African horse sickness virus (AHSV: Sedoreoviridae; Orbivirus) causes a severe and often fatal disease in horses (African horse sickness: AHS) and is transmitted almost exclusively by Culicoides biting midges (Diptera: Ceratopogonidae). In recent years, unprecedented outbreaks of AHSV have occurred in new geographical foci in Thailand and other related Culicoides-borne viruses continue to emerge unexpectedly, causing disease outbreaks in northern Europe. This study investigated Culicoides abundance and diversity at a donkey (Equus asinus) sanctuary in southern England. The incidence and severity of AHS in infected donkeys are lower than in horses, with concerns, therefore, that these species could act as potential reservoirs in the event of an incursion of AHSV. A total of 21,350 Culicoides of 20 species were collected over 14 nights during spring and summer 2019 using three Onderstepoort Veterinary Institute ultraviolet light-suction traps. The most abundant species were identified within the subgenus Avaritia (19,574; 91.7%), which are known vectors of other Orbiviruses in northern Europe and have been previously identified as putative vectors of AHSV in southern Europe. Furthermore, Culicoides blood-feeding on donkeys was confirmed for the subgenus Avaritia through polymerase chain reaction of blood-fed female Culicoides using a 685 bp region of the cytochrome c oxidase subunit 1 gene. Data on the size and distribution of the donkey population and the potential impact of infection with AHSV on donkeys within the United Kingdom are scarce. This study demonstrates that large populations of Culicoides can exist near these hosts and that they regularly take blood meals from them. There is a potential risk that donkeys could play a significant role in transmission and persistence of AHSV in the event of an incursion into the United Kingdom, which could complicate disease control.

Understanding how life is adapting to urban environments represents an important challenge in evolutionary biology. In this work, we investigate a widely cited example of urban adaptation, Culex pipiens form molestus, also known as the London Underground mosquito. Population genomic analysis of ~350 contemporary and historical samples counters the popular hypothesis that molestus originated belowground in London <200 years ago. Instead, we show that molestus first adapted to human environments aboveground in the Mediterranean or Middle East over the course of more than 1000 years, possibly in association with ancient agricultural civilizations of the Middle East. Our results highlight the role of early human society in priming taxa for contemporary urban evolution. They also provide insight into whether and how molestus contributes to West Nile virus transmission in modern cities.

IntroductionBluetongue virus (BTV) is an arthropod-borne Orbivirus that is almost solely transmitted by Culicoides biting midges and causes a globally important haemorrhagic disease, bluetongue (BT), in susceptible ruminants. Infection with BTV is characterised by immunosuppression and substantial lymphopenia at peak viraemia in the host.MethodsIn this study, the role of cell-mediated immunity and specific T-cell subsets in BTV pathogenesis, clinical outcome, viral dynamics, immune protection, and onwards transmission to a susceptible Culicoides vector is defined in unprecedented detail for the first time, using an in vivo arboviral infection model system that closely mirrors natural infection and transmission of BTV. Individual circulating CD4+, CD8+, or WC1+ γδ T-cell subsets in sheep were depleted through the administration of specific monoclonal antibodies.ResultsThe absence of cytotoxic CD8+ T cells was consistently associated with less severe clinical signs of BT, whilst the absence of CD4+ and WC1+ γδ T cells both resulted in an increased clinical severity. The absence of CD4+ T cells also impaired both a timely protective neutralising antibody response and the production of IgG antibodies targeting BTV non-structural protein, NS2, highlighting that the CD4+ T-cell subset is important for a timely protective immune response. T cells did not influence viral replication characteristics, including onset/dynamics of viraemia, shedding, or onwards transmission of BTV to Culicoides. We also highlight differences in T-cell dependency for the generation of immunoglobulin subclasses targeting BTV NS2 and the structural protein, VP7.DiscussionThis study identifies a diverse repertoire of T-cell functions during BTV infection in sheep, particularly in inducing specific anti-viral immune responses and disease manifestation, and will support more effective vaccination strategies.

