Estimating the Impact of Consecutive Blood Meals on Vector Competence of Aedes albopictus for Chikungunya Virus.

The continuous expansion of Aedes albopictus in Europe and the increases in autochthonous arboviruses transmissions in the region urge a better understanding of the virus transmission dynamic. Recent work described enhanced chikungunya virus (CHIKV) dissemination in Aedes aegypti mosquitoes exposed to a virus-free blood meal three days after their infection with CHIKV. Our study investigated the impact of a second blood meal on the vector competence of Ae. albopictus from southern Switzerland infected with CHIKV. Seven-day-old Ae. albopictus females were exposed to CHIKV-spiked blood and incubated at constant (27 °C) and fluctuating (14-28 °C) temperatures. Four days post-infection (dpi), some of these females were re-fed with a non-infectious blood meal. Virus infectivity, dissemination, transmission rate, and efficiency were investigated at seven and ten dpi. No enhanced dissemination rate was observed among females fed a second time; however, re-fed females have shown higher transmission efficiency than those fed only once after seven days post-infection and incubated under a fluctuating temperature regime. Vector competence for CHIKV was confirmed in Ae. albopictus from southern Switzerland. We did not observe an increase in dissemination rates among mosquitoes fed a second time (second blood meal), regardless of the temperature regime.

Infravec2 guidelines for the design and operation of containment level 2 and 3 insectaries in Europe.

With the current expansion of vector-based research and an increasing number of facilities rearing arthropod vectors and infecting them with pathogens, common measures for containment of arthropods as well as manipulation of pathogens are becoming essential for the design and running of such research facilities to ensure safe work and reproducibility, without compromising experimental feasibility. These guidelines and comments were written by experts of the Infravec2 consortium, a Horizon 2020-funded consortium integrating the most sophisticated European infrastructures for research on arthropod vectors of human and animal diseases. They reflect current good practice across European laboratories with experience of safely handling different mosquito species and the pathogens they transmit. As such, they provide experience-based advice to assess and manage the risks to work safely with mosquitoes and the pathogens they transmit. This document can also form the basis for research with other arthropods, for example, midges, ticks or sandflies, with some modification to reflect specific requirements.

Zika vector competence data reveals risks of outbreaks: the contribution of the European ZIKAlliance project.

First identified in 1947, Zika virus took roughly 70 years to cause a pandemic unusually associated with virus-induced brain damage in newborns. Zika virus is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus, both colonizing a large strip encompassing tropical and temperate regions. As part of the international project ZIKAlliance initiated in 2016, 50 mosquito populations from six species collected in 12 countries were experimentally infected with different Zika viruses. Here, we show that Ae. aegypti is mainly responsible for Zika virus transmission having the highest susceptibility to viral infections. Other species play a secondary role in transmission while Culex mosquitoes are largely non-susceptible. Zika strain is expected to significantly modulate transmission efficiency with African strains being more likely to cause an outbreak. As the distribution of Ae. aegypti will doubtless expand with climate change and without new marketed vaccines, all the ingredients are in place to relive a new pandemic of Zika.

Impact of Irradiation on Vector Competence of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) for Dengue and Chikungunya Viruses.

Effective control strategies against arthropod disease vectors are amongst the most powerful tools to prevent the spread of vector-borne diseases. The sterile insect technique (SIT) is an effective and sustainable autocidal control method that has recently shown effective population suppression against different Aedes vector species worldwide. The SIT approach for mosquito vectors requires the release of radio-sterilized male mosquitoes only, but currently available sex separation techniques cannot ensure the complete elimination of females resulting in short-term risk of increased biting rate and arboviral disease transmission. In this study, we compared for the first time the transmission of dengue and chikungunya viruses in Aedes aegypti and Aedes albopictus females exposed as pupae to an irradiation dose of 40 Gy. Females of both species were fed on blood spiked with either dengue or chikungunya viruses, and body parts were tested for virus presence by real-time RT-PCR at different time points. No differences were observed in the dissemination efficiency of the dengue virus in irradiated and unirradiated Ae. albopictus and Ae. aegypti mosquitoes. The dissemination of the chikungunya virus was higher in Ae. albopictus than in Ae. Aegypti, and irradiation increased the virus load in both species. However, we did not observe differences in the transmission efficiency for chikungunya (100%) and dengue (8-27%) between mosquito species, and irradiation did not impact transmissibility. Further implications of these results on the epidemiology of vector-borne diseases in the field are discussed.

Field-Reassortment of Bluetongue Virus Illustrates Plasticity of Virus Associated Phenotypic Traits in the Arthropod Vector and Mammalian Host In Vivo.

