Biology subjects

Beard, P. M.

Publications and source records attributed to Beard, P. M..

8 recordsLinked to original sources

bioRxiv2025

Defining the transmission parameters of lumpy skin disease virus by the three model insect vector species Aedes aegypti, Stomoxys calcitrans and Culicoides nubeculosus reveals important differences in their likely role in transmission

Lumpy skin disease virus (LSDV) is a poxvirus that can cause severe, systemic disease in cattle. By far the most important route of transmission of LSDV is mechanical transmission via haematophagous arthropod vectors. However we lack detailed information on this process including the likelihood of transmission by different vector species. This study used an experimental bovine model of LSDV transmission to quantify the transmission of LSDV from an infected donor to a naive recipient calf. Three species of Diptera representing different vector groups were included (Stomoxys calcitrans, Aedes aegypti and Culicoides nubeculosus, respectively a large biting fly, a mosquito and a midge), and the clinical, virological and immunological outcomes in the recipient calves studied. The ability of Ae. aegypti to mechanically transmit LSDV following feeding on an artificial membrane feeding system was also examined. Both Ae. aegypti and S. calcitrans were able to transmit LSDV, resulting in disease in recipient calves. Bites from virus-positive C. nubeculosus did not result in disease in recipient calves, though the presence of neutralising antibodies in these recipients indicated exposure to virus or virus components. Ae. aegypti successfully transmitted LSDV following feeding on LSDV-spiked blood through an artificial membrane feeding system, validating this laboratory model as a future replacement for donor cattle. Mathematical models of the data were generated and predicted S. calcitrans to be the most efficient vector of LSDV of the insects tested with a reproduction number (R0) of 5.8. Impact statementLumpy skin disease virus (LSDV) is a neglected, rapidly emerging pathogen of cattle that has spread into Europe and throughout the Middle East and Asia over the past ten years. Lack of understanding of the mechanism of transmission of LSDV has hampered efforts to control its rapid spread. This study compares the ability of three model species of Diptera (Stomoxys calcitrans, Aedes aegypti and Culicoides nubeculosus) to mechanically transmit the virus to cattle, generating high quality quantitative data to facilitate mathematical modelling of virus transmission. S. calcitrans was identified as a potential driver of LSDV transmission with a R0 of 5.8. This work provides new insights into the vector transmission of LSDV that can be used to design more targeted and effective disease control programmes.

microbiology↗

bioRxiv2023

The mechanical arthropod vector Stomoxys calcitrans influences the outcome of lumpy skin disease virus infection in cattle

The poxvirus lumpy skin disease virus (LSDV) is the etiological agent of lumpy skin disease (LSD), a severe disease of cattle and water buffalo that is characterised by numerous necrotic cutaneous nodules. LSD is a rapidly emerging disease, spreading into and across the Middle East, eastern Europe, and Asia in the past decade. The disease causes substantial production and economic losses in rural communities and affected regions. LSDV is mechanically transmitted by haematophagous arthropods including stable flies (Stomoxys calcitrans), however our understanding of this mechanical transmission method is sparse. A secreted saliva collection methodology using a modified artificial membrane feeding system was optimised for S. calcitrans and used to collect and characterise secreted S. calcitrans saliva. Saliva was mixed with LSDV and shown not to affect virus growth in primary bovine fibroblasts. S. calcitrans saliva or spot-feeding by S. calcitrans was then incorporated into a bovine in vivo experimental model of LSD to determine if either influenced disease pathogenesis. S. calcitrans saliva resulted in fewer animals developing disease, however this difference was not statistically significant. Spot-feeding with S. calcitrans prior to inoculation did not alter the number of animals that developed disease or the overall severity of disease however disease progression was accelerated as demonstrated by the appearance of cutaneous nodules, detection of viral DNA in the blood stream, and production of neutralising antibodies. This shows that S. calcitrans influence disease kinetics through co-incident bite trauma and/or saliva deposition. This increases our understanding of LSDV pathogenesis and highlights the overlooked importance of mechanical vectors in pathogen transmission. Author summaryInsect vectors are important conduits for the transmission of pathogens that cause diseases such as Zika, dengue, malaria, and lumpy skin disease. Biological vector-borne transmission incorporates a replication phase for the pathogen in the insect, whereas no replication occurs in the vector during mechanical transmission. When the insect bites the host it inoculates a pathogen whilst also delivering arthropod-derived factors such as saliva components and causing tissue trauma through biting and probing. Arthropod saliva and/or bite trauma have been shown to enhance the speed and severity of disease following inoculation with a range of biologically transmitted viruses. This study examined if this was true also for the mechanically transmitted pathogen lumpy skin disease virus (LSDV). LSDV is a neglected pathogen that causes severe systemic disease in cattle and is transmitted mechanically by the stable fly Stomoxys calcitrans. Using an experimental bovine model of LSD, we found that disease occurred more rapidly when virus was delivered in association with the bites of uninfected flies. This work has increased our knowledge of lumpy skin disease virus transmission, and the discovery that disease outcome can be impacted by previously overlooked mechanical insect vectors should prompt further investigation into this mechanism of transmission.

