Indiscriminate activities of different henipavirus polymerase complex proteins allow for efficient minigenome replication in hybrid systems.

The henipaviruses, including Nipah virus (NiV) and Hendra virus (HeV), are biosafety level 4 (BSL-4) zoonotic pathogens that cause severe neurological and respiratory disease in humans. To study the replication machinery of these viruses, we developed robust minigenome systems that can be safely used in BSL-2 conditions. The nucleocapsid (N), phosphoprotein (P), and large protein (L) of henipaviruses are critical elements of their replication machinery and thus essential support components of the minigenome systems. Here, we tested the effects of diverse combinations of the replication support proteins on the replication capacity of the NiV and HeV minigenomes by exchanging the helper plasmids coding for these proteins among the two viruses. We demonstrate that all combinations including one or more heterologous proteins were capable of replicating both the NiV and HeV minigenomes. Sequence alignment showed identities of 92% for the N protein, 67% for P, and 87% for L. Notably, variations in amino acid residues were not concentrated in the N-P and P-L interacting regions implying that dissimilarities in amino acid composition among NiV and HeV polymerase complex proteins may not impact their interactions. The observed indiscriminate activity of NiV and HeV polymerase complex proteins is different from related viruses, which can support the replication of heterologous genomes only when the whole polymerase complex belongs to the same virus. This newly observed promiscuous property of the henipavirus polymerase complex proteins likely attributed to their conserved interaction regions could potentially be harnessed to develop universal anti-henipavirus antivirals.IMPORTANCEGiven the severity of disease induced by Hendra and Nipah viruses in humans and the continuous emergence of new henipaviruses as well as henipa-like viruses, it is necessary to conduct a more comprehensive investigation of the biology of henipaviruses and their interaction with the host. The replication of henipaviruses and the development of antiviral agents can be studied in systems that allow experiments to be performed under biosafety level 2 conditions. Here, we developed robust minigenome systems for the Nipah virus (NiV) and Hendra virus (HeV) that provide a convenient alternative for studying NiV and HeV replication. Using these systems, we demonstrate that any combination of the three polymerase complex proteins of NiV and HeV could effectively initiate the replication of both viral minigenomes, which suggests that the interaction regions of the polymerase complex proteins could be effective targets for universal and effective anti-henipavirus interventions.

Prefusion stabilization of the Hendra and Langya virus F proteins.

Nipah virus (NiV) and Hendra virus (HeV) are pathogenic paramyxoviruses that cause mild-to-severe disease in humans. As members of the Henipavirus genus, NiV and HeV use an attachment (G) glycoprotein and a class I fusion (F) glycoprotein to invade host cells. The F protein rearranges from a metastable prefusion form to an extended postfusion form to facilitate host cell entry. Prefusion NiV F elicits higher neutralizing antibody titers than postfusion NiV F, indicating that stabilization of prefusion F may aid vaccine development. A combination of amino acid substitutions (L104C/I114C, L172F, and S191P) is known to stabilize NiV F in its prefusion conformation, although the extent to which substitutions transfer to other henipavirus F proteins is not known. Here, we perform biophysical and structural studies to investigate the mechanism of prefusion stabilization in F proteins from three henipaviruses: NiV, HeV, and Langya virus (LayV). Three known stabilizing substitutions from NiV F transfer to HeV F and exert similar structural and functional effects. One engineered disulfide bond, located near the fusion peptide, is sufficient to stabilize the prefusion conformations of both HeV F and LayV F. Although LayV F shares low overall sequence identity with NiV F and HeV F, the region around the fusion peptide exhibits high sequence conservation across all henipaviruses. Our findings indicate that substitutions targeting this site of conformational change might be applicable to prefusion stabilization of other henipavirus F proteins and support the use of NiV as a prototypical pathogen for henipavirus vaccine antigen design.IMPORTANCEPathogenic henipaviruses such as Nipah virus (NiV) and Hendra virus (HeV) cause respiratory symptoms, with severe cases resulting in encephalitis, seizures, and coma. The work described here shows that the NiV and HeV fusion (F) proteins share common structural features with the F protein from an emerging henipavirus, Langya virus (LayV). Sequence alignment alone was sufficient to predict which known prefusion-stabilizing amino acid substitutions from NiV F would stabilize the prefusion conformations of HeV F and LayV F. This work also reveals an unexpected oligomeric interface shared by prefusion HeV F and NiV F. Together, these advances lay a foundation for future antigen design targeting henipavirus F proteins. In this way, Nipah virus can serve as a prototypical pathogen for the development of protective vaccines and monoclonal antibodies to prepare for potential henipavirus outbreaks.

