Flying-Fox Roost Disturbance and Hendra Virus Spillover Risk

Bats of the genus Pteropus (flying-foxes) are the natural host of Hendra virus (HeV) which periodically causes fatal disease in horses and humans in Australia. The increased urban presence of flying-foxes often provokes negative community sentiments because of reduced social amenity and concerns of HeV exposure risk, and has resulted in calls for the dispersal of urban flying-fox roosts. However, it has been hypothesised that disturbance of urban roosts may result in a stress-mediated increase in HeV infection in flying-foxes, and an increased spillover risk. We sought to examine the impact of roost modification and dispersal on HeV infection dynamics and cortisol concentration dynamics in flying-foxes. The data were analysed in generalised linear mixed models using restricted maximum likelihood (REML). The difference in mean HeV prevalence in samples collected before (4.9%), during (4.7%) and after (3.4%) roost disturbance was small and non-significant (P = 0.440). Similarly, the difference in mean urine specific gravity-corrected urinary cortisol concentrations was small and non-significant (before = 22.71 ng/mL, during = 27.17, after = 18.39) (P= 0.550). We did find an underlying association between cortisol concentration and season, and cortisol concentration and region, suggesting that other (plausibly biological or environmental) variables play a role in cortisol concentration dynamics. The effect of roost disturbance on cortisol concentration approached statistical significance for region, suggesting that the relationship is not fixed, and plausibly reflecting the nature and timing of disturbance. We also found a small positive statistical association between HeV excretion status and urinary cortisol concentration. Finally, we found that the level of flying-fox distress associated with roost disturbance reflected the nature and timing of the activity, highlighting the need for a ‘best practice’ approach to dispersal or roost modification activities. The findings usefully inform public discussion and policy development in relation to Hendra virus and flying-fox management.     

Routes of Hendra Virus Excretion in Naturally-Infected Flying-Foxes: Implications for Viral Transmission and Spillover Risk

Pteropid bats or flying-foxes (Chiroptera: Pteropodidae) are the natural host of Hendra virus (HeV) which sporadically causes fatal disease in horses and humans in eastern Australia. While there is strong evidence that urine is an important infectious medium that likely drives bat to bat transmission and bat to horse transmission, there is uncertainty about the relative importance of alternative routes of excretion such as nasal and oral secretions, and faeces. Identifying the potential routes of HeV excretion in flying-foxes is important to effectively mitigate equine exposure risk at the bat-horse interface, and in determining transmission rates in host-pathogen models. The aim of this study was to identify the major routes of HeV excretion in naturally infected flying-foxes, and secondarily, to identify between-species variation in excretion prevalence. A total of 2840 flying-foxes from three of the four Australian mainland species (Pteropus alecto, P. poliocephalus and P. scapulatus) were captured and sampled at multiple roost locations in the eastern states of Queensland and New South Wales between 2012 and 2014. A range of biological samples (urine and serum, and urogenital, nasal, oral and rectal swabs) were collected from anaesthetized bats, and tested for HeV RNA using a qRT-PCR assay targeting the M gene. Forty-two P. alecto (n = 1410) had HeV RNA detected in at least one sample, and yielded a total of 78 positive samples, at an overall detection rate of 1.76% across all samples tested in this species (78/4436). The rate of detection, and the amount of viral RNA, was highest in urine samples (>serum, packed haemocytes >faecal >nasal >oral), identifying urine as the most plausible source of infection for flying-foxes and for horses. Detection in a urine sample was more efficient than detection in urogenital swabs, identifying the former as the preferred diagnostic sample. The detection of HeV RNA in serum is consistent with haematogenous spread, and with hypothesised latency and recrudesence in flying-foxes. There were no detections in P. poliocephalus (n = 1168 animals; n = 2958 samples) or P. scapulatus (n = 262 animals; n = 985 samples), suggesting (consistent with other recent studies) that these species are epidemiologically less important than P. alecto in HeV infection dynamics. The study is unprecedented in terms of the individual animal approach, the large sample size, and the use of a molecular assay to directly determine infection status. These features provide a high level of confidence in the veracity of our findings, and a sound basis from which to more precisely target equine risk mitigation strategies.     