Bluetongue virus (BTV, Sedoreoviridae: Orbivirus) causes an economically important disease, namely, bluetongue (BT), in domestic and wild ruminants worldwide. BTV is endemic to South India and has occurred with varying severity every year since the virus was first reported in 1963. BT can cause high morbidity and mortality to sheep flocks in this region, resulting in serious economic losses to subsistence farmers, with impacts on food security. The epidemiology of BTV in South India is complex, characterized by an unusually wide diversity of susceptible ruminant hosts, multiple vector species biting midges (Culicoides spp., Diptera: Ceratopogonidae), which have been implicated in the transmission of BTV and numerous co-circulating virus serotypes and strains. BT presence data (1997–2011) for South India were obtained from multiple sources to develop a presence/absence model for the disease. A non-linear discriminant analysis (NLDA) was carried out using temporal Fourier transformed variables that were remotely sensed as potential predictors of BT distribution. Predictive performance was then characterized using a range of different accuracy statistics (sensitivity, specificity, and Kappa). The top ten variables selected to explain BT distribution were primarily thermal metrics (land surface temperature, i.e., LST, and middle infrared, i.e., MIR) and a measure of plant photosynthetic activity (the Normalized Difference Vegetation Index, i.e., NDVI). A model that used pseudo-absence points, with three presence and absence clusters each, outperformed the model that used only the recorded absence points and showed high correspondence with past BTV outbreaks. The resulting risk maps may be suitable for informing disease managers concerned with vaccination, prevention, and control of BT in high-risk areas and for planning future state-wide vector and virus surveillance activities.

Abstract Background Culex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay. Results Genomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55–80%. Conclusions This study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.

Background Arthropods transmit a wide range of pathogens of importance for the global health of humans, animals, and plants. One group of these arthropod vectors, Culicoides biting midges (Diptera: Ceratopogonidae), is the biological vector of several human and animal pathogens, including economically important livestock viruses like bluetongue virus (BTV). Like other arthropod-borne viruses (arboviruses), Culicoides -borne viruses must reach and replicate in the salivary apparatus, from where they can be transmitted to susceptible hosts through the saliva during subsequent blood feeding. Despite the importance of the salivary gland apparatus for pathogen transmission to susceptible animals from the bite of infected Culicoides , these structures have received relatively little attention, perhaps due to the small size and fragility of these vectors. Results In this study, we developed techniques to visualize the infection of the salivary glands and other soft tissues with BTV, in some of the smallest known arbovirus vectors, Culicoides biting midges, using three-dimensional immunofluorescence confocal microscopy. We showed BTV infection of specific structures of the salivary gland apparatus of female Culicoides vectors following oral virus uptake, related visualisation of viral infection in the salivary apparatus to high viral RNA copies in the body, and demonstrated for the first time, that the accessory glands are a primary site for BTV replication within the salivary apparatus. Conclusions Our work has revealed a novel site of virus-vector interactions, and a novel role of the accessory glands of Culicoides in arbovirus amplification and transmission. Our approach would also be applicable to a wide range of arbovirus vector groups including sand flies (Diptera: Psychodidae), as well as provide a powerful tool to investigate arbovirus infection and dissemination, particularly where there are practical challenges in the visualization of small size and delicate tissues of arthropods.

