In vivo delivery of bovine viral diahorrea virus, E2 protein using hollow mesoporous silica nanoparticles

Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 [small mu ]g Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 [small mu ]g dose of E2 adsorbed to 250 [small mu ]g HMSA was compared to immunisation with Opti-E2 (50 [small mu ]g) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 [small mu ]g). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine.

Flying-Fox Species Density-A Spatial Risk Factor for Hendra Virus Infection in Horses in Eastern Australia

Hendra virus causes sporadic but typically fatal infection in horses and humans in eastern Australia. Fruit-bats of the genus Pteropus (commonly known as flying-foxes) are the natural host of the virus, and the putative source of infection in horses; infected horses are the source of human infection. Effective treatment is lacking in both horses and humans, and notwithstanding the recent availability of a vaccine for horses, exposure risk mitigation remains an important infection control strategy. This study sought to inform risk mitigation by identifying spatial and environmental risk factors for equine infection using multiple analytical approaches to investigate the relationship between plausible variables and reported Hendra virus infection in horses. Spatial autocorrelation (Global Moran’s I) showed significant clustering of equine cases at a distance of 40 km, a distance consistent with the foraging ‘footprint’ of a flying-fox roost, suggesting the latter as a biologically plausible basis for the clustering. Getis-Ord Gi* analysis identified multiple equine infection hot spots along the eastern Australia coast from far north Queensland to central New South Wales, with the largest extending for nearly 300 km from southern Queensland to northern New South Wales. Geographically weighted regression (GWR) showed the density of P. alecto and P. conspicillatus to have the strongest positive correlation with equine case locations, suggesting these species are more likely a source of infection of Hendra virus for horses than P. poliocephalus or P. scapulatus. The density of horses, climate variables and vegetation variables were not found to be a significant risk factors, but the residuals from the GWR suggest that additional unidentified risk factors exist at the property level. Further investigations and comparisons between case and control properties are needed to identify these local risk factors.

Applications of a sugar-based surveillance system to track arboviruses in wild mosquito populations

Effective arbovirus surveillance is essential to ensure the implementation of control strategies, such as mosquito suppression, vaccination, or dissemination of public warnings. Traditional strategies employed for arbovirus surveillance, such as detection of virus or virus-specific antibodies in sentinel animals, or detection of virus in hematophagous arthropods, have limitations as an early-warning system. A system was recently developed that involves collecting mosquitoes in CO2-baited traps, where the insects expectorate virus on sugar-baited nucleic acid preservation cards. The cards are then submitted for virus detection using molecular assays. We report the application of this system for detecting flaviviruses and alphaviruses in wild mosquito populations in northern Australia. This study was the first to employ nonpowered passive box traps (PBTs) that were designed to house cards baited with honey as the sugar source. Overall, 20/144 (13.9%) of PBTs from different weeks contained at least one virus-positive card. West Nile virus Kunjin subtype (WNVKUN), Ross River virus (RRV), and Barmah Forest virus (BFV) were detected, being identified in 13/20, 5/20, and 2/20 of positive PBTs, respectively. Importantly, sentinel chickens deployed to detect flavivirus activity did not seroconvert at two Northern Territory sites where four PBTs yielded WNVKUN. Sufficient WNVKUN and RRV RNA was expectorated onto some of the honey-soaked cards to provide a template for gene sequencing, enhancing the utility of the sugar-bait surveillance system for investigating the ecology, emergence, and movement of arboviruses. © 2014, Mary Ann Liebert, Inc.