Hendra Virus Spillover is a Bimodal System Driven by Climatic Factors

Understanding environmental factors driving spatiotemporal patterns of disease can improve risk mitigation strategies. Hendra virus (HeV), discovered in Australia in 1994, spills over from bats (Pteropus sp.) to horses and thence to humans. Below latitude ? 22°, almost all spillover events to horses occur during winter, and above this latitude spillover is aseasonal. We generated a statistical model of environmental drivers of HeV spillover per month. The model reproduced the spatiotemporal pattern of spillover risk between 1994 and 2015. The model was generated with an ensemble of methods for presence–absence data (boosted regression trees, random forests and logistic regression). Presences were the locations of horse cases, and absences per spatial unit (2.7 × 2.7 km pixels without spillover) were sampled with the horse census of Queensland and New South Wales. The most influential factors indicate that spillover is associated with both cold-dry and wet conditions. Bimodal responses to several variables suggest spillover involves two systems: one above and one below a latitudinal area close to ? 22°. Northern spillovers are associated with cold-dry and wet conditions, and southern with cold-dry conditions. Biologically, these patterns could be driven by immune or behavioural changes in response to food shortage in bats and horse husbandry. Future research should look for differences in these traits between seasons in the two latitudinal regions. Based on the predicted risk patterns by latitude, we recommend enhanced preventive management for horses from March to November below latitude 22° south.     

Landscape Utilisation,Animal Behaviour and Hendra Virus Risk

Hendra virus causes sporadic fatal disease in horses and humans in eastern Australia. Pteropid bats (flying-foxes) are the natural host of the virus. The mode of flying-fox to horse transmission remains unclear, but oro-nasal contact with flying-fox urine, faeces or saliva is the most plausible. We used GPS data logger technology to explore the landscape utilisation of black flying-foxes and horses to gain new insight into equine exposure risk. Flying-fox foraging was repetitious, with individuals returning night after night to the same location. There was a preference for fragmented arboreal landscape and non-native plant species, resulting in increased flying-fox activity around rural infrastructure. Our preliminary equine data logger study identified significant variation between diurnal and nocturnal grazing behaviour that, combined with the observed flying-fox foraging behaviour, could contribute to Hendra virus exposure risk. While we found no significant risk-exposing difference in individual horse movement behaviour in this study, the prospect warrants further investigation, as does the broader role of animal behaviour and landscape utilisation on the transmission dynamics of Hendra virus.     

Climate Change and the Geographic Distribution of Infectious Diseases

Our ability to predict the effects of climate change on the spread of infectious diseases is in its infancy. Numerous, and in some cases conflicting, predictions have been developed, principally based on models of biological processes or mapping of current and historical disease statistics. Current debates on whether climate change, relative to socioeconomic determinants, will be a major influence on human disease distributions are useful to help identify research needs but are probably artificially polarized. We have at least identified many of the critical geophysical constraints, transport opportunities, biotic requirements for some disease systems, and some of the socioeconomic factors that govern the process of migration and establishment of parasites and pathogens. Furthermore, we are beginning to develop a mechanistic understanding of many of these variables at specific sites. Better predictive understanding will emerge in the coming years from analyses regarding how these variables interact with each other.     

Quantitative Risk Assessment of the Pathways by Which West Nile Virus Could Reach Hawaii

The introduction of West Nile virus (WNV) to Hawaii could have severe impacts on human health, wildlife health and, as a result, Hawaiis tourism-based economy. To provide guidance for management agencies seeking to prevent the introduction of WNV, we performed a quantitative assessment of the pathways by which WNV could reach Hawaii from North America. We estimated the rate of infectious individuals reaching Hawaii by the following means (1) humans on aircraft, (2) wind-transported mosquitoes, (3) human-transported mosquitoes, (4) human-transported birds or other vertebrates, and (5) migratory birds. We found that pathways 3 and 4 represented the highest risk. We estimated that each year, 7–70 WNV infectious mosquitoes will reach Hawaii by airplane when WNV becomes well established in the Western U.S. Exemptions in current quarantine regulations will also result in the import of birds that will be infectious with WNV for 0.3–2.2 bird-days each year. We propose actions that would substantially reduce the risk of WNV reaching Hawaii by these means, including reinstating aircraft disinsection in cargo holds and altering bird quarantine rules. Finally, research is urgently needed to determine whether a migratory bird can survive the migration from North America to Hawaii with a viremic WNV infection.     

Climate Change Influences on the Global Potential Distribution of Bluetongue Virus

The geographic distribution of arboviruses has received considerable attention after several dramatic emergence events around the world. Bluetongue virus (BTV) is classified among category “A” diseases notifiable to the World Organization of Animal Health (OIE), and is transmitted among ruminants by biting midges of the genus Culicoides. Here, we developed a comprehensive occurrence data set to map the current distribution, estimate the ecological niche, and explore the future potential distribution of BTV globally using ecological niche modeling and based on diverse future climate scenarios from general circulation models (GCMs) for four representative concentration pathways (RCPs). The broad ecological niche and potential geographic distribution of BTV under present-day conditions reflected the disease’s current distribution across the world in tropical, subtropical, and temperate regions. All model predictions were significantly better than random expectations. As a further evaluation of model robustness, we compared our model predictions to 331 independent records from most recent outbreaks from the Food and Agriculture Organization Emergency Prevention System for Transboundary Animal and Plant Pests and Diseases Information System (EMPRES-i); all were successfully anticipated by the BTV model. Finally, we tested ecological niche similarity among possible vectors and BTV, and could not reject hypotheses of niche similarity. Under future-climate conditions, the potential distribution of BTV was predicted to broaden, especially in central Africa, United States, and western Russia.     