microbiology↗

bioRxiv2021

Stem cell-derived macrophages as a new platform for studying host-pathogen interactions in livestock

Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. We explored how pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages from livestock. Porcine and bovine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages. Pig PSCdMs were productively infected by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and African Swine Fever Virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, Pig PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied in parental stem cells, or directly by lentiviral vector transduction. PSCs and differentiated derivatives therefore provide a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in livestock.

immunology↗

bioRxiv2020

Quantifying the acquisition and retention of lumpy skin disease virus by haematophagus insects and the implications for transmission and control.

Lumpy skin disease virus (LSDV), a poxvirus that causes severe disease in cattle, has in the last few years rapidly extended its distribution from Africa and the Middle East into Europe, Russia, and across Asia. LSDV is believed to be primarily spread mechanically by blood-feeding arthropods, however the exact mode of arthropod transmission, the relative ability of different arthropod species to acquire and retain the virus, as well as their comparative importance for LSDV transmission, remain poorly characterised. Since the vector-borne nature of LSDV transmission is believed to have enabled the rapid geographic expansion of this virus, the lack of quantitative evidence on LSDV transmission has impeded effective control of the disease during the current epidemic. Obtaining high quality data on virus transmission by arthropods is challenging, and practical limitations often result in inadequate arthropod numbers or model hosts, limiting the transferability of experimental findings to the natural transmission scenario. We have addressed these limitations in this study. Using a highly representative bovine experimental model of lumpy skin disease we allowed four representative vector species (Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus) to blood-feed on LSDV-inoculated cattle in order to examine the acquisition and retention of LSDV by these species in unprecedented detail. We found the probability of LSDV transmission from clinical cattle to vector correlated with disease severity. Subclinical disease was more common than clinical disease in the inoculated cattle, however the probability of vectors acquiring LSDV from subclinical animals was very low. All four potential vector species studied had a similar rate of acquisition of LSDV after feeding on the host, but Aedes aegypti and Stomoxys calcitrans retained the virus for a longer time, up to 8 days. There was no evidence of virus replication in the vector, consistent with mechanical rather than biological transmission. The parameters obtained in the in-vivo transmission experiments subsequently enabled enhanced modelling approaches to determine the basic reproduction number of LSDV in cattle mediated by each of the insect species. This was highest for Stomoxys calcitrans (19.1), C. nubeculosus (7.4), and Ae. aegypti (2.4), surprisingly indicating these three species are all potentially efficient transmitters of LSDV. These results reveal that currently applied LSDV control measures such as stamping out of all cattle on affected premises or insect control measures targeting single species need to be urgently reconsidered. Overall our studies have highlighted that the combination of highly relevant in-vivo experiments and mathematical modelling can be directly applied to devise evidence-based proportionate and targeted control programmes.

microbiology↗

bioRxiv2019

Identification of a functional small non-coding RNA encoded by African swine fever virus

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of domestic pigs, to which there is no vaccine available. ASFV has a large, double-stranded DNA genome that encodes over 150 proteins. Replication takes place in the cytoplasm of the cell and involves complex interactions with host cellular components including small non-coding RNAs (sncRNAs). A number of DNA viruses are known to manipulate sncRNA either by encoding their own or disrupting host sncRNA. In order to investigate the interplay between ASFV and sncRNAs, study of host and viral small RNAs extracted from ASFV-infected primary porcine macrophages (PAMs) was undertaken. We discovered that ASFV infection had only a modest effect on host miRNAs, with only 6 miRNAs differentially expressed during infection. The data also revealed 3 potential novel small RNAs encoded by ASFV, ASFVsRNA1-3. Further investigation of ASFVsRNA2 detected it in lymphoid tissue from pigs with ASF. Overexpression of ASFVsRNA2 led to up to a 1 log reduction in ASFV growth indicating that ASFV utilises a virally-encoded small RNA to disrupt its own replication. This study describes the modest impact of ASFV on host sncRNAs and the identification of a functional ASFV-encoded sncRNA. ImportanceAfrican swine fever (ASF) poses a major threat to pig populations and food security worldwide. The disease is endemic in Africa and Eastern Europe and rapidly emerging into Asia where it has led to the deaths of millions of pigs in the past 12 months. The development of safe and effective vaccines to protect pigs against ASF has been hindered by lack of understanding of the complex interactions between ASFV and the host cell. We focused our work on characterising the interactions between ASFV and sncRNAs. We found only modest changes to host sncRNA abundance after ASFV infection, and discovered a functional ASFV-encoded sncRNA. The knowledge from this study can be exploited to develop more effective ASFV vaccines that take advantage of the sncRNA system.

molecular biology↗

bioRxiv2019

An alternative AUG codon that produces an N-terminally extended form of the influenza A virus NP is a virulence factor for a swine-derived virus.