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.

Functional benefit of structural disorder for the replication of measles, Nipah and Hendra viruses.

Measles, Nipah and Hendra viruses are severe human pathogens within the Paramyxoviridae family. Their non-segmented, single-stranded, negative-sense RNA genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the viral RNA-dependent-RNA-polymerase (RpRd) for transcription and replication. The RpRd is a complex made of the large protein (L) and of the phosphoprotein (P), the latter serving as an obligate polymerase cofactor and as a chaperon for N. Both the N and P proteins are enriched in intrinsically disordered regions (IDRs), i.e. regions devoid of stable secondary and tertiary structure. N possesses a C-terminal IDR (NTAIL), while P consists of a large, intrinsically disordered N-terminal domain (NTD) and a C-terminal domain (CTD) encompassing alternating disordered and ordered regions. The V and W proteins, two non-structural proteins that are encoded by the P gene via a mechanism of co-transcriptional edition of the P mRNA, are prevalently disordered too, sharing with P the disordered NTD. They are key players in the evasion of the host antiviral response and were shown to phase separate and to form amyloid-like fibrils in vitro. In this review, we summarize the available information on IDRs within the N, P, V and W proteins from these three model paramyxoviruses and describe their molecular partnership. We discuss the functional benefit of disorder to virus replication in light of the critical role of IDRs in affording promiscuity, multifunctionality, fine regulation of interaction strength, scaffolding functions and in promoting liquid-liquid phase separation and fibrillation.

Batting below Average: Failure to Manage Fatal Zoonotic Diseases.

Zoonotic diseases are those which originate in animals but are transmitted to humans often through an intermediate host such as a wild animal. In Australia Hendra virus (HeV) is a disease of horses with occasional human fatalities and which is spread by the fruit bat. This article explores the lessons learnt from managing the Queensland outbreak of HeV in 1994. The legal framework for the notification and management of prohibited matter including zoonotic diseases in Queensland and New South Wales has been strengthened by provisions in the Biosecurity Act 2015 (NSW) which create strong penalties for failure to notify outbreaks and failure to isolate infected stock and prevent their removal from premises within 24 hours. The response of at least 20% of Queensland equine veterinarians to the new legal obligations has been to cease practising equine medicine. There may be scope for enhanced education of veterinary students in legal obligations under the biosecurity legislation.

Structural Dynamics of the C-terminal X Domain of Nipah and Hendra Viruses Controls the Attachment to the C-terminal Tail of the Nucleocapsid Protein.

To understand the dynamic interactions between the phosphoprotein (P) and the nucleoprotein (N) within the transcription/replication complex of the Paramyxoviridae and to decipher their roles in regulating viral multiplication, we characterized the structural properties of the C-terminal X domain (PXD) of Nipah (NiV) and Hendra virus (HeV) P protein. In crystals, isolated NiV PXD adopted a two-helix dimeric conformation, which was incompetent for binding its partners, but in complex with the C-terminal intrinsically disordered tail of the N protein (NTAIL), it folded into a canonical 3H bundle conformation. In solution, SEC-MALLS, SAXS and NMR spectroscopy experiments indicated that both NiV and HeV PXD were larger in size than expected for compact proteins of the same molecular mass and were in conformational exchange between a compact three-helix (3H) bundle and partially unfolded conformations, where helix α3 is detached from the other two. Some measurements also provided strong evidence for dimerization of NiV PXD in solution but not for HeV PXD. Ensemble modeling of experimental SAXS data and statistical-dynamical modeling reconciled all these data, yielding a model where NiV and HeV PXD exchanged between different conformations, and where NiV but not HeV PXD formed dimers. Finally, recombinant NiV comprising a chimeric P carrying HeV PXD was rescued and compared with parental NiV. Experiments carried out in cellula demonstrated that the replacement of PXD did not significantly affect the replication dynamics while caused a slight virus attenuation, suggesting a possible role of the dimerization of NiV PXD in viral replication.