Climate Change Expands the Spatial Extent and Duration of Preferred Thermal Habitat for Lake Superior Fishes

Climate change is expected to alter species distributions and habitat suitability across the globe. Understanding these shifting distributions is critical for adaptive resource management. The role of temperature in fish habitat and energetics is well established and can be used to evaluate climate change effects on habitat distributions and food web interactions. Lake Superior water temperatures are rising rapidly in response to climate change and this is likely influencing species distributions and interactions. We use a three-dimensional hydrodynamic model that captures temperature changes in Lake Superior over the last 3 decades to investigate shifts in habitat size and duration of preferred temperatures for four different fishes. We evaluated habitat changes in two native lake trout (Salvelinus namaycush) ecotypes, siscowet and lean lake trout, Chinook salmon (Oncorhynchus tshawytscha), and walleye (Sander vitreus). Between 1979 and 2006, days with available preferred thermal habitat increased at a mean rate of 6, 7, and 5 days per decade for lean lake trout, Chinook salmon, and walleye, respectively. Siscowet lake trout lost 3 days per decade. Consequently, preferred habitat spatial extents increased at a rate of 579, 495 and 419 km² per year for the lean lake trout, Chinook salmon, and walleye while siscowet lost 161 km² per year during the modeled period. Habitat increases could lead to increased growth and production for three of the four fishes. Consequently, greater habitat overlap may intensify interguild competition and food web interactions. Loss of cold-water habitat for siscowet, having the coldest thermal preference, could forecast potential changes from continued warming. Additionally, continued warming may render more suitable conditions for some invasive species.     

Survival of Hendra Virus in the Environment: Modelling the Effect of Temperature

EcoHealth - Hendra virus (HeV), a highly pathogenic zoonotic paramyxovirus recently emerged from bats, is a major concern to the horse industry in Australia. Previous research has shown that higher...     

Duration of Maternal Antibodies against Canine Distemper Virus and Hendra Virus in Pteropid Bats

Old World frugivorous bats have been identified as natural hosts for emerging zoonotic viruses of significant public health concern, including henipaviruses (Nipah and Hendra virus), Ebola virus, and Marburg virus. Epidemiological studies of these viruses in bats often utilize serology to describe viral dynamics, with particular attention paid to juveniles, whose birth increases the overall susceptibility of the population to a viral outbreak once maternal immunity wanes. However, little is understood about bat immunology, including the duration of maternal antibodies in neonates. Understanding duration of maternally derived immunity is critical for characterizing viral dynamics in bat populations, which may help assess the risk of spillover to humans. We conducted two separate studies of pregnant Pteropus bat species and their offspring to measure the half-life and duration of antibodies to 1) canine distemper virus antigen in vaccinated captive Pteropus hypomelanus; and 2) Hendra virus in wild-caught, naturally infected Pteropus alecto. Both of these pteropid bat species are known reservoirs for henipaviruses. We found that in both species, antibodies were transferred from dam to pup. In P. hypomelanus pups, titers against CDV waned over a mean period of 228.6 days (95% CI: 185.4–271.8) and had a mean terminal phase half-life of 96.0 days (CI 95%: 30.7–299.7). In P. alecto pups, antibodies waned over 255.13 days (95% CI: 221.0–289.3) and had a mean terminal phase half-life of 52.24 days (CI 95%: 33.76–80.83). Each species showed a duration of transferred maternal immunity of between 7.5 and 8.5 months, which was longer than has been previously estimated. These data will allow for more accurate interpretation of age-related Henipavirus serological data collected from wild pteropid bats.     

Australia's Dengue Risk Driven by Human Adaptation to Climate Change

Background

The reduced rainfall in southeast Australia has placed this region''s urban and rural communities on escalating water restrictions, with anthropogenic climate change forecasts suggesting that this drying trend will continue. To mitigate the stress this may place on domestic water supply, governments have encouraged the installation of large domestic water tanks in towns and cities throughout this region. These prospective stable mosquito larval sites create the possibility of the reintroduction of Ae. aegypti from Queensland, where it remains endemic, back into New South Wales and other populated centres in Australia, along with the associated emerging and re-emerging dengue risk if the virus was to be introduced.