Cotton bunchy top virus and other relatives

Cotton bunchy top virus (CBTV) and the related Cotton leafroll dwarf virus (CLRDV) have caused sporadic disease outbreaks in most cotton regions of the world. Until recently, little was known about the diversity of CBTV or its natural host range. Seven natural field hosts and one experimental host of CBTV have now been identified. These include cotton, Malva parviflora (Marshmallow weed), Abutilon theophrasti (Velvetleaf), Anoda cristata (Spurred anoda), Hibiscus sabdariffa (Rosella), Sida rhombifolia (Paddy’s lucerne), Chamaesyce hirta (Asthma plant) and Gossypium australe. These are currently the only eight known hosts of CBTV. However the virus may have a wider host range than originally thought and include further non-Malvaceae species like asthma plant (family Euphorbiaceae). There are two distinct strains of CBTV in Australia, -A and -B, which have been detected in cotton from numerous locations across almost all growing regions. From 105 samples of cotton that have been positive for CBTV, 6 were infections of strain A only, 60 were strain B only and 64 were a mixed infection of strains A and B. These results indicate the symptoms of cotton bunchy top disease are closely associated with the presence of strain CBTV-B. A diagnostic assay for Cotton leafroll dwarf virus (CLRDV - cotton blue disease) is being developed and applied successfully for the detection of CLRDV samples from Brazil and Thailand. This is the first confirmation of CLRDV from SE-Asia, which may pose an increased biosecurity threat to the Australian industry.

Number and proportion of selenocucleosides in the transfer RNA of escherichia-coli

tRNA isolated from escherichia-coli grown in a medium containing [75Se] sodium selenosulfate was converted to nucleosides and analysed for selenonucleosides on a phosphocellulose column. Upon chromatography of the nucleosides on phosphocellulose column, the radioactivity resolved into three peaks. The first peak consisted of free selenium and traces of undigested nucleotides. The second peak was identified as 4-selenouridine by co-chromatographing with an authentic sample of 4-selenouridine. The identity of the third peak was not established. The second and third peaks represented 93% and 7% of the selenium present in nucleosides respectively.

Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein

Bovine Viral Diarrhoea Virus (BVDV) is widely distributed in cattle industries and causes significant economic losses worldwide annually. A limiting factor in the development of subunit vaccines for BVDV is the need to elicit both antibody and T-cell-mediated immunity as well as addressing the toxicity of adjuvants. In this study, we have prepared novel silica vesicles (SV) as the new generation antigen carriers and adjuvants. With small particle size of 50 nm, thin wall (similar to 6 nm), large cavity (similar to 40 nm) and large entrance size (5.9 nm for SV-100 and 16 nm for SV-140), the SV showed high loading capacity (similar to 250 mu g/mg) and controlled release of codon-optimised E2 (oE2) protein, a major immunogenic determinant of BVDV. The in vivo functionality of the system was validated in mice immunisation trials comparing oE2 plus Quil A (50 mu g of oE2 plus 10 mu g of Quil A, a conventional adjuvant) to the oE2/SV-140 (50 mu g of oE2 adsorbed to 250 mu g of SV-140) or oE2/SV-140 together with 10 mu g of Quil A. Compared to the oE2 plus Quil A, which generated BVDV specific antibody responses at a titre of 10(4), the oE2/SV-140 group induced a 10 times higher antibody response. In addition, the cell-mediated response, which is essential to recognise and eliminate the invading pathogens, was also found to be higher [1954-2628 spot forming units (SFU)/million cells] in mice immunised with oE2/SV-140 in comparison to oE2 plus Quil A (512-1369 SFU/million cells). Our study has demonstrated that SV can be used as the next-generation nanocarriers and adjuvants for enhanced veterinary vaccine delivery. (C) 2014 Elsevier Ltd. All rights reserved.

Ecological dynamics of emerging bat virus spillover

Viruses that originate in bats may be the most notorious emerging zoonoses that spill over from wildlife into domestic animals and humans. Understanding how these infections filter through ecological systems to cause disease in humans is of profound importance to public health. Transmission of viruses from bats to humans requires a hierarchy of enabling conditions that connect the distribution of reservoir hosts, viral infection within these hosts, and exposure and susceptibility of recipient hosts. For many emerging bat viruses, spillover also requires viral shedding from bats, and survival of the virus in the environment. Focusing on Hendra virus, but also addressing Nipah virus, Ebola virus, Marburg virus and coronaviruses, we delineate this cross-species spillover dynamic from the within-host processes that drive virus excretion to land-use changes that increase interaction among species. We describe how land-use changes may affect co-occurrence and contact between bats and recipient hosts. Two hypotheses may explain temporal and spatial pulses of virus shedding in bat populations: episodic shedding from persistently infected bats or transient epidemics that occur as virus is transmitted among bat populations. Management of livestock also may affect the probability of exposure and disease. Interventions to decrease the probability of virus spillover can be implemented at multiple levels from targeting the reservoir host to managing recipient host exposure and susceptibility.

