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Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness with fatality rates of 50–100%. No vaccines or licensed therapeutics currently exist to protect humans against NiV or HeV. HNVs enter host cells by fusing the viral and cellular membranes via the concerted action of the attachment (G) and fusion (F) glycoproteins, the main targets of the humoral immune response. Here, we describe the isolation and humanization of a potent monoclonal antibody cross-neutralizing NiV and HeV. Cryo-electron microscopy, triggering and fusion studies show the antibody binds to a prefusion-specific quaternary epitope, conserved in NiV F and HeV F glycoproteins, and prevents membrane fusion and viral entry. This work supports the importance of the HNV prefusion F conformation for eliciting a robust immune response and paves the way for using this antibody for prophylaxis and post-exposure therapy with NiV- and HeV-infected individuals.

Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

Hendra virus (HeV) belongs to the paramyxoviridae family of viruses which is associated with the respiratory distress, neurological illness, and potential fatality of the affected individuals. So far, no competitive approved therapeutic substance is available for HeV. For that reason, the current research work was conducted to propose some novel compounds, by adopting a Computer Aided Drug Discovery approach, which could be used to combat HeV. The G attachment Glycoprotein (Ggp) of HeV was selected to achieve the primary objective of this study, as this protein makes the entry of HeV possible in the host cells. Briefly, a library of 6000 antiviral compounds was screened for potential drug-like properties, followed by the molecular docking of short-listed compounds with the Protein Data Bank (PDB) structure of Ggp. Docked complexes of top two hits, having maximum binding affinities with the active sites of Ggp, were further considered for molecular dynamic simulations of 200 ns to elucidate the results of molecular docking analysis. MD simulations and Molecular Mechanics Energies combined with the Generalized Born and Surface Area (MMGBSA) or Poisson–Boltzmann and Surface Area (MMPBSA) revealed that both docked complexes are stable in nature. Furthermore, the same methodology was used between lead compounds and HeV Ggp in complex with its functional receptor in human, Ephrin-B2. Surprisingly, no major differences were found in the results, which demonstrates that our identified compounds can also perform their action even when the Ggp is attached to the Ephrin-B2 ligand. Therefore, in light of all of these results, we strongly suggest that compounds (S)-5-(benzylcarbamoyl)-1-(2-(4-methyl-2-phenylpiperazin-1-yl)-2-oxoethyl)-6-oxo-3,6-dihydropyridin-1-ium-3-ide and 5-(cyclohexylcarbamoyl)-1-(2-((2-(3-fluorophenyl)-2-methylpropyl)amino)-2-oxoethyl)-6-oxo-3,6-dihydropyridin-1-ium-3-ide could be considered as potential therapeutic agents against HeV; however, further in vitro and in vivo experiments are required to validate this study.

Hendra virus (HeV) is a paramyxovirus that causes lethal disease in humans, for which no vaccine or antiviral agent is available. HeV V protein is central to pathogenesis through its ability to interact with cytoplasmic host proteins, playing key antiviral roles. Here we use immunoprecipitation, siRNA knockdown and confocal laser scanning microscopy to show that HeV V shuttles to and from the nucleus through specific host nuclear transporters. Spectroscopic and small angle X-ray scattering studies reveal HeV V undergoes a disorder-to-order transition upon binding to either importin α/β1 or exportin-1/Ran-GTP, dependent on the V N-terminus. Importantly, we show that specific inhibitors of nuclear transport prevent interaction with host transporters, and reduce HeV infection. These findings emphasize the critical role of host-virus interactions in HeV infection, and potential use of compounds targeting nuclear transport, such as the FDA-approved agent ivermectin, as anti-HeV agents.

Background Using a recently described monolayer assay amenable to high throughput screening format for the identification of potential Nipah virus and Hendra virus antivirals, we have partially screened a low molecular weight compound library (>8,000 compounds) directly against live virus infection and identified twenty eight promising lead molecules. Initial single blind screens were conducted with 10 μM compound in triplicate with a minimum efficacy of 90% required for lead selection. Lead compounds were then further characterised to determine the median efficacy (IC 50 ), cytotoxicity (CC 50 ) and the in vitro therapeutic index in live virus and pseudotype assay formats. Results While a number of leads were identified, the current work describes three commercially available compounds: brilliant green, gentian violet and gliotoxin, identified as having potent antiviral activity against Nipah and Hendra virus. Similar efficacy was observed against pseudotyped Nipah and Hendra virus, vesicular stomatitis virus and human parainfluenza virus type 3 while only gliotoxin inhibited an influenza A virus suggesting a non-specific, broad spectrum activity for this compound. Conclusion All three of these compounds have been used previously for various aspects of anti-bacterial and anti-fungal therapy and the current results suggest that while unsuitable for internal administration, they may be amenable to topical antiviral applications, or as disinfectants and provide excellent positive controls for future studies.