Methodology/Principal Findings

Having collated the known distribution of Ae. aegypti in Australia, we built distributional models using a genetic algorithm to project Ae. aegypti''s distribution under today''s climate and under climate change scenarios for 2030 and 2050 and compared the outputs to published theoretical temperature limits. Incongruence identified between the models and theoretical temperature limits highlighted the difficulty of using point occurrence data to study a species whose distribution is mediated more by human activity than by climate. Synthesis of this data with dengue transmission climate limits in Australia derived from historical dengue epidemics suggested that a proliferation of domestic water storage tanks in Australia could result in another range expansion of Ae. aegypti which would present a risk of dengue transmission in most major cities during their warm summer months.

Conclusions/Significance

In the debate of the role climate change will play in the future range of dengue in Australia, we conclude that the increased risk of an Ae. aegypti range expansion in Australia would be due not directly to climate change but rather to human adaptation to the current and forecasted regional drying through the installation of large domestic water storing containers. The expansion of this efficient dengue vector presents both an emerging and re-emerging disease risk to Australia. Therefore, if the installation and maintenance of domestic water storage tanks is not tightly controlled, Ae. aegypti could expand its range again and cohabit with the majority of Australia''s population, presenting a high potential dengue transmission risk during our warm summers.     

Effect of Climate Change on Lyme Disease Risk in North America

An understanding of the influence of climate change on Ixodes scapularis, the main vector of Lyme disease in North America, is a fundamental component in assessing changes in the spatial distribution of human risk for the disease. We used a climate suitability model of I. scapularis to examine the potential effects of global climate change on future Lyme disease risk in North America. A climate-based logistic model was first used to explain the current distribution of I. scapularis in North America. Climate-change scenarios were then applied to extrapolate the model in time and to forecast vector establishment. The spatially modeled relationship between I. scapularis presence and large-scale environmental data generated the current pattern of I. scapularis across North America with an accuracy of 89% (P < 0.0001). Extrapolation of the model revealed a significant expansion of I. scapularis north into Canada with an increase in suitable habitat of 213% by the 2080s. Climate change will also result in a retraction of the vector from the southern U.S. and movement into the central U.S. This report predicts the effect of climate change on Lyme disease risk and specifically forecasts the emergence of a tickborne infectious disease in Canada. Our modeling approach could thus be used to outline where future control strategies and prevention efforts need to be applied.     

Climate Change Could Negate Positive Tree Diversity Effects on Forest Productivity: A Study Across Five Climate Types in Spain and Canada

A positive relationship between tree diversity and forest productivity is reported for many forested biomes of the world. However, whether tree diversity is able to increase the stability of forest growth to changes in climate is still an open question. We addressed this question using 36,378 permanent forest plots from National Forest Inventories of Spain and Québec (Eastern Canada), covering five of the most important climate types where forests grow on Earth and a large temperature and precipitation gradient. The plots were used to compute forest productivity (aboveground woody biomass increment) and functional diversity (based on the functional traits of species). Divergence from normal levels of precipitation (dryer or wetter than 30-year means) and temperature (warmer or colder) were computed for each plot from monthly temperature and precipitation means. Other expected drivers of forest growth were also included. Our results show a significant impact of climate divergences on forest productivity, but not always in the expected direction. Furthermore, although functional trait diversity had a general positive impact on forest productivity under normal conditions, this effect was not maintained in stands having suffered from temperature divergence (i.e., warmer conditions). Contrary to our expectations, we found that tree diversity did not result in more stable forest’s growth conditions during changes in climate. These results could have important implications for the future dynamics and management of mixed forests worldwide under climate change.     

Side Chain Packing below the Fusion Peptide Strongly Modulates Triggering of the Hendra Virus F Protein