Segmented RNA viruses are a taxonomically diverse group that can infect plant, wildlife, livestock and human hosts. A shared feature of these viruses is the ability to exchange genome segments during coinfection of a host by a process termed "reassortment." Reassortment enables rapid evolutionary change, but where transmission involves a biological arthropod vector, this change is constrained by the selection pressures imposed by the requirement for replication in two evolutionarily distant hosts. In this study, we use an in vivo, host-arbovirus-vector model to investigate the impact of reassortment on two phenotypic traits, virus infection rate in the vector and virulence in the host. Bluetongue virus (BTV) (Reoviridae) is the causative agent of bluetongue (BT), an economically important disease of domestic and wild ruminants and deer. The genome of BTV comprises 10 linear segments of dsRNA, and the virus is transmitted between ruminants by Culicoides biting midges (Diptera: Ceratopogonidae). Five strains of BTV representing three serotypes (BTV-1, BTV-4, and BTV-8) were isolated from naturally infected ruminants in Europe and ancestral/reassortant lineage status assigned through full genome sequencing. Each strain was then assessed in parallel for the ability to replicate in vector Culicoides and to cause BT in sheep. Our results demonstrate that two reassortment strains, which themselves became established in the field, had obtained high replication ability in C. sonorensis from one of the ancestral virus strains, which allowed inferences of the genome segments conferring this phenotypic trait. IMPORTANCE Reassortment between virus strains can lead to major shifts in the transmission parameters and virulence of segmented RNA viruses, with consequences for spread, persistence, and impact. The ability of these pathogens to adapt rapidly to their environment through this mechanism presents a major challenge in defining the conditions under which emergence can occur. Utilizing a representative mammalian host-insect vector infection and transmission model, we provide direct evidence of this phenomenon in closely related ancestral and reassortant strains of BTV. Our results demonstrate that efficient infection of Culicoides observed for one of three ancestral BTV strains was also evident in two reassortant strains that had subsequently emerged in the same ecosystem.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) cause economically important diseases that are currently exotic to the United Kingdom (UK), but have significant potential for introduction and onward transmission. Given the susceptibility of animals kept in zoo collections to vector-borne diseases, a qualitative risk assessment for the introduction of BTV and AHSV to ZSL London Zoo was performed. Risk pathways for each virus were identified and assessed using published literature, animal import data and outputs from epidemiological models. Direct imports of infected animals, as well as wind-borne infected Culicoides, were considered as routes of incursion. The proximity of ongoing disease events in mainland Europe and proven capability of transmission to the UK places ZSL London Zoo at higher risk of BTV release and exposure (estimated as low to medium) than AHSV (estimated as very low to low). The recent long-range expansion of AHSV into Thailand from southern Africa highlights the need for vector competence studies of Palearctic Culicoides for AHSV to assess the risk of transmission in this region.

Background Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of livestock arboviruses that cause diseases with significant economic, social and welfare impacts. Within temperate regions, livestock movement during arbovirus outbreaks can be facilitated by declaring a ‘seasonal vector-free period’ (SVFP) during winter when adult Culicoides are not active. In this study we carry out long-term monitoring of Culicoides adult emergence from larval development habitats at two farms in the UK to validate current definitions of the SVFP and to provide novel bionomic data for known vector species. Methods Standard emergence traps were used to collect emerging adult Culicoides from dung heaps at two cattle farms in the south-east of England from June to November 2016 and March 2017 to May 2018. Culicoides were morphologically identified to species or complex level and count data were analysed using a simple population dynamic model for pre-adult Culicoides that included meteorological components. Results More than 96,000 Culicoides were identified from 267 emergence trapping events across 2 years, revealing clear evidence of bivoltinism from peaks of male populations of Culicoides obsoletus emerging from dung heaps. This pattern was also reflected in the emergence of adult female Obsoletus complex populations, which dominated the collections (64.4% of total catch) and emerged throughout the adult active period. Adult male C. obsoletus were observed emerging earlier than females (protandry) and emergence of both sexes occurred throughout the year. Culicoides chiopterus and Culicoides scoticus were also identified in spring emergence collections, providing the first evidence for the overwintering of larvae in dung heaps for these species. Conclusions This study demonstrates continual and highly variable rates of emergence of Culicoides throughout the year. A lack of evidence for mass emergence in spring along with the ability to observe male generations highlights the need for complementary surveillance techniques in addition to light-trap data when investigating seasonality and phenology. Evidence was found of other vector species, C. chiopterus and C. scoticus , utilising cattle dung heaps as an overwintering habitat, further highlighting the importance of these habitats on farms. Graphical Abstract