Increased virulence of rabbit haemorrhagic disease virus associated with genetic resistance in wild Australian rabbits (Oryctolagus cuniculus)

The release of myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV) in Australia with the aim of controlling overabundant rabbits has provided a unique opportunity to study the initial spread and establishment of emerging pathogens, as well as their co-evolution with their mammalian hosts. In contrast to MYXV, which attenuated shortly after its introduction, rapid attenuation of RHDV has not been observed. By studying the change in virulence of recent field isolates at a single field site we show, for the first time, that RHDV virulence has increased through time, likely because of selection to overcome developing genetic resistance in Australian wild rabbits. High virulence also appears to be favoured as rabbit carcasses, rather than diseased animals, are the likely source of mechanical insect transmission. These findings not only help elucidate the co-evolutionary interaction between rabbits and RHDV, but reveal some of the key factors shaping virulence evolution.

Molecular diversity of Chickpea chlorotic dwarf virus in Sudan: High rates of intra-species recombination – a driving force in the emergence of new strains

In Sudan Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus, family Geminiviridae) is an important pathogen of pulses that are grown both for local consumption, and for export. Although a few studies have characterised CpCDV genomes from countries in the Middle East, Africa and the Indian subcontinent, little is known about CpCDV diversity in any of the major chickpea production areas in these regions. Here we analyse the diversity of 146 CpCDV isolates characterised from pulses collected across the chickpea growing regions of Sudan. Although we find that seven of the twelve known CpCDV strains are present within the country, strain CpCDV-H alone accounted for ∼73% of the infections analysed. Additionally we identified four new strains (CpCDV-M, -N, -O and -P) and show that recombination has played a significant role in the diversification of CpCDV, at least in this region. Accounting for observed recombination events, we use the large amounts of data generated here to compare patterns of natural selection within protein coding regions of CpCDV and other dicot-infecting mastrevirus species.

Number and proportion of selenocucleosides in the transfer RNA of Image Image

tRNA isolated from . grown in a medium containing [75Se] sodium selenosulfate was converted to nucleosides and analysed for selenonucleosides on a phosphocellulose column. Upon chromatography of the nucleosides on phosphocellulose column, the radioactivity resolved into three peaks. The first peak consisted of free selenium and traces of undigested nucleotides. The second peak was identified as 4-selenouridine by co-chromatographing with an authentic sample of 4-selenouridine. The identity of the third peak was not established. The second and third peaks represented 93% and 7% of the selenium present in nucleosides respectively.

Ecological dynamics of emerging bat virus spillover

Viruses that originate in bats may be the most notorious emerging zoonoses that spill over from wildlife into domestic animals and humans. Understanding how these infections filter through ecological systems to cause disease in humans is of profound importance to public health. Transmission of viruses from bats to humans requires a hierarchy of enabling conditions that connect the distribution of reservoir hosts, viral infection within these hosts, and exposure and susceptibility of recipient hosts. For many emerging bat viruses, spillover also requires viral shedding from bats, and survival of the virus in the environment. Focusing on Hendra virus, but also addressing Nipah virus, Ebola virus, Marburg virus and coronaviruses, we delineate this cross-species spillover dynamic from the within-host processes that drive virus excretion to land-use changes that increase interaction among species. We describe how land-use changes may affect co-occurrence and contact between bats and recipient hosts. Two hypotheses may explain temporal and spatial pulses of virus shedding in bat populations: episodic shedding from persistently infected bats or transient epidemics that occur as virus is transmitted among bat populations. Management of livestock also may affect the probability of exposure and disease. Interventions to decrease the probability of virus spillover can be implemented at multiple levels from targeting the reservoir host to managing recipient host exposure and susceptibility.

