other henipavirus detection methods, these two newly developed AgELISAs can rapidly detect NiV and HeV in a large number of samples and are suitable for use in remote areas where other tests are not available.

Horse populations are severely underestimated in a region at risk of Hendra virus spillover.

OBJECTIVE: To identify the size and distribution of the horse population in the Northern Rivers Region of NSW, including changes from 2007 to 2021, to better understand populations at risk of Hendra virus transmission. METHODS: Census data from the 2007 Equine Influenza (EI) outbreak were compared with data collected annually by New South Wales Local Land Services (LLS) (2011-2021), and with field observations via road line transects (2021). RESULTS: The horse populations reported to LLS in 2011 (3000 horses; 0.77 horses/km2) was 145% larger than that reported during the EI outbreak in 2007 (1225 horses; 0.32 horses/km2). This was inconsistent with the 6% increase in horses recorded from 2011 to 2020 within the longitudinal LLS dataset. Linear modelling suggested the true horse population of this region in 2007 was at least double that reported at the time. Distance sampling in 2021 estimated the region's population at 10,185 horses (3.89 per km2; 95% CI = 4854-21,372). Field sampling and modelling identified higher horse densities in rural cropland, with the percentage of conservation land, modified grazing, and rural residential land identified as the best predictors of horse densities. CONCLUSIONS: Data from the 2007 EI outbreak no longer correlates to the current horse population in size or distribution and was likely not a true representation at the time. Current LLS data also likely underestimates horse populations. Ongoing efforts to further quantify and map horse populations in Australia are important for estimating and managing the risk of equine zoonoses.

Infection and transmission of henipavirus in animals.

Henipavirus (HNV) is well known for two zoonotic viruses in the genus, Hendra virus (HeV) and Nipah virus (NiV), which pose serious threat to human and animal health. In August 2022, a third zoonotic virus in the genus Henipavirus, Langya virus (LayV), was discovered in China. The emergence of HeV, NiV, and LayV highlights the persistent threat of HNV to human and animal health. In addition to the above three HNVs, new species within this genus are still being discovered. Although they have not yet caused a pandemic in humans or livestock, they still have the risk of spillover as a potential threat to the health of humans and animals. It's important to understand the infection and transmission of different HNV in animals for the prevention and control of current or future HNV epidemics. Therefore, this review mainly summarizes the animal origin, animal infection and transmission of HNV that have been found worldwide, and further analyzes and summarizes the rules of infection and transmission, so as to provide a reference for relevant scientific researchers. Furthermore, it can provide a direction for epidemic prevention and control, and animal surveillance to reduce the risk of the global pandemic of HNV.

Structure-guided mutagenesis of Henipavirus receptor-binding proteins reveals molecular determinants of receptor usage and antibody-binding epitopes.

Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.

Nipah Virus: A Multidimensional Update.

Nipah virus (NiV) is an emerging zoonotic paramyxovirus to which is attributed numerous high mortality outbreaks in South and South-East Asia; Bangladesh's Nipah belt accounts for the vast majority of human outbreaks, reporting regular viral emergency events. The natural reservoir of NiV is the Pteropus bat species, which covers a wide geographical distribution extending over Asia, Oceania, and Africa. Occasionally, human outbreaks have required the presence of an intermediate amplification mammal host between bat and humans. However, in Bangladesh, the viral transmission occurs directly from bat to human mainly by ingestion of contaminated fresh date palm sap. Human infection manifests as a rapidly progressive encephalitis accounting for extremely high mortality rates. Despite that, no therapeutic agents or vaccines have been approved for human use. An updated review of the main NiV infection determinants and current potential therapeutic and preventive strategies is exposed.

Sequence basis for selectivity of ephrin-B2 ligand for Eph receptors and pathogenic henipavirus G glycoproteins.

IMPORTANCE: Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and regulates multiple cell developmental and signaling processes. It also functions as the cell entry receptor for Nipah virus and Hendra virus, zoonotic viruses that can cause respiratory and/or neurological symptoms in humans with high mortality. Here, we investigate the sequence basis of EFNB2 specificity for binding the Nipah virus attachment G glycoprotein over Eph receptors. We then use this information to engineer EFNB2 as a soluble decoy receptor that specifically binds the attachment glycoproteins of the Nipah virus and other related henipaviruses to neutralize infection. These findings further mechanistic understanding of protein selectivity and may facilitate the development of diagnostics or therapeutics against henipavirus infection.