Leishmania parasites, causative agents of leishmaniasis, are currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia. The recently established subgenus Mundinia has a wide geographical distribution and contains five species, three of which have the potential to infect and cause disease in humans. While the other Leishmania subgenera are transmitted exclusively by phlebotomine sand flies (Diptera: Psychodidae), natural vectors of Mundinia remain uncertain. This study investigates the potential of sand flies and biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to transmit Leishmania parasites of the subgenus Mundinia. Sand flies (Phlebotomus argentipes, P. duboscqi and Lutzomyia migonei) and Culicoides biting midges (Culicoides sonorensis) were exposed to five Mundinia species through a chicken skin membrane and dissected at specific time intervals post bloodmeal. Potentially infected insects were also allowed to feed on ear pinnae of anaesthetized BALB/c mice and the presence of Leishmania DNA was subsequently confirmed in the mice using polymerase chain reaction analyses. In C. sonorensis, all Mundinia species tested were able to establish infection at a high rate, successfully colonize the stomodeal valve and produce a higher proportion of metacyclic forms than in sand flies. Subsequently, three parasite species, L. martiniquensis, L. orientalis and L. sp. from Ghana, were transmitted to the host mouse ear by C. sonorensis bite. In contrast, transmission experiments entirely failed with P. argentipes, although colonisation of the stomodeal valve was observed for L. orientalis and L. martiniquensis and metacyclic forms of L. orientalis were recorded. This laboratory-based transmission of Mundinia species highlights that Culicoides are potential vectors of members of this ancestral subgenus of Leishmania and we suggest further studies in endemic areas to confirm their role in the lifecycles of neglected pathogens.

Background Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses and inflict biting nuisance on humans, companion animals and livestock. In temperate regions, transmission of arboviruses is limited by temperature thresholds, both in replication and dissemination of arboviruses within the vector and in the flight activity of adult Culicoides . This study aims to determine the cold-temperature thresholds for flight activity of Culicoides from the United Kingdom under laboratory conditions.MethodsOver 18,000 Culicoides adults were collected from the field using 4W down-draught miniature ultraviolet Centers for Disease Control traps. Populations of Culicoides were sampled at three different geographical locations within the United Kingdom during the summer months, and again in the autumn at one geographical location. Activity at constant temperatures was assessed using a bioassay that detected movement of adult Culicoides towards an ultraviolet light source over a 24-hour period.ResultsThe proportion of active adult Culicoides increased with temperature but cold temperature thresholds for activity varied significantly according to collection season and location. Populations dominated by the subgenus Avaritia collected in South East England had a lower activity threshold temperature in the autumn (4°C), when compared with populations collected in the summer (10°C). Within the subgenus Avaritia , Culicoides scoticus was significantly more active across all temperatures tested than Culicoides obsoletus within the experimental setup Populations of Culicoides impunctatus collected in the North East of England , were only active once temperatures reached 14°C. Preliminary data suggested flight activity of the subgenus Avaritia does not differ between populations in South East England and those in the Scottish Borders.ConclusionsThese findings demonstrate seasonal changes in temperature thresholds for flight and across different populations of Culicoides. This data, alongside that defining thresholds for virus replication within Culicoides, provides a primary tool for risk assessment of arbovirus transmission in temperate regions. In addition, the study also provides a comparison with thermal limits derived directly from light-suction trapping data, which is currently used as the main method to define adult Culicoides activity during surveillance.

Segmented RNA viruses are a taxonomically diverse group of 11 families that can infect plant, wildlife, livestock and human hosts. A shared feature of these viruses is the ability to exchange genome segments during co-infection of a host by a process termed ‘reassortment’. Reassortment enables rapid evolutionary change, but in the case of segmented RNA viruses utilising an arthropod vector is set against the constraint of purifying selection and genetic bottlenecks imposed by replication in two evolutionarily distant hosts. In this study, we use an in vivo host: arbovirus: vector model to investigate the impact of reassortment on two phenotypic traits: vector competence and virulence in the host. Bluetongue virus (BTV) ( Reoviridae ) is the causative agent of bluetongue (BT), an economically important disease of domestic and wild ruminants and deer. The genome of BTV is comprised of 10 linear segments of dsRNA and the virus is transmitted between ruminants by Culicoides biting midges (Diptera: Ceratopogonidae). Five strains of BTV representing three serotypes (BTV-1, BTV-4 and BTV-8) were isolated from naturally infected ruminants in Europe and parental/reassortant lineage status assigned through full genome sequencing. Each strain was then assessed in parallel for the ability to infect Culicoides and to cause BT in sheep. Our results demonstrate that two reassortment strains, which themselves became established in the field, had obtained high replication ability in C. sonorensis from one of the parental virus strains which allowed inferences of the genome segments conferring this phenotypic trait.IMPORTANCE Reassortment between strains can lead to major shifts in the transmission parameters and virulence of segmented RNA viruses with consequences for spread, persistence and impact. The ability of these pathogens to change their phenotypes rapidly in response to selection pressure in new environments presents a major challenge in understanding factors driving emergence. Utilising a natural mammalian host-insect vector infection and transmission model, we demonstrated for the first time the genetic basis for a phenotypic trait of BTV within strains directly isolated from the field and, hence, selected and relevant for natural transmission.