Efficacy of acibenzolar-S-methyl (Bion®) treatment of Australian commercial passionfruit, Passiflora edulis f. sp. flavicarpa, on resistance to Passionfruit woodiness virus (PWV) and activities of chitinase & β-1,3-glucanase

This greenhouse study investigated the efficacy of acibenzolar-S-methyl (Bion®) treatment of lower leaves of passionfruit, (Passiflora edulis f. sp. flavicarpa), on Passionfruit woodiness disease and activities of two pathogenesis-related proteins, chitinase and β-1,3-glucanase after inoculation with passionfruit woodiness virus (PWV). All Bion® concentrations reduced disease symptoms, but the concentration of 0.025 g active ingredient (a.i.)/l was the most effective, reducing disease severity in systemic leaves by 23, 29 and 30 compared with water-treated controls at 30, 40 and 50 days post inoculation (dpi) with PWV, respectively. Correspondingly, relative virus concentration as determined by DAS-ELISA in the upper, untreated leaves (new growth) above the site of inoculation at 50 dpi was reduced by 17 and 22 in plants treated with 0.025 and 0.05 g a.i./l, respectively. Bion® treatment and subsequent inoculation with PWV increased chitinase and β-1,3-glucanase activities in the new leaves above the site of inoculation at 30 dpi with PWV. It was concluded that optimal protective Bion® treatment concentrations were 0.025 and 0.05 g a.i./l.

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

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 temperatures led to lower virus survival rates in the laboratory. We develop a model of survival of HeV in the environment as influenced by temperature. We used 20 years of daily temperature at six locations spanning the geographic range of reported HeV incidents to simulate the temporal and spatial impacts of temperature on HeV survival. At any location, simulated virus survival was greater in winter than in summer, and in any month of the year, survival was higher in higher latitudes. At any location, year-to-year variation in virus survival 24 h post-excretion was substantial and was as large as the difference between locations. Survival was higher in microhabitats with lower than ambient temperature, and when environmental exposure was shorter. The within-year pattern of virus survival mirrored the cumulative within-year occurrence of reported HeV cases, although there were no overall differences in survival in HeV case years and non-case years. The model examines the effect of temperature in isolation; actual virus survivability will reflect the effect of additional environmental factors

Evaluation of potential candidate genes involved in salinity tolerance in striped catfish (Pangasianodon hypophthalmus) using an RNA-Seq approach

Increasing salinity levels in freshwater and coastal environments caused by sea level rise linked to climate change is now recognized to be a major factor that can impact fish growth negatively, especially for freshwater teleost species. Striped catfish (Pangasianodon hypophthalmus) is an important freshwater teleost that is now widely farmed across the Mekong River Delta in Vietnam. Understanding the basis for tolerance and adaptation to raised environmental salinity conditions can assist the regional culture industry to mitigate predicted impacts of climate change across this region. Attempt of next generation sequencing using the ion proton platform results in more than 174 million raw reads from three tissue libraries (gill, kidney and intestine). Reads were filtered and de novo assembled using a variety of assemblers and then clustered together to generate a combined reference transcriptome. Downstream analysis resulted in a final reference transcriptome that contained 60,585 transcripts with an N50 of 683 bp. This resource was further annotated using a variety of bioinformatics databases, followed by differential gene expression analysis that resulted in 3062 transcripts that were differentially expressed in catfish samples raised under two experimental conditions (0 and 15 ppt). A number of transcripts with a potential role in salinity tolerance were then classified into six different functional gene categories based on their gene ontology assignments. These included; energy metabolism, ion transportation, detoxification, signal transduction, structural organization and detoxification. Finally, we combined the data on functional salinity tolerance genes into a hypothetical schematic model that attempted to describe potential relationships and interactions among target genes to explain the molecular pathways that control adaptive salinity responses in P. hypophthalmus. Our results indicate that P. hypophthalmus exhibit predictable plastic regulatory responses to elevated salinity by means of characteristic gene expression patterns, providing numerous candidate genes for future investigations.

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.