Analysis of Hendra Virus Fusion Protein N-Terminal Transmembrane Residues.

Hendra virus (HeV) is a zoonotic enveloped member of the family Paramyoxviridae. To successfully infect a host cell, HeV utilizes two surface glycoproteins: the attachment (G) protein to bind, and the trimeric fusion (F) protein to merge the viral envelope with the membrane of the host cell. The transmembrane (TM) region of HeV F has been shown to have roles in F protein stability and the overall trimeric association of F. Previously, alanine scanning mutagenesis has been performed on the C-terminal end of the protein, revealing the importance of ß-branched residues in this region. Additionally, residues S490 and Y498 have been demonstrated to be important for F protein endocytosis, needed for the proteolytic processing of F required for fusion. To complete the analysis of the HeV F TM, we performed alanine scanning mutagenesis to explore the residues in the N-terminus of this region (residues 487-506). In addition to confirming the critical roles for S490 and Y498, we demonstrate that mutations at residues M491 and L492 alter F protein function, suggesting a role for these residues in the fusion process.

Information delivery and the veterinarian-horse owner relationship in the context of Hendra virus in Australia.

Hendra virus (HeV) is an emerging bat-borne virus endemic in Australia that can be transmitted from horses to humans and has a high fatality rate for horses and people. Controversy surrounding HeV risk mitigation measures have strained the veterinarian-horse owner relationship. This study aimed to characterise the veterinarian-horse owner relationship in general and also in the context of HeV by analysing data derived from the 'Horse Owners and Hendra Virus: A Longitudinal Study to Evaluate Risk' (HHALTER) study. Australian horse owners were recruited via emails, social media and word-of-mouth for a series of five surveys that were administered online at six-monthly intervals over a two-year period to capture baseline knowledge, attitudes and practices of horse owners regarding HeV and any changes over time. In the current study, descriptive analyses of information sources were performed to understand the use of veterinarians as a HeV information source (Surveys 1 and 5; n = 1195 and n = 617). Ordinal logistic regression analyses were conducted to determine factors associated with the frequency of horse owner contact with a veterinarian (Survey 3; n = 636). This study found a relative increase over the study period in the proportion of horse owners who had used veterinarians as HeV information source in the last 12 months (from 51.9% to 88.3%). Owning more horses, being older, having a 'duty of care' for other people working with horses and deriving the main income from horse related business were factors associated with more frequent veterinary contact. Results suggest that traditional information sources such as workshops, information packs and risk training are likely to be used by horse owners. Smart phone applications should be considered for use in the future and require further investigation for horse health communication. The findings of this study may be helpful in optimising strategies for horse health information delivery.

A structural basis for antibody-mediated neutralization of Nipah virus reveals a site of vulnerability at the fusion glycoprotein apex.

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes frequent outbreaks of severe neurologic and respiratory disease in humans with high case fatality rates. The 2 glycoproteins displayed on the surface of the virus, NiV-G and NiV-F, mediate host-cell attachment and membrane fusion, respectively, and are targets of the host antibody response. Here, we provide a molecular basis for neutralization of NiV through antibody-mediated targeting of NiV-F. Structural characterization of a neutralizing antibody (nAb) in complex with trimeric prefusion NiV-F reveals an epitope at the membrane-distal domain III (DIII) of the molecule, a region that undergoes substantial refolding during host-cell entry. The epitope of this monoclonal antibody (mAb66) is primarily protein-specific and we observe that glycosylation at the periphery of the interface likely does not inhibit mAb66 binding to NiV-F. Further characterization reveals that a Hendra virus-F-specific nAb (mAb36) and many antibodies in an antihenipavirus-F polyclonal antibody mixture (pAb835) also target this region of the molecule. Integrated with previously reported paramyxovirus F-nAb structures, these data support a model whereby the membrane-distal region of the F protein is targeted by the antibody-mediated immune response across henipaviruses. Notably, our domain-specific sequence analysis reveals no evidence of selective pressure at this region of the molecule, suggestive that functional constraints prevent immune-driven sequence variation. Combined, our data reveal the membrane-distal region of NiV-F as a site of vulnerability on the NiV surface.