The biting midge Culicoides imicola is a small, haematophagous fly that plays a key role in the biological transmission of viral diseases including bluetongue and African horse sickness (AHS) that inflict damage on both subsistence and intensive livestock as well as companion animals and wildlife. Within the subgenus Avaritia, the monophyletic Imicola group includes ten species, although cryptic diversity may hide the existence of additional species closely related to C. imicola. C. imicola has the broadest geographic range in the genus Culicoides, including most of the African continent, the Mediterranean basin and parts of southern Europe, the Middle East, India, and Southeast Asia to Taiwan. The broad distribution of C. imicola illustrates exploitation of anthropogenic habitats and may allow future expansion into new areas. Long-distance movement of adults, especially over the sea, has been proposed and may facilitate this geographic expansion.

Background: Bluetongue (BT) is a viral disease of ruminants and camelids which can have a significant impact on animal health and welfare and cause severe economic loss. The UK has been officially free of bluetongue virus (BTV) since 2011. In 2015, BTV-8 re-emerged in France and since then BTV has been spreading throughout Europe. In response to this outbreak, risk-based active surveillance was carried out at the end of the vector seasons in 2017 and 2018 to assess the risk of incursion of BTV into Great Britain. Method: Atmospheric dispersion modelling identified counties on the south coast of England at higher risk of an incursion. Blood samples were collected from cattle in five counties based on a sample size designed to detect at least one positive if the prevalence was 5 per cent or greater, with 95 per cent confidence. Results: No virus was detected in the 478 samples collected from 32 farms at the end of the 2017 vector season or in the 646 samples collected from 43 farms at the end of the 2018 vector season, when tested by RT-qPCR. Conclusion: The negative results from this risk-based survey provided evidence to support the continuation of the UK's official BTV-free status.

Bovine ephemeral fever virus (Rhabdoviridae: Ephemerovirus) (BEFV) causes bovine ephemeral fever (BEF), an economically important disease of cattle and water buffalo. Outbreaks of BEF in Africa, Australia, Asia and the Middle East are characterized by high rates of morbidity and highly efficient transmission between cattle hosts. Despite this, the vectors of BEFV remain poorly defined. Colony lines of biting midges (Culicoides sonorensis) and mosquitoes (Aedes aegypti, Culex pipiens and Culex quinquefasciatus) were infected with a strain of BEFV originating from Israel by feeding on blood–virus suspensions and by intrathoracic inoculation. In addition, in vivo transmission of BEFV was also assessed by allowing C. sonorensis inoculated by the intrathoracic route to feed on male 6 month-old Holstein-Friesian calves. There was no evidence of BEFV replication within mosquitoes fed on blood/virus suspensions for mosquitoes of any species tested for each of the three colony lines. In 170 C. sonorensis fed on the blood/virus suspension, BEFV RNA was detected in the bodies of 13 individuals and in the heads of two individuals, indicative of fully disseminated infections and an oral susceptibility rate of 1.2%. BEFV RNA replication was further demonstrated in all C. sonorensis that were inoculated by the intrathoracic route with virus after 5, 6 or 7 days post-infection. Despite this, transmission of BEFV could not be demonstrated when infected C. sonorensis were allowed to feed on calves. No evidence for infection or dissemination of BEFV (bovine/Israel/2005-6) in mosquitoes of three different species was found. Evidence was found for infection of C. sonorensis by the oral route. However, attempts to transmit BEFV to calves from infected C. sonorensis failed. These results highlight the challenge of defining the natural vector of BEFV and of establishing an in vivo transmission model. The results are discussed with reference to the translation of laboratory-based studies to inference of vector competence in the field.