Animal Models for Henipavirus Research.

Hendra virus (HeV) and Nipah virus (NiV) are zoonotic paramyxoviruses in the genus Henipavirus (HNV) that emerged nearly thirty years ago. Outbreaks of HeV and NiV have led to severe respiratory disease and encephalitis in humans and animals characterized by a high mortality rate. Despite the grave threat HNVs pose to public health and global biosecurity, no approved medical countermeasures for human use currently exist against HeV or NiV. To develop candidate vaccines and therapeutics and advance the field's understanding of HNV pathogenesis, animal models of HeV and NiV have been instrumental and remain indispensable. Various species, including rodents, ferrets, and nonhuman primates (NHPs), have been employed for HNV investigations. Among these, NHPs have demonstrated the closest resemblance to human HNV disease, although other animal models replicate some key disease features. Here, we provide a comprehensive review of the currently available animal models (mice, hamsters, guinea pigs, ferrets, cats, dogs, nonhuman primates, horses, and swine) to support HNV research. We also discuss the strengths and limitations of each model for conducting pathogenesis and transmission studies on HeV and NiV and for the evaluation of medical countermeasures.

Enhanced broad spectrum in vitro antiviral efficacy of 3-F-4-MeO-Bn, 3-CN, and 4-CN derivatives of lipid remdesivir nucleoside monophosphate prodrugs.

Broad spectrum oral antivirals are urgently needed for the early treatment of many RNA viruses of clinical concern. We previously described the synthesis of 1-O-octadecyl-2-O-benzyl-glycero-3-phospho-RVn (V2043), an orally bioavailable lipid prodrug of remdesivir nucleoside (RVn, GS-441524) with broad spectrum antiviral activity against viruses with pandemic potential. Here we compared the relative activity of V2043 with new RVn lipid prodrugs containing sn-1 alkyl ether or sn-2 glycerol modifications. We found that 3-F-4-MeO-Bn, 3-CN-Bn, and 4-CN-Bn sn-2 glycerol modifications improved antiviral activity compared to V2043 when tested in vitro against clinically important RNA viruses from 5 virus families. These results support the continued development of V2043 and sn-2 glycerol modified RVn lipid prodrugs for the treatment of a broad range of RNA viruses for which there are limited therapies.

Recombinant Soluble Henipavirus Glycoprotein Preparation.

Henipaviruses possess two envelope glycoproteins, the attachment (G) and the fusion (F) proteins that mediate cellular entry and are the major targets of virus-neutralizing antibody responses. Recombinant expression technologies have been used to produce soluble G and F proteins (sG and sF) that retain native-like oligomeric conformations and epitopes, which are advantageous for the development and characterization of vaccines and antiviral antibody therapeutics. In addition to Hendra virus and Nipah virus tetrameric sG and trimeric sF production, we also describe the expression and purification of Cedar virus tetrameric sG and Ghana virus trimeric sF glycoproteins. These henipavirus glycoproteins were also used as immunizing antigens to generate monoclonal antibodies, and binding was demonstrated with a pan-henipavirus multiplex microsphere immunoassay.

In Vitro Antiviral Screening for Henipaviruses at BSL4.

In vitro screening for antivirals is an essential step in the development of effective treatments against new and emerging pathogens. Here, we describe a simple, cell-based screening assay for evaluating antiviral effectiveness against Hendra and Nipah live virus infection under BSL4 conditions.

Nipah virus attachment glycoprotein ectodomain delivered by type 5 adenovirus vector elicits broad immune response against NiV and HeV.

Nipah virus (NiV) and Hendra virus (HeV) are newly emerging dangerous zoonotic pathogens of the Henipavirus genus of the Paramyxoviridae family. NiV and HeV (HNVs) which are transmitted by bats cause acute respiratory disease and fatal encephalitis in humans. To date, as there is a lack of antiviral drugs or effective antiviral therapies, the development of vaccines against those two viruses is of primary importance, and the immunogen design is crucial to the success of vaccines. In this study, the full-length protein (G), the ectodomain (Ge) and the head domain (Gs) of NiV attachment glycoprotein were delivered by the replication-defective type 5 adenovirus vector (Ad5) respectively, and the recombinant Ad5-NiV vaccine candidates (Ad5-NiVG, Ad5-NiVGe and Ad5-NiVGs) were constructed and their immunogenicity were evaluated in mice. The results showed that all the vaccine candidates stimulated specific humoral and cellular immune responses efficiently and rapidly against both NiV and HeV, and the Ad5-NiVGe elicited the strongest immune responses after a single-dose immunization. Furthermore, the potent conserved T-cell epitope DTLYFPAVGFL shared by NiV and HeV was identified in the study, which may provide valid information on the mechanism of HNVs-specific cellular immunity. In summary, this study demonstrates that the Ad5-NiVGe could be a potent vaccine candidate against HNVs by inducing robust humoral and cellular immune responses.