Triggering of the Hendra virus fusion (F) protein is required to initiate the conformational changes which drive membrane fusion, but the factors which control triggering remain poorly understood. Mutation of a histidine predicted to lie near the fusion peptide to alanine greatly reduced fusion despite wild-type cell surface expression levels, while asparagine substitution resulted in a moderate restoration in fusion levels. Slowed kinetics of six-helix bundle formation, as judged by sensitivity to heptad repeat B-derived peptides, was observed for all H372 mutants. These data suggest that side chain packing beneath the fusion peptide is an important regulator of Hendra virus F triggering.Hendra virus and Nipah virus are highly pathogenic paramyxoviruses infecting humans. They were identified in 1994 and 1999, respectively, as the etiological agents behind cases of severe encephalitis and respiratory disease in Australia and Malaysia (7, 10, 17-18). Owing to their unusually high virulence, broad host range, and genetic similarity, Hendra virus and Nipah virus (NiV) have been classified into the new genus Henipavirus (31). Henipavirus membrane fusion requires the concerted effort of two viral surface glycoproteins (3-4, 30): the attachment protein (G), which binds receptor, and the fusion (F) protein, which drives membrane merger through vast conformational changes. Paramyxovirus F proteins are synthesized as inactive F₀ precursors which are subsequently cleaved into fusogenic disulfide-linked heterodimers, F₁+F₂. Despite a conserved requirement for cleavage, protease usage varies among paramyxoviruses, with henipavirus F being cleaved by the endosomal/lysosomal cysteine protease cathepsin L (19-20). This cleavage event positions the fusion peptide (FP) at the newly created N terminus and acts to prime the F protein. Following cleavage, the primed F protein must be triggered to begin the sequence of conformational changes required for membrane fusion. Like most F proteins, triggering of the henipavirus F proteins likely involves the henipavirus attachment proteins, though the mechanism remains poorly understood (reviewed in reference 28). F triggering facilitates refolding and extension of heptad repeat A (HRA) toward the target cell membrane, resulting in FP insertion into the bilayer (2). Further rearrangement brings HRA and HRB into close proximity, resulting in the formation of a stable six-helix bundle and culminating in a fully formed fusion pore (reviewed in reference 32).Cathepsin L cleavage of F does not require specific residues upstream of or at the cleavage site (K109) itself (5, 16), and the mechanism by which cathepsin L recognizes and specifically cleaves F is unclear. Modeling of the Hendra virus F amino acid sequence onto the prefusion structure of parainfluenza virus 5 (PIV5) (34) indicates that two of the three ectodomain histidine residues (H102 and H372) are positioned near the cathepsin L cleavage site following residue K109 (Fig. ​(Fig.11 A). In the monomer, H372 is located distally from K109, yet trimerization places H372 from one monomer directly beneath the FP and cleavage site of the neighboring monomer (Fig. ​(Fig.1A,1A, inset). We hypothesized that protonation of histidine residues could cause local conformational changes, potentially modulating cathepsin L cleavage, though these hypothesized conformational changes would not be due to direct modulation of K109 interactions since the predicted distances from K109 to either H102 or H372 are 12 Å and 27 Å, respectively (α-carbon to α-carbon distances). To test the role of H102 and H372 in cathepsin L cleavage, each was mutated individually or together to alanine (A) or asparagine (N), which has a side chain volume similar to that of histidine. Surface expression levels and cleavage of wild-type (WT) and mutant F proteins were examined by cell surface biotinylation as previously described (8). All mutant F proteins were surface expressed and cleaved, though levels of the H102A/H372A (AA) and H102N/H372N (NN) proteins were decreased compared to those of the wild type (Fig. ​(Fig.1B).1B). To examine cleavage kinetics, Vero cells transiently transfected with wild-type or mutant pCAGGS-Hendra virus F were metabolically labeled for 30 min and chased for 0 to 24 h. Band density corresponding to F₀ and F₁ was quantitated, and percent cleavage was defined as the density of F₁/(F₁+F₀). Cleavage kinetics of all Hendra virus F mutants were not significantly different from wild-type levels (Fig. ​(Fig.2).2). These data suggest that protonation of histidines in the region of the cleavage site is not involved in cathepsin L processing of Hendra virus F.Open in a separate windowFIG. 1.Structural modeling and surface expression of Hendra virus F H102 and H372 mutants. (A) Homology model of the Hendra virus F monomer based on the crystal structure of PIV5 F, shown as a ribbon diagram (image generated using PyMOL; Delano Scientific [www.pymol.org]): red, H102 and H372; blue, fusion peptide (FP); green, P1 cleavage site residue K109. The locations of H102 and H372 in the trimeric protein are shown in the box insert using the same color scheme except with an additional monomer shown in teal. (B) Surface expression of transiently transfected wild-type and Hendra virus F mutants in Vero cells following metabolic labeling (3 h). Surface proteins were biotinylated prior to immunoprecipitation, and the total and surface populations were separated by streptavidin pull-down. Proteins were analyzed via 15% SDS-PAGE and visualized using autoradiography. F₁ band quantitation via densitometry is shown normalized to wild-type levels plus or minus one standard deviation. The surface expression levels represent the averages of data from three independent experiments, with one representative gel shown.Open in a separate windowFIG. 