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase 1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens.

This is the third in a planned series of data papers presenting modelled vector distributions produced during the ECDC and EFSA funded VectorNet project. The data package presented here includes those 'Culicoides' vectors species first modelled in 2015 as part of the VectorNet gap analysis work namely 'C. imicola, C. obsoletus, C. scoticus, C. dewulfi, C. chiopterus, C. pulicaris, C. lupicaris, C. punctatus, and C. newsteadi'. The known distributions of these species within the Project area (Europe, the Mediterranean Basin, North Africa, and Eurasia) are currently incomplete to a greater or lesser degree. The models are designed to fill the gaps with predicted distributions, to provide a) first indication of vector species distributions across the project geographical extent, and b) assistance in targeting surveys to collect distribution data for those areas with no field validated information. The models are based on input data from light trap surveillance of adult 'Culicoides' across continental Europe and surrounding regions (71.8°N –33.5°S, – 11.2°W – 62°E), concentrated in Western countries, supplemented by transect samples in eastern and northern Europe. Data from central EU are relatively sparse. Funding statement: This work was carried out with support from the VectorNet framework contract OC/EFSA/AHAW/2013/02-FWC1 funded by the European Centre for Disease prevention and Control (ECDC) and the European Food Safety Agency (EFSA) and the PALE-Blu H2020 Project ID: 727393.

Background Culicoides biting midges are biological vectors of arboviruses including bluetongue virus (BTV), Schmallenberg virus (SBV) and African horse sickness virus (AHSV). Zoos are home to a wide range of ‘at risk’ exotic and native species of animals. These animals have a high value both in monetary terms, conservation significance and breeding potential. To understand the risk these viruses pose to zoo animals, it is necessary to characterise the Culicoides fauna at zoos and determine which potential vector species are feeding on which hosts. Methods Light-suction traps were used at two UK zoos: the Zoological Society of London (ZSL) London Zoo (LZ) and ZSL Whipsnade Zoo (WZ). Traps were run one night each week from June 2014 to June 2015. Culicoides were morphologically identified to the species level and any blood-fed Culicoides were processed for blood-meal analysis. DNA from blood meals was extracted and amplified using previously published primers. Sequencing was then carried out to determine the host species. Results A total of 11,648 Culicoides were trapped and identified ( n = 5880 from ZSL WZ; n = 5768 from ZSL LZ), constituting 25 different species. The six putative vectors of BTV, SBV and AHSV in northern Europe were found at both zoos and made up the majority of the total catch ( n = 10,701). A total of 31 host sequences were obtained from blood-fed Culicoides. Culicoides obsoletus / C. scoticus , Culicoides dewulfi , Culicoides parroti and Culicoides punctatus were found to be biting a wide range of mammals including Bactrian camels, Indian rhinoceros, Asian elephants and humans, with Culicoides obsoletus/C. scoticus also biting Darwin’s rhea. The bird-biting species, Culicoides achrayi , was found to be feeding on blackbirds, blue tits, magpies and carrion crows. Conclusions To our knowledge, this is the first study to directly confirm blood-feeding of Culicoides on exotic zoo animals in the UK and shows that they are able to utilise a wide range of exotic as well as native host species. Due to the susceptibility of some zoo animals to Culicoides -borne arboviruses, this study demonstrates that in the event of an outbreak of one of these viruses in the UK, preventative and mitigating measures would need to be taken.

African horse sickness was confirmed in horses in Thailand during March 2020. The virus was determined to belong to serotype 1 and is phylogenetically closely related to isolates from South Africa. This is the first incidence of African horse sickness occurring in South East Asia and of serotype 1 outside of Africa.

An amendment to this paper has been published and can be accessed via the original article.