The problem of scale in the prediction and management of pathogen spillover.

Disease emergence events, epidemics and pandemics all underscore the need to predict zoonotic pathogen spillover. Because cross-species transmission is inherently hierarchical, involving processes that occur at varying levels of biological organization, such predictive efforts can be complicated by the many scales and vastness of data potentially required for forecasting. A wide range of approaches are currently used to forecast spillover risk (e.g. macroecology, pathogen discovery, surveillance of human populations, among others), each of which is bound within particular phylogenetic, spatial and temporal scales of prediction. Here, we contextualize these diverse approaches within their forecasting goals and resulting scales of prediction to illustrate critical areas of conceptual and pragmatic overlap. Specifically, we focus on an ecological perspective to envision a research pipeline that connects these different scales of data and predictions from the aims of discovery to intervention. Pathogen discovery and predictions focused at the phylogenetic scale can first provide coarse and pattern-based guidance for which reservoirs, vectors and pathogens are likely to be involved in spillover, thereby narrowing surveillance targets and where such efforts should be conducted. Next, these predictions can be followed with ecologically driven spatio-temporal studies of reservoirs and vectors to quantify spatio-temporal fluctuations in infection and to mechanistically understand how pathogens circulate and are transmitted to humans. This approach can also help identify general regions and periods for which spillover is most likely. We illustrate this point by highlighting several case studies where long-term, ecologically focused studies (e.g. Lyme disease in the northeast USA, Hendra virus in eastern Australia, Plasmodium knowlesi in Southeast Asia) have facilitated predicting spillover in space and time and facilitated the design of possible intervention strategies. Such studies can in turn help narrow human surveillance efforts and help refine and improve future large-scale, phylogenetic predictions. We conclude by discussing how greater integration and exchange between data and predictions generated across these varying scales could ultimately help generate more actionable forecasts and interventions. This article is part of the theme issue 'Dynamic and integrative approaches to understanding pathogen spillover'.

Nipah and Hendra Virus Glycoproteins Induce Comparable Homologous but Distinct Heterologous Fusion Phenotypes.

Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are biosafety level 4 (BSL-4) paramyxoviruses in the growing genus Henipavirus The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, which is pathognomonic of henipaviral infections. Here, we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (hemagglutinin [HA] tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly, however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F-G interactions, supporting the F/G dissociation model of henipavirus-induced membrane fusion, even in the context of heterologous glycoprotein pairs.IMPORTANCE The NiV and HeV henipaviruses are BSL-4 pathogens transmitted from bats. NiV and HeV often lead to human death and animal diseases. The formation of multinucleated cells (syncytia) is a hallmark of henipaviral infections and is caused by fusion of cells coordinated by interactions of the viral attachment (G) and fusion (F) glycoproteins. We found via various assays that viral entry and syncytium formation depend on the viral origin of the glycoproteins, with HeV F and NiV G promoting higher membrane fusion levels than their counterparts. This is important knowledge, since both viruses use the same bat vector species and potential coinfections of these or subsequent hosts may alter the outcome of disease.

Viral regulation of host cell biology by hijacking of the nucleolar DNA-damage response.