2.Total protein cleavage time points for WT and mutant F protein. Vero cells transiently transfected with wild-type or mutant Hendra virus F were metabolically labeled (3 h) and chased for the indicated times. The total protein population was immunoprecipitated and analyzed by 15% SDS-PAGE and autoradiography. Band intensity was quantitated using the ImageQuant 5.2 software program (GE Healthcare, Piscataway, NJ), and percent cleavage was defined as the intensity of F₁/(F₁+F₀). Error bars are plus or minus one standard deviation and represent the average of data from three independent experiments.Since the mutants were expressed on the cell surface in the mature, cleaved form, fusion was examined using a syncytium assay (Fig. ​(Fig.33 A). Mutations at H102 did not significantly alter syncytium formation, but large reductions in fusion were observed for F proteins containing an H372 mutation. To quantitatively analyze fusion, a reporter gene assay was utilized. Since the single mutations all resulted in increased surface density (Fig. ​(Fig.1B)1B) while decreases were observed for the double mutants, analysis of the effects of surface density on WT Hendra virus F fusion was first performed. Previous work with other class I viral fusion proteins, including PIV5 F and influenza virus hemagglutinin (HA), has shown that surface density correlates with the final extent of fusion over a range of densities (6). Aguilar et al. (1) demonstrated that increasing the amount of NiV G and NiV F DNA transfected results in an increase in syncytium formation. However, a direct correlation between F surface expression and fusion has not been previously reported for henipaviruses. To assess this, Vero cells were transfected with various amounts of wild-type pCAGGS-Hendra virus F and biotinylated as described previously (8); reporter gene analyses using the same DNA amounts were performed alongside the biotinylation experiments. Increased surface densities clearly led to increases in fusion, though the correlation was not completely linear (Fig. ​(Fig.3B).3B). These data were then utilized to generate a percent WT fusion level for each mutant normalized for the observed cell surface expression levels. Mutations at H102 did not significantly alter fusion (Fig. ​(Fig.3C).3C). However, cell-cell fusion was dramatically reduced (10% to 20% of wild-type values) with the H372A and AA mutant F proteins. A partial restoration in fusion was seen for the H372N and NN mutants, suggesting that side chain volume plays a role; however, fusion levels were only 30% to 60% of those of the wild type (Fig. ​(Fig.3C).3C). While histidine residues proximal to the influenza virus HA fusion peptide have been shown to regulate low-pH triggering, Hendra virus F-mediated fusion occurs at neutral pH, and incubation at low pH has no effect on fusogenicity (A. Chang and R. E. Dutch, unpublished results). These data indicate that mutations at H372 result in a hypofusogenic protein, suggesting that side chain packing within this region may strongly modulate F protein triggering, potentially by altering protein stability.Open in a separate windowFIG. 3.Syncytium assay, reporter gene analysis, and correlation of wild-type and mutant F protein surface expression versus fusion activity. (A) Representative syncytium images from three independent experiments for wild-type and mutant Hendra virus F proteins. Vero cells transiently transfected with wild-type pCAGGS-Hendra virus G and wild-type or mutant pCAGGS-Hendra virus F were kept at 37°C for 24 to 48 h posttransfection, and photographs were taken using a Nikon digital camera mounted atop a Nikon TS100 microscope with a 10× objective. (B) Correlation between surface expression and fusogenicity for wild-type Hendra virus F. Vero cells transiently transfected with various amounts of wild-type Hendra virus F DNA were biotinylated, and reporter gene analysis was performed simultaneously using the same DNA quantities. (C) Reporter gene analysis of equal μg of wild-type or mutant Hendra virus F in pCAGGS normalized to average cell surface expression levels. Vero cells transiently transfected with wild-type Hendra virus G, wild-type or mutant Hendra virus F, and T7 luciferase were overlaid with BSR cells 18 h posttransfection, lysed, and assayed for luciferase activity (n = 5 to 8; ±95% confidence interval [CI]).The hypofusogenic phenotype of the H372 mutants could be explained by changes in the stability of the prefusion form following cleavage, resulting in altered fusion kinetics. To examine fusion kinetics, sensitivity over time to peptides which block formation of the postfusion six-helix bundle was examined (NiV C2; corresponding to HRB of the highly homologous Nipah virus F protein; generously provided by Chris Broder [Uniformed Services University of the Health Sciences]). One hundred nM NiV C2 has been shown to inhibit Henipavirus F-mediated cell-cell fusion (4). Similar peptides inhibit many other class I fusion proteins (11-13, 23, 33, 35-36). Vero cells transfected with wild-type pCAGGS-Hendra virus F and G and T7 luciferase were overlaid with target BSR cells on ice for 1 h. Prewarmed Dulbecco''s modified Eagle medium (DMEM) was added to initiate fusion, and at the indicated time points, DMEM containing 100 nM NiV C2 or 100 nM NiV SC (scrambled control peptide) was added. Cells were kept at 37°C for 3 h, and luciferase activity was assayed (Fig. ​(Fig.44 A). Cell-cell fusion kinetics for the wild-type Hendra virus F protein