Area 1. Dispersal and spatial ecology of large biting flies (years 1 and 2) Experiments will be undertaken to quantify the dispersal potential of LBFs in the UK. Initial site LBF trapping surveys and fauna characterization, including data from sites used under SE4115 to inform selection, will be used to select suitable sites for dispersal trials as well as examining factors that determine local abundance and diversity of LBFs across years. Dispersal trials will validate established and novel techniques to determine the distances moved by LBFs between larval habitats, hosts and holdings. The dispersal potential and abundance data collected will be used to define the potential role of LBFs and calculate the risk of mechanical transmission of viruses to livestock in the UK. Objective 1.1: Site recruitment and preliminary surveys: Recruit 10 UK livestock holdings and stables across an East/West transect of the south of the UK and two additional sites in the north of England, Wales and/or Scotland. Carry out site characterization of LBF presence including mammalian host availability and habitat classification Objective 1.2: LBF trapping and fauna characterization: Carry out trapping and identify flies that could act as mechanical vectors including Stomoxys and Tabanids using suitable traps at these sites over two field seasons to examine inter-year variation in fly abundance and diversity. Objective 1.3: Marking technique validation: Validate techniques used to study dispersal of other insect species, including capture-mark-recapture, mark-capture and direct observation techniques for study of dispersal in LBFs. Using colony-derived (Stomoxys only) and field-collected specimens, quantify of marker acquisition, retention and longevity and impacts on survival and behaviour. Marking individuals with fluorescent pigments in dust and paint form will be tested, alongside the use of protein markers and associated detection ELISAs. Photographic images of LBFs feeding on hosts will be trialed for direct observation of movement between hosts and assessment of biting rates on hosts. Objective 1.4: Quantify LBF dispersal behaviour: Conduct dispersal field trials with at least two key LBF species, including Stomoxys calcitrans, as the LBF most relevant to study due to abundance, implication as vector for mechanically transmitted viruses and data from colony individuals, using selected techniques from 1.3 at up to two sites with a large abundance of flies in up to three replicates of each dispersal study. Trials will involve the release of large numbers of marked individual LBFs (Stomoxys calcitrans) or marking of individuals within a resting or breeding site. At time intervals post release/marking, insects collected in traps on radial transects from the release site will be assessed (visually or by ELISA, depending on marking technique) for the marker and distance travelled by marked individuals calculated. Objective 1.5: Host use and blood meal analysis: Quantify dispersal with regard to habitat and use molecular techniques of blood meal analysis to determine host use by collected individuals where a blood meal is present. Objective 1.6: Incorporation of dispersal data into estimates of R0 for LSDV in UK: Link field data from objectives 1.1 to 1.5 to the current epidemiological parameters used to estimate R0 for transmission of LSDV in the UK. Objectives 1.1-1.6 are interdependent but low risk, as they utilize methods and techniques used by the group in previous studies with other even less malleable insect species. Data from traditional capture-mark -recapture trials will provide quantification of dispersal distances achieved by LBFs. The use of more novel mark-capture techniques, as used previously with Culicoides, may increase the recovery of marked insects and therefore increase the robustness of dispersal estimates. Area 2. Impact of temperature and humidity extremes on Culicoides flight activity and survival (years 1, 2 and 3) The flight activity and survival of Culicoides vectors at different humidities and temperatures will be explored in bioassays using field-collected individuals across the year to ascertain the impact of season or timing of adult emergence on the tolerance of adult Culicoides for these climatic factors. Temperature and humidity thresholds for activity and survival will be incorporated into the NAME model of Culicoides dispersal for the prediction of Culicoides-borne virus incursion into the UK. Objective 2.1: Site selection and characterization of Culicoides fauna: Establish Culicoides trapping at three sites local to The Pirbright Institute and ensure populations of >100 individuals/night are collected under conditions conducive to adult flight. Objective 2.2: Enhancement of phototaxis assays to investigate the effect of environmental