Recent studies indicate that nucleoli play critical roles in the DNA-damage response (DDR) via interaction of DDR machinery including NBS1 with nucleolar Treacle protein, a key mediator of ribosomal RNA (rRNA) transcription and processing. Here, using proteomics, confocal and single molecule super-resolution imaging, and infection under biosafety level-4 containment, we show that this nucleolar DDR pathway is targeted by infectious pathogens. We find that the matrix proteins of Hendra virus and Nipah virus, highly pathogenic viruses of the Henipavirus genus in the order Mononegavirales, interact with Treacle and inhibit its function, thereby silencing rRNA biogenesis, consistent with mimicking NBS1-Treacle interaction during a DDR. Furthermore, inhibition of Treacle expression/function enhances henipavirus production. These data identify a mechanism for viral modulation of host cells by appropriating the nucleolar DDR and represent, to our knowledge, the first direct intranucleolar function for proteins of any mononegavirus.

Environmental drivers of spatiotemporal foraging intensity in fruit bats and implications for Hendra virus ecology.

In the Australian subtropics, flying-foxes (family Pteropididae) play a fundamental ecological role as forest pollinators. Flying-foxes are also reservoirs of the fatal zoonosis, Hendra virus. Understanding flying fox foraging ecology, particularly in agricultural areas during winter, is critical to determine their role in transmitting Hendra virus to horses and humans. We developed a spatiotemporal model of flying-fox foraging intensity based on foraging patterns of 37 grey-headed flying-foxes (Pteropus poliocephalus) using GPS tracking devices and boosted regression trees. We validated the model with independent population counts and summarized temporal patterns in terms of spatial resource concentration. We found that spatial resource concentration was highest in late-summer and lowest in winter, with lowest values in winter 2011, the same year an unprecedented cluster of spillover events occurred in Queensland and New South Wales. Spatial resource concentration was positively correlated with El Niño Southern Oscillation at 3-8 month time lags. Based on shared foraging traits with the primary reservoir of Hendra virus (Pteropus alecto), we used our results to develop hypotheses on how regional climatic history, eucalypt phenology, and foraging behaviour may contribute to the predominance of winter spillovers, and how these phenomena connote foraging habitat conservation as a public health intervention.

Favipiravir (T-705) protects against Nipah virus infection in the hamster model.

Nipah and Hendra viruses are recently emerged bat-borne paramyxoviruses (genus Henipavirus) causing severe encephalitis and respiratory disease in humans with fatality rates ranging from 40-75%. Despite the severe pathogenicity of these viruses and their pandemic potential, no therapeutics or vaccines are currently approved for use in humans. Favipiravir (T-705) is a purine analogue antiviral approved for use in Japan against emerging influenza strains; and several phase 2 and 3 clinical trials are ongoing in the United States and Europe. Favipiravir has demonstrated efficacy against a broad spectrum of RNA viruses, including members of the Paramyxoviridae, Filoviridae, Arenaviridae families, and the Bunyavirales order. We now demonstrate that favipiravir has potent antiviral activity against henipaviruses. In vitro, favipiravir inhibited Nipah and Hendra virus replication and transcription at micromolar concentrations. In the Syrian hamster model, either twice daily oral or once daily subcutaneous administration of favipiravir for 14 days fully protected animals challenged with a lethal dose of Nipah virus. This first successful treatment of henipavirus infection in vivo with a small molecule drug suggests that favipiravir should be further evaluated as an antiviral treatment option for henipavirus infections.

Electrostatic Interactions between Hendra Virus Matrix Proteins Are Required for Efficient Virus-Like-Particle Assembly.