showed a steep increase in membrane fusion events from the 5- to 20-min time points (Fig. ​(Fig.4B,4B, solid line). Approximately 50% of cell-cell fusion events became insensitive to the addition of NiV C2 by 30 min, with the majority of membrane fusion events complete by 60 min (Fig. ​(Fig.4B,4B, solid line). Fusion kinetics for the H102A and H102N proteins closely resembled wild-type kinetics, consistent with overall fusion levels (Fig. 4B and C). In contrast, cell-cell fusion was dramatically slowed for F proteins containing mutations at H372. For all H372 mutants, no fusion was observed during the first 30 min, in stark contrast to results for the wild type. After 30 min, fusion was observed for the H372N and NN proteins, which reach 20 to 40% of maximal fusion within 60 min (Fig. ​(Fig.4C).4C). The small amount of fusion observed for the H372A protein occurred long after fusion was complete for the wild-type protein. Combined, these data demonstrate that mutation of H372 to either alanine or asparagine decreases the initial rate of membrane fusion potentially by increasing the energetic barrier for Hendra virus F triggering.Open in a separate windowFIG. 4.Cell-cell reporter gene fusion kinetics for wild-type and mutant Hendra virus F proteins. (A) Diagram of the experimental setup: a, binding of BSR cells; b, addition of DMEM, NiV-SC peptide, or NiV-C2 peptides (0-min time point); c, addition of NiV-C2 at indicated time points; d, continued incubation of cells. (B and C) Cell-cell fusion kinetics for wild-type and mutant F proteins. Reporter gene analysis was performed as described above following binding of BSR cells at 4°C, addition of either DMEM, NiV-SC, or NiV-C2 at the indicated times, and continued incubation for 3 h at 37°C. Percent maximal fusion is defined as the amount of fusion which occurred during a given time point as a fraction of membrane fusion for a given construct over the duration of the experiment (3 h) in the absence of any peptide. Error bars are 95% confidence intervals (n = 3 to 6).While most paramyxovirus F proteins, including the Hendra virus F protein, are thought to be triggered by specific interactions with a homotypic attachment protein (reviewed in reference 28), mutations within paramyxovirus F proteins which alter stability of the prefusion form (8, 15, 21-22, 27) can also strongly modulate triggering. H372 is modeled to be near a conserved domain, termed CBF₁, in the Hendra virus F prefusion structure. Studies suggest that CBF₁, which is structurally composed of β-sheets, is important for F protein folding (9), likely playing a critical role in stabilization of the fusion peptide following proteolytic cleavage, with the CBF₁ domain from one monomer interacting with the fusion peptide from the neighboring monomer. Mutations in CBF₁ in Hendra virus F resulted in folding defects which could not be rescued at decreased temperatures. Given the proximity of H372 to CBF₁ in Hendra virus F, changes in side chain packing could stabilize the fusion peptide following cleavage and thus decrease the ability of the protein to trigger efficiently. In the model structure, H372 is predicted to be surrounded by polar and nonpolar residues (within 5 Å of the side chain), including I425, N423, Q342, F376, and two FP residues, A125 and T129. Extension out to 10 Å reveals that H372 is also near four additional FP residues (A126, I128, T129, and V132), suggesting that substitution of H372 with a smaller residue (H372A) could alter the packing depth of the more C-terminal portion of the FP following cleavage.Studies from other systems also implicate the fusion peptide and surrounding residues as regulators of F-promoted membrane fusion (14, 24, 26, 29). The paramyxovirus fusion peptide is an important regulator of triggering, since conserved glycine residues within the FP have been shown to play a role in regulation and activation of F (26). Numerous mutations within the fusion peptide pocket of H5N1 influenza virus HA were shown to regulate the pH needed for HA activation (25), with only one mutation causing significant changes to HA expression or cleavage. Similar experiments using the H3 subtype of influenza virus HA also demonstrated changes in pH requirements upon mutation of certain fusion peptide-proximal residues (29). While influenza virus HA requires low pH for fusion promotion, the data presented here show that regulation of interactions with and around the fusion peptide is also critically important for triggering and fusion promotion of neutral-pH fusing systems. Thus, the decrease in the rate of triggering observed for the H372A mutant is consistent with a model in which residues surrounding the fusion peptide can act to regulate F-mediated fusion promotion independently of large changes in protein expression or cleavage.Our data, therefore, strongly indicate that side chain packing near the fusion peptide (Fig. ​(Fig.1A,1A, inset) is a strong modulator of Hendra virus F triggering, with a dramatic slowing in the rate of six-helix bundle formation observed when H372 is replaced with residues with smaller side chain volumes (Fig. ​(Fig.4).4). Modulation of side chain packing proximal to the FP could change the positioning of paramyxovirus FPs, thus altering the kinetics and efficiency of later conformational changes. Mutation of H372 may well stabilize interactions of the FP with the ectodomain following cleavage and thus affect triggering by substantially increasing the energy needed to project the FP toward the target cell membrane. Together, these data suggest a model by which packing around the fusion peptide affects both the rate and extent of F triggering.     