The 2009 influenza A virus (IAV) pandemic (pdm2009) was caused by a swine H1N1 virus with several atypical genetic features. Here, we investigate the origin and significance of an upstream AUG (uAUG) codon in the 5-untranslated region of the NP gene. Phylogeny indicated that the uAUG codon arose in the classical swine IAV lineage in the mid 20th Century, and has become fixed in the current triple reassortant, variant pdm2009 swine IAV and human pdm2009 lineages. Functionally, it supports leaky ribosomal initiation in vitro and in vivo to produce two isoforms of NP: canonical, and a longer \"eNP\". The uAUG codon had little effect on viral gene expression or replication in vitro. However, in both murine and porcine models of IAV infection, removing the uAUG codon gene attenuated pdm2009 virus pathogenicity. Thus, the NP uAUG codon is a virulence factor for swine IAVs with proven zoonotic ability.

microbiology↗

bioRxiv2018

Mutation of influenza A virus PA-X decreases pathogenicity in chicken embryos and can increase the yield of reassortant candidate vaccine viruses

The PA-X protein of influenza A virus has roles in host cell shut-off and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression compared with PA-Xs from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X-ORF, had little effect on the infectious virus titre of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology; effects that were more pronounced in the PR8 strain than the T/E reassortant, despite the low shut-off activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of HA to NP and M1 compared to their WT counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and NA segments from H1N1 2009 pandemic viruses or an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbour a function unrelated to host cell shut-off and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.\n\nIMPORTANCEInfluenza A virus is a widespread pathogen that affects both man and a variety of animal species, causing regular epidemics and sporadic pandemics with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans, mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.

microbiology↗

bioRxiv2017

Isolation of a natural DNA virus of Drosophila melanogaster, and characterisation of host resistance and immune responses

Drosophila melanogaster has played a key role in our understanding of invertebrate immunity. However, both functional and evolutionary studies of host-virus interaction in Drosophila have been limited by a dearth of native virus isolates. In particular, despite a long history of virus research, DNA viruses of D. melanogaster have only recently been described, and none have been available for experimental study. Here we report the isolation and comprehensive characterisation of Kallithea virus, a large double-stranded DNA virus, and the first DNA virus to have been reported from wild populations of D. melanogaster. We find that Kallithea virus infection is costly for adult flies, reaching high titres in both sexes and disproportionately reducing survival in males and movement and late fecundity in females. Using the Drosophila Genetic Reference Panel, we quantify host genetic variance for virus-induced mortality and viral titre and identify candidate host genes that may underlie this variation, including Cdc42-interacting protein 4. Using full transcriptome sequencing of infected males and females, we examine the transcriptional response of flies to Kallithea virus infection, and describe differential regulation of virus-responsive genes. This work establishes Kallithea virus as a new tractable model to study the natural interaction between D. melanogaster and DNA viruses, and we hope it will serve as a basis for future studies of immune responses to DNA viruses in insects.\n\nAuthor SummaryThe fruit fly Drosophila melanogaster is a useful model species to study host-virus interaction and innate immunity. However, few natural viruses of Drosophila have been available for experiments, and no natural DNA viruses of Drosophila melanogaster have been available at all. Although infecting flies with viruses from other insects has been useful to uncover general immune mechanisms, viruses that naturally infect wild flies could help us to learn more about the coevolutionary process, and more about the genes that underlie the host-virus interaction. Here we present an isolate of a DNA virus (named Kallithea Virus) that naturally infects the model species Drosophila melanogaster in the wild. We describe the basic biology of infection by this virus, finding that both male and females flies die from infection, but females are more tolerant of infection than males, while laying lay fewer eggs than uninfected females. We quantify genetic variation for virus resistance in the flies, and we use RNA sequencing to see which genes are expressed in male and female flies in response to infection. These results will form the basis for further research to understand how insects defend themselves against infection by DNA viruses, and how DNA viruses can overcome antiviral defence.

microbiology↗