Interferon (IFN) can activate Signal Transducer and Activator of Transcription (STAT) proteins to establish a cellular antiviral response and inhibit virus replication. Many viruses have evolved strategies to inhibit this antiviral mechanism, but paramyxoviruses are unique in their abilities to directly target the IFN-responsive STAT proteins. Hendra virus and Nipah virus (Henipaviruses) are recently emerged paramyxoviruses that are the causative agents of fatal disease outbreaks in Australia and peninsular Malaysia. Similar to other paramyxoviruses, Henipaviruses inhibit IFN signal transduction through a virus-encoded protein called V. Recent studies have shown that Henipavirus V proteins target STAT proteins by inducing the formation of cytoplasmically localized high molecular weight STAT-containing complexes. This sequestration of STAT1 and STAT2 prevents STAT activation and blocks antiviral IFN signaling. As the V proteins are important factors for host evasion, they represent logical targets for therapeutics directed against Henipavirus epidemics.

Hendra virus is one of two virus species within the newly-formed genus Henipavirus, subfamily Paramxyovirinae. It is a designated select agent with potential biosecurity threat to both human and animal health. Quantitative real-time PCR was used to measure viral RNA synthesis in Vero cells infected by Hendra virus, and to examine the inhibitory effect of ribavirin. It was also used to determine the points of attenuation during transcription of the six viral genes N, P, M, F, G and L by targeting amplicons located towards the 3' end of each gene. Major increases in viral RNA and virus yield occurred between 4 to 8 h and 8 to 10 h post infection, respectively. The effect of ribavirin was examined at a range of concentrations up to 400 microm. At 50 microm, RNA synthesis was reduced 9 fold, and virus yield 58 fold. As expected for a member of the order Mononegavirales, a gradient of transcription was observed in Hendra virus-infected cells. There was significant attenuation at the M-F and G-L junctions, more closely resembling Sendai virus (genus Respirovirus) than measles virus (genus Morbillivirus).

Hendra and Nipah virus are closely related emerging viruses comprising the Henipavirus genus of the subfamily Paramyxovirinae and are distinguished by their ability to cause fatal disease in both animal and human hosts. 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 and necessity of developing effective therapeutic interventions. Much research conducted on the henipaviruses over the past several years has focused on virus entry, including the attachment of virus to the host cell, the identification of the virus receptor and the membrane fusion process between the viral and host cell membranes. These findings have led to the development of possible vaccine candidates, as well as potential antiviral therapeutics. The common link among all of the possible antiviral agents discussed here, which have also been developed and tested, is that they target very early stages of the infection process. The establishment and validation of suitable animal models of Henipavirus infection and pathogenesis are also discussed as they will be crucial in the assessment of the effectiveness of any treatments for Hendra and Nipah virus infection.

The Hendra and Nipah viruses (HNVs) are highly pathogenic pathogens without approved interventions for human use. In addition, the interaction pattern between the attachment (G) and fusion (F) glycoproteins required for virus entry remains unclear. Here, we isolate a panel of Macaca-derived G-specific antibodies that cross-neutralize HNVs via multiple mechanisms. The most potent antibody, 1E5, confers adequate protection against the Nipah virus challenge in female hamsters. Crystallography demonstrates that 1E5 has a highly similar binding pattern to the receptor. In cryo-electron microscopy studies, the tendency of 1E5 to bind to the upper or lower heads results in two distinct quaternary structures of G. Furthermore, we identify the extended outer loop β1S2-β1S3 of G and two pockets on the apical region of fusion (F) glycoprotein as the essential sites for G-F interactions. This work highlights promising drug candidates against HNVs and contributes deeper insights into the viruses. There are no approved interventions for Hendra or Nipah viruses. Here, the authors isolate a G glycoprotein-specific antibody with cross-neutralizing and in vivo protective activities, and structurally resolve its binding pattern to the G protein.

Most paramyxoviruses circumvent the IFN response by blocking IFN signaling and limiting the production of IFN by virus-infected cells. Here we report that the highly conserved cysteine-rich C-terminal domain of the V proteins of a wide variety of paramyxoviruses binds melanoma differentiation-associated gene 5 (mda-5) product. mda-5 is an IFN-inducible host cell DExD/H box helicase that contains a caspase recruitment domain at its N terminus. Overexpression of mda-5 stimulated the basal activity of the IFN-β promoter in reporter gene assays and significantly enhanced the activation of the IFN-β promoter by intracellular dsRNA. Both these activities were repressed by coexpression of the V proteins of simian virus 5, human parainfluenza virus 2, mumps virus, Sendai virus, and Hendra virus. Similar results to the reporter assays were obtained by measuring IFN production. Inhibition of mda-5 by RNA interference or by dominant interfering forms of mda-5 significantly inhibited the activation of the IFN-β promoter by dsRNA. It thus appears that mda-5 plays a central role in an intracellular signal transduction pathway that can lead to the activation of the IFN-β promoter, and that the V proteins of paramyxoviruses interact with mda-5 to block its activity.