Dimeric Architecture of the Hendra Virus Attachment Glycoprotein: Evidence for a Conserved Mode of Assembly

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed β-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to α-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60°) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses.The zoonotic Hendra virus (HeV) uses the flying fox as a natural host and is highly virulent in humans and horses. Reported cases of HeV transmission in humans have, to date, been sporadic and restricted to Australia (23, 32, 42). HeV was originally discovered in 1994 following the infection and subsequent death of a horse trainer and numerous horses (42, 49). Infection was characterized by the swift onset (7 to 10 days) of acute respiratory disease with clinical symptoms including fever, dizziness, hypotension, and respiratory illness. HeV is closely related to Nipah virus (NiV) which, in its largest outbreak, was responsible for the death of approximately 105 people in Malaysia (46). HeV and NiV (referred to as HNV) collectively form the Henipavirus genus which belongs to the Paramyxoviridae family of single-stranded, negative-sense RNA viruses. Putative new members of this genus have been recently identified in African bats (19).HeV contains attachment (HeV-G) and fusion (HeV-F) membrane glycoproteins which extend from the viral envelope and are required for efficient entry (20). HeV and NiV exhibit a broad tissue tropism which correlates with the use of the widely expressed cell surface glycoproteins ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as functional receptors for HeV-G and NiV-G (referred to as HNV-G). Oligomerization of HeV-G is also critical for productive attachment (1, 4). Despite the shared underlying molecular architecture of the attachment glycoprotein across the paramyxoviruses, there is marked divergence in their cellular receptors. Newcastle disease virus (NDV) and parainfluenza viruses (PIVs) attach to cell surface sialic acid (neuraminic acid), a negatively charged nine-carbon saccharide that is located at the nonreducing termini of glycolipids and N- and O-linked glycans of glycoproteins. In contrast, morbilliviruses such as measles virus (MV), canine distemper virus, and rinderpest virus have been shown to require surface lymphocyte activation molecule (SLAM; CD150) (51, 52) or the complement regulator CD46 for viral attachment (18, 44, 48). Structure-based phylogenetic analysis of available paramyxovirus structures shows that NiV-G and HeV-G are structurally more similar to attachment glycoproteins of sialic acid-binding viruses such as NDV and PIVs than to that of MV (7). We have suggested that the protein-binding capacities of henipaviruses and morbilliviruses have evolved independently, indicating an innate propensity for viruses to acquire novel protein receptor specificity, a characteristic that presents a natural route for the emergence of new pathogenic viruses (7).HeV-G and NiV-G are closely related by sequence (81% identity) and structure (7). This similarity is underscored by the observation that vaccination of cats with recombinant HeV-G can protect against challenge with NiV (39). The similarity is evident in the crystal structures of NiV-G and HeV-G in complex with EFNB2, which both display a common binding mode (7). Knowledge of the molecular determinants of HeV attachment and fusion is of value for the rational development of immunotherapeutic and antiviral reagents with which to target these viruses. Such a structure-based approach to drug design has led to the development of active antivirals against influenza (2). However, in contrast to the rigid carbohydrate binding cleft of influenza neuraminidase, crystallographic analysis of the unbound NiV-G revealed significant plasticity in many of the EFNB2-binding loops which extend from the β-propeller scaffold (10). This information redefines the structural template for rational antiviral drug design against the HNV-G family of viral glycoproteins. Here, we have sought to further refine this template by identifying structural characteristics of HeV attachment.We have crystallized and solved the structure of the globular six-bladed β-propeller domain of HeV-G. Analysis of this structure reveals that, as previously observed for the closely related NiV-G, significant conformational changes occur in the EFNB2 and EFNB3 receptor binding loops between unliganded and receptor-bound structures (10). Additionally, based on the packing of identical HeV-G molecules in the crystal asymmetric unit, we provide a model for the dimeric component of the native oligomeric assembly of the attachment glycoprotein. This model is consistent with oligomeric structures from other paramyxovirus attachment glycoproteins, and we propose that it may represent a component of the biological assembly. Furthermore, we observe that the angle of association between monomeric subunits is much more conserved between glycan-binding hemagglutinin-neuraminidases (HN) than the protein-binding HeV and MV attachment glycoproteins. As a result, we suggest that the acquisition of protein-binding functionality by paramyxoviruses may require structural adaptation, which perturbs the conserved dimeric packing. Together these data support the hypothesis of a globally conserved mode of oligomerization among the paramyxoviruses.