Hendra virus (HeV) is a zoonotic paramyxovirus belonging to the genus Henipavirus HeV is highly pathogenic, and it can cause severe neurological and respiratory illnesses in both humans and animals, with an extremely high mortality rate of up to 70%. Among the genes that HeV encodes, the matrix (M) protein forms an integral part of the virion structure and plays critical roles in coordinating viral assembly and budding. Nevertheless, the molecular mechanism of this process is not fully elucidated. Here, we determined the crystal structure of HeV M to 2.5-Å resolution. The dimeric structural configuration of HeV M is similar to that of Newcastle disease virus (NDV) M and is fundamental to protein stability and effective virus-like-particle (VLP) formation. Analysis of the crystal packing revealed a notable interface between the α1 and α2 helices of neighboring HeV M dimers, with key residues sharing degrees of sequence conservation among henipavirus M proteins. Structurally, a network of electrostatic interactions dominates the α1-α2 interactions, involving residues Arg57 from the α1 helix and Asp105 and Glu108 from the α2 helix. The disruption of the α1-α2 interactions using engineered charge reversal substitutions (R57E, R57D, and E108R) resulted in significant reduction or abrogation of VLP production. This phenotype was reversible with an R57E E108R mutant that was designed to partly restore salt bridge contacts. Collectively, our results define and validate previously underappreciated regions of henipavirus M proteins that are crucial for productive VLP assembly.IMPORTANCE Hendra virus is a henipavirus associated with lethal infections in humans. It is classified as a biosafety level 4 (BSL4) agent, and there are currently no preventive or therapeutic treatments available against HeV. Vital to henipavirus pathogenesis, the structural protein M has been implicated in viral assembly and budding, as well as host-virus interactions. However, there is no structural information available for henipavirus M, and the basis of M-driven viral assembly is not fully elucidated. We demonstrate the first three-dimensional structure of a henipavirus M protein. We show the dimeric organization of HeV M as a basic unit for higher-order oligomerization. Additionally, we define key regions/residues of HeV M that are required for productive virus-like-particle formation. These findings provide the first insight into the mechanism of M-driven assembly in henipavirus.

Recognition by host nuclear transport proteins drives disorder-to-order transition in Hendra virus V.

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/ß1 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.

Australian horse owners and their biosecurity practices in the context of Hendra virus.

In recent years, outbreaks of exotic as well as newly emerging infectious diseases have highlighted the importance of biosecurity for the Australian horse industry. As the first potentially fatal zoonosis transmissible from horses to humans in Australia, Hendra virus has emphasised the need to incorporate sound hygiene and general biosecurity practices into day-to-day horse management. Recommended measures are widely publicised, but implementation is at the discretion of the individual owner. This cross-sectional study aimed to determine current levels of biosecurity of horse owners and to identify factors influencing the uptake of practices utilising data from an online survey. Level of biosecurity (low, medium, high), as determined by horse owners' responses to a set of questions on the frequency of various biosecurity practices performed around healthy (9 items) and sick horses (10 items), was used as a composite outcome variable in ordinal logistic regression analyses. The majority of horse owners surveyed were female (90%), from the states of Queensland (45%) or New South Wales (37%), and were involved in either mainly competitive/equestrian sports (37%) or recreational horse activities (35%). Seventy-five percent of owners indicated that they follow at least one-third of the recommended practices regularly when handling their horses, resulting in medium to high levels of biosecurity. Main factors associated with a higher level of biosecurity were high self-rated standard of biosecurity, access to personal protective equipment, absence of flying foxes in the local area, a good sense of control over Hendra virus risk, likelihood of discussing a sick horse with a veterinarian and likelihood of suspecting Hendra virus in a sick horse. Comparison of the outcome variable with the self-rated standard of biosecurity showed that over- as well as underestimation occurred. This highlights the need for continuous communication and education to enhance awareness and understanding of what biosecurity is and how it aligns with good horsemanship. Overall, strengthened biosecurity practices will help to improve animal as well as human health and increase preparedness for future disease outbreaks.

Conditions affecting the timing and magnitude of Hendra virus shedding across pteropodid bat populations in Australia.

Understanding infection dynamics in animal hosts is fundamental to managing spillover and emergence of zoonotic infections. Hendra virus is endemic in Australian pteropodid bat populations and can be lethal to horses and humans. However, we know little about the factors driving Hendra virus prevalence in resevoir bat populations, making spillover difficult to predict. We use Hendra virus prevalence data collected from 13 000 pooled bat urine samples across space and time to determine if pulses of prevalence are periodic and synchronized across sites. We also test whether site-specific precipitation and temperature affect the amplitude of the largest annual prevalence pulses. We found little evidence for a periodic signal in Hendra virus prevalence. Although the largest amplitude pulses tended to occur over winter, pulses could also occur in other seasons. We found that Hendra virus prevalence was weakly synchronized across sites over short distances, suggesting that prevalence is driven by local-scale effects. Finally, we found that drier conditions in previous seasons and the abundance of Pteropus alecto were positively correlated with the peak annual values of Hendra virus prevalence. Our results suggest that in addition to seasonal effects, bat density and local climatic conditions interact to drive Hendra virus infection dynamics.