parameters on flight activity: Previously phototaxis bioassays were developed to assess Culicoides activity by monitoring their flight response towards a UV light source. Here we will further enhance these assays to allow studying the effect of additional single (humidity, high temperatures) and combined (humidity and temperature) parameters on Culicoides activity Objective 2.3: Determine humidity threshold for Culicoides flight activity: Determine activity quantified as the proportion of individuals responding with phototaxis towards the light source and survival of field-collected Culicoides under a range of humidities observed in the UK (23,33,55, 80 and 90% relative humidity) generated using saturated salt solutions within climate chambers. Test populations collected at three timepoints through the year (spring, summer and autumn) Objective 2.4: Determine warm temperature thresholds for Culicoides flight activity: Assess the interaction of temperature and humidity in determining activity and survival of field-collected Culicoides using the humidities at a range of temperatures from 10 to 35°C at three time points during the year (spring, summer and autumn) Objective 2.5: Incorporation of thresholds into NAME midge dispersion model: Provide humidity and temperature activity threshold data to the UK Met Office for integration into the NAME model via addition to the midge species parametrisation. The UK Met Office will be employed as a sub-contractor for the implementation and assessment of the impact of the findings from objective 2.4 in the non-operational NAME model. Should changes made to this research version of the model prove advantageous, changes to the operational model will be made outside the scope of this project. Objectives 2.1-2.5 are interdependent but low risk, as they utilize methods and techniques used by the group in previous studies. Area 3. Transmission parameters of an emerging arbovirus threat to the UK (year 3) The proximity of known BTV outbreaks to the UK, the changing epidemiology of BTV in southern Europe and probability of emergence of other Culicoides-borne virus strains (such as BTV or potentially EHDV) requires a flexible approach so that the greatest arbovirus threat is selected for impact and transmission potential characterization. The characterization of clinical severity and transmission parameters of high-consequence arboviruses of livestock is a key capacity of the Pirbright Institute and is nationally unique. Feeding of both field-collected and colony-derived Culicoides on viremic animals will allow assessment of the infection susceptibility of UK vector populations and direct comparison of transmission likelihood with previous studies. The approach outlined here describes objectives assuming the selection of a BTV4 strain currently circulating in mainland Europe as as the virus of current highest concern. However the objective allows for flexibility in the selection of the specific Culicoides-borne virus should a virus of higher concern emerge with the proviso that if a cattle virus such as EHDV is selected, the hosts used will be adjusted to suit. Objective 3.1: Selection of emergent Culicoides-borne virus: Select exotic and emergent Culicoides-borne virus of interest to UK policy Possible candidates currently circulating in Europe include BTV4 from France and BTV4 from Italy. Virus selection criteria will include proximity to UK, time of emergence, speed of spread and reported virulence in country of origin in consultation with national reference laboratory and policy. Objective 3.2: Define sites for collection of Culicoides from the field: Establish Culicoides trapping at sites used for Objective 2.1 Objective 3.3: Infection of ruminant hosts with a Culicoides-borne virus: Infect 6 sheep using Culicoides sonorensis from the colony maintained at Pirbright infected via intrathoracic inoculation. Objective 3.4: Assessment of susceptibility of UK Culicoides for a Culicoides-borne virus: Feed populations of both UK and colony-derived Culicoides on infected sheep at the peak of the vireamic period as monitored by PCR of the sheep blood. Incubation of blood fed females for 7-10 days and subsequently define infection status by qPCR. Objective 3.5: Assessment of clinical impact of a Culicoides-borne virus strain: Record clinical impact of infection in sheep, according to the clinical signs of disease associated with BTV infection observed throughout the experiment and at post-mortem, using an established scoring system. As an animal experiment these objectives are interdependent, however the study could be run solely with colony derived Culicoides in the event of weather not conducive to adult flight in the field. All objectives are low risk and use techniques extensively defined in previous studies.