Henipavirus zoonosis: outbreaks, animal hosts and potential new emergence.

Hendra virus (HeV) and Nipah virus (NiV) are biosafety level 4 zoonotic pathogens causing severe and often fatal neurological and respiratory disease. These agents have been recognized by the World Health Organization as top priority pathogens expected to result in severe future outbreaks. HeV has caused sporadic infections in horses and a small number of human cases in Australia since 1994. The NiV Malaysia genotype (NiV-M) was responsible for the 1998-1999 epizootic outbreak in pigs with spillover to humans in Malaysia and Singapore. Since 2001, the NiV Bangladesh genotype (NiV-B) has been the predominant strain leading to outbreaks almost every year in Bangladesh and India, with hundreds of infections in humans. The natural reservoir hosts of HeV and NiV are fruit bats, which carry the viruses without clinical manifestation. The transmission pathways of henipaviruses from bats to humans remain poorly understood. Transmissions are often bridged by an intermediate animal host, which amplifies and spreads the viruses to humans. Horses and pigs are known intermediate hosts for the HeV outbreaks in Australia and NiV-M epidemic in Malaysia and Singapore, respectively. During the NiV-B outbreaks in Bangladesh, following initial spillover thought to be through the consumption of date palm sap, the spread of infection was largely human-to-human transmission. Spillover of NiV-B in recent outbreaks in India is less understood, with the primary route of transmission from bat reservoir to the initial human infection case(s) unknown and no intermediate host established. This review aims to provide a concise update on the epidemiology of henipaviruses covering their previous and current outbreaks with emphasis on the known and potential role of livestock as intermediate hosts in disease transmission. Also included is an up-to-date summary of newly emerging henipa-like viruses and animal hosts. In these contexts we discuss knowledge gaps and new challenges in the field and propose potential future directions.

Rapid, sensitive, and specific, low-resource molecular detection of Hendra virus.

Efficient and accurate diagnosis of Hendra virus (HeV), a biosafety level 4 (BSL-4) pathogen and zoonotic disease, is of primary importance for surveillance and outbreak control in the Australian equine industry. Sporadic HeV spillover events pose a serious public health concern and are predicted to expand geographically, aligning with the moving distribution of the main reservoir hosts, the flying-foxes. Here we describe the development of a low-resource rapid Hendra test. The test used a fast and simple sample processing protocol followed by reverse transcription isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results were obtained in 30 min and required only a heating block, ice, and pipettes for liquid handling. The one-step sample processing protocol inactivated HeV in 2 min, providing a simple protocol that could enable safe testing outside of a laboratory. Analytical sensitivity testing demonstrated a detection limit of 1000 copies/µL of synthetic HeV RNA, and analytical specificity testing indicated assays did not detect other pathogens. Gamma-irradiated HeV-spiked in viral transport medium was detected with high sensitivity, down to 10,000 TCID50/mL, the equivalent of 18 RNA copies per reaction. Collectively, our data suggests that our rapid Hendra test offers a potential first-line screening on-site alternative to gold-standard RT-PCR detection, which requires samples to be shipped to central containment laboratories, thermocyclers and labour-intensive viral RNA purification, with testing time of approximately four hours. Our rapid Hendra test provided performance and speed without compromising sensitivity and specificity, and could become a promising more accessible tool for testing under resource-limited conditions for the veterinary community and thoroughbred industry.