Physiological stress and Hendra virus in flying-foxes (Pteropus spp.), Australia.

Pteropid bats (flying-foxes) are the natural reservoir of Hendra virus, an emergent paramyxovirus responsible for fatal infection in horses and humans in Australia. Pteropus alecto (the Black flying-fox) and the paraphyletic P. conspicillatus (the Spectacled flying-fox) appear to be the primary reservoir hosts. Previous studies have suggested that physiological and ecological factors may underpin infection dynamics in flying-foxes, and subsequent spillover to horses and in turn humans. We sought to examine temporal trends in urinary cortisol concentration in wild Australian flying-fox populations, to elucidate the putative relationship between Hendra virus infection and physiological stress. Pooled and individual urine samples were non-invasively collected from under roosting flying-foxes at two latitudinally disparate regions in the eastern Australian state of Queensland. Hendra virus detection, and (in individual urine samples) sex and species determination were PCR-based. Urinary cortisol measurement used a validated enzyme immunoassay. We found no direct correlation between increased urinary cortisol and Hendra virus excretion, but our findings do suggest a biologically plausible association between low winter temperatures and elevated cortisol levels in P. alecto in the lower latitude Southeast Queensland roosts. We hypothesize an indirect association between low winter temperatures and increased Hendra virus infection and excretion, mediated by the physiological cost of thermoregulation. Our findings and our approach are directly relevant to elaboration of the disease ecology of Nipah virus and other emerging henipaviruses in bats. More broadly, they inform investigation of emerging disease infection dynamics across the wildlife/livestock/human interface.

Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation.

Henipaviruses, such as Nipah (NiV) and Hendra (HeV) viruses, are highly pathogenic zoonotic agents within the Paramyxoviridae family. The phosphoprotein (P) gene products of the paramyxoviruses have been well characterized for their interferon (IFN) antagonist activity and their contribution to viral pathogenicity. In this study, we demonstrated that the nucleoprotein (N) of henipaviruses also prevents the host IFN signaling response. Reporter assays demonstrated that the NiV and HeV N proteins (NiV-N and HeV-N, respectively) dose-dependently suppressed both type I and type II IFN responses and that the inhibitory effect was mediated by their core domains. Additionally, NiV-N prevented the nuclear transport of signal transducer and activator of transcription 1 (STAT1) and STAT2. However, NiV-N did not associate with Impα5, Impß1, or Ran, which are members of the nuclear transport system for STATs. Although P protein is known as a binding partner of N protein and actively retains N protein in the cytoplasm, the IFN antagonist activity of N protein was not abolished by the coexpression of P protein. This suggests that the IFN inhibition by N protein occurs in the cytoplasm. Furthermore, we demonstrated that the complex formation of STATs was hampered in the N protein-expressing cells. As a result, STAT nuclear accumulation was reduced, causing a subsequent downregulation of interferon-stimulated genes (ISGs) due to low promoter occupancy by STAT complexes. This novel route for preventing host IFN responses by henipavirus N proteins provides new insight into the pathogenesis of these viruses.IMPORTANCE Paramyxoviruses are well known for suppressing interferon (IFN)-mediated innate immunity with their phosphoprotein (P) gene products, and the henipaviruses also possess P, V, W, and C proteins for evading host antiviral responses. There are numerous studies providing evidence for the relationship between viral pathogenicity and antagonistic activities against IFN responses by P gene products. Meanwhile, little attention has been paid to the influence of nucleoprotein (N) on host innate immune responses. In this study, we demonstrated that both the NiV and HeV N proteins have antagonistic activity against the JAK/STAT signaling pathway by preventing the nucleocytoplasmic trafficking of STAT1 and STAT2. This inhibitory effect is due to an impairment of the ability of STATs to form complexes. These results provide new insight into the involvement of N protein in viral pathogenicity via its IFN antagonism.