Henipavirus Matrix Protein Employs a Non-Classical Nuclear Localization Signal Binding Mechanism.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic species from the Henipavirus genus within the paramyxovirus family and are harbored by Pteropus Flying Fox species. Henipaviruses cause severe respiratory disease, neural symptoms, and encephalitis in various animals and humans, with human mortality rates exceeding 70% in some NiV outbreaks. The henipavirus matrix protein (M), which drives viral assembly and budding of the virion, also performs non-structural functions as a type I interferon antagonist. Interestingly, M also undergoes nuclear trafficking that mediates critical monoubiquitination for downstream cell sorting, membrane association, and budding processes. Based on the NiV and HeV M X-ray crystal structures and cell-based assays, M possesses a putative monopartite nuclear localization signal (NLS) (residues 82KRKKIR87; NLS1 HeV), positioned on an exposed flexible loop and typical of how many NLSs bind importin alpha (IMPα), and a putative bipartite NLS (244RR-10X-KRK258; NLS2 HeV), positioned within an α-helix that is far less typical. Here, we employed X-ray crystallography to determine the binding interface of these M NLSs and IMPα. The interaction of both NLS peptides with IMPα was established, with NLS1 binding the IMPα major binding site, and NLS2 binding as a non-classical NLS to the minor site. Co-immunoprecipitation (co-IP) and immunofluorescence assays (IFA) confirm the critical role of NLS2, and specifically K258. Additionally, localization studies demonstrated a supportive role for NLS1 in M nuclear localization. These studies provide additional insight into the critical mechanisms of M nucleocytoplasmic transport, the study of which can provide a greater understanding of viral pathogenesis and uncover a potential target for novel therapeutics for henipaviral diseases.

Communication Interventions and Assessment of Drivers for Hendra Virus Vaccination Uptake.

Hendra virus disease (HeVD) is an emerging zoonosis in Australia, resulting from the transmission of Hendra virus (HeV) to horses from Pteropus bats. Vaccine uptake for horses is low despite the high case fatality rate of HeVD in both horses and people. We reviewed evidence-based communication interventions to promote and improve HeV vaccine uptake for horses by horse owners and conducted a preliminary evaluation of potential drivers for HeV vaccine uptake using the Behavioural and Social Drivers of Vaccination (BeSD) framework developed by the World Health Organization. Six records were eligible for review following a comprehensive search and review strategy of peer-reviewed literature, but evidence-based communication interventions to promote and improve HeV vaccine uptake for horses were lacking. An evaluation of potential drivers for HeV vaccine uptake using the BeSD framework indicated that horse owners' perceptions, beliefs, social processes, and practical issues are similar to those experienced by parents making decisions about childhood vaccines, although the overall motivation to vaccinate is lower amongst horse owners. Some aspects of HeV vaccine uptake are not accounted for in the BeSD framework (for example, alternative mitigation strategies such as covered feeding stations or the zoonotic risk of HeV). Overall, problems associated with HeV vaccine uptake appear well-documented. We, therefore, propose to move from a problems-focused to a solutions-focused approach to reduce the risk of HeV for humans and horses. Following our findings, we suggest that the BeSD framework could be modified and used to develop and evaluate communication interventions to promote and improve HeV vaccine uptake by horse owners, which could have a global application to promote vaccine uptake for other zoonotic diseases in animals, such as rabies.

A Recombinant Chimeric Cedar Virus-Based Surrogate Neutralization Assay Platform for Pathogenic Henipaviruses.

The henipaviruses, Nipah virus (NiV), and Hendra virus (HeV) can cause fatal diseases in humans and animals, whereas Cedar virus is a nonpathogenic henipavirus. Here, using a recombinant Cedar virus (rCedV) reverse genetics platform, the fusion (F) and attachment (G) glycoprotein genes of rCedV were replaced with those of NiV-Bangladesh (NiV-B) or HeV, generating replication-competent chimeric viruses (rCedV-NiV-B and rCedV-HeV), both with and without green fluorescent protein (GFP) or luciferase protein genes. The rCedV chimeras induced a Type I interferon response and utilized only ephrin-B2 and ephrin-B3 as entry receptors compared to rCedV. The neutralizing potencies of well-characterized cross-reactive NiV/HeV F and G specific monoclonal antibodies against rCedV-NiV-B-GFP and rCedV-HeV-GFP highly correlated with measurements obtained using authentic NiV-B and HeV when tested in parallel by plaque reduction neutralization tests (PRNT). A rapid, high-throughput, and quantitative fluorescence reduction neutralization test (FRNT) using the GFP-encoding chimeras was established, and monoclonal antibody neutralization data derived by FRNT highly correlated with data derived by PRNT. The FRNT assay could also measure serum neutralization titers from henipavirus G glycoprotein immunized animals. These rCedV chimeras are an authentic henipavirus-based surrogate neutralization assay that is rapid, cost-effective, and can be utilized outside high containment.