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.

Putative roles of mosquitoes (Culicidae) and biting midges (Culicoides spp.) as mechanical or biological vectors of lumpy skin disease virus.

The stable fly Stomoxys calcitrans (Diptera: Muscidae) is considered as the main mechanical vector of the lumpy skin disease virus (LSDV). In addition, the mosquito species Aedes aegypti (Diptera: Culicidae) was shown to transmit the virus from donor to receptor animals. Retention of the virus for several days was shown for two additional tropical mosquito species and the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae). In the present study, viral retention for 10- or 7-days post feeding on virus-spiked blood through a membrane was shown for field-collected Aedes japonicus and laboratory-reared Culex pipiens, two widely distributed mosquito species in temperate regions. Viral DNA could be detected from honey-coated Flinders Technology Associates (FTA) cards and shedded faeces for 1 or 4 days after an infectious blood meal was given to Ae. aegypti. Virus increase over time and virus dissemination was observed in laboratory-reared C. nubeculosus, but the virus could be isolated from field-collected biting midges only from the day of exposure to the blood meal. Thus, mosquitoes might serve as mechanical vectors of LSDV in case of interrupted feeding. A putative biological virus transmission by Culicoides biting midges, as suspected from field observations, deserves further investigations.

Potential mechanical transmission of Lumpy skin disease virus (LSDV) by the stable fly (Stomoxys calcitrans) through regurgitation and defecation.

Lumpy skin disease (LSD) is a viral disorder of cattle caused by the lumpy skin disease virus (LSDV) which can induce severe infections leading to high economic losses. Being of African origin, the first LSD outbreaks in Europe occurred in Greece and later in the Balkan region. Little is known about the mode of transmission, especially in relation to the potential role of arthropods vectors. The purpose of our study was to investigate the role of Stomoxys calcitrans in the transmission of LSDV and their presence at different farms in Switzerland. Laboratory-reared flies were exposed to LSDV spiked-blood and incubated under a realistic fluctuating temperature regime. Body parts, regurgitated blood, and faecal samples were analysed by qPCR for the presence of viral DNA and infectious virus at different time points post-feeding (p.f.). LSDV DNA was detected in heads, bodies, and regurgitated blood up to three days p.f. and up to two days p.f. in the faeces. Infectious virus was isolated from bodies and faeces up to two days and in the regurgitated blood up to 12 h p.f. There was no increase in viral load, consolidating the role of S. calcitrans as mechanical vectors for LSDV. Stomoxys flies were present at all eight farms investigated, including a farm located at 2128 m asl. The persistence of LSDV in S. calcitrans in combination with the long flight ranges of this abundant and widespread fly might have implications on LSD epidemiology and on implementing control measures during disease outbreaks.

Assessing the role of two populations of Aedes japonicus japonicus for Zika virus transmission under a constant and a fluctuating temperature regime.

BACKGROUND: Since the huge epidemic of Zika virus (ZIKV) in Brazil in 2015, questions were raised to understand which mosquito species could transmit the virus. Aedes aegypti has been described as the main vector. However, other Aedes species (e.g. Ae. albopictus and Ae. japonicus) proven to be competent for other flaviviruses (e.g. West Nile, dengue and yellow fever), have been described as potential vectors for ZIKV under laboratory conditions. One of these, the Asian bush mosquito, Ae. japonicus, is widely distributed with high abundances in central-western Europe. In the present study, infection, dissemination and transmission rates of ZIKV (Dak84 strain) in two populations of Ae. japonicus from Switzerland (Zürich) and France (Steinbach, Haut-Rhin) were investigated under constant (27 °C) and fluctuating (14-27 °C, mean 23 °C) temperature regimes. RESULTS: The two populations were each able to transmit ZIKV under both temperature regimes. Infectious virus particles were detected in the saliva of females from both populations, regardless of the incubation temperature regime, from 7 days post-exposure to infectious rabbit blood. The highest amount of plaque forming units (PFU) (400/ml) were recorded 14 days post-oral infection in the Swiss population incubated at a constant temperature. No difference in terms of infection, dissemination and transmission rate were found between mosquito populations. Temperature had no effect on infection rate but the fluctuating temperature regime resulted in higher dissemination rates compared to constant temperature, regardless of the population. Finally, transmission efficiency ranged between 7-23% and 7-10% for the constant temperature and 0-10% and 3-27% under fluctuating temperatures for the Swiss and the French populations, respectively. CONCLUSIONS: To the best of our knowledge, this is the first study confirming vector competence for ZIKV of Ae. japonicus originating from Switzerland and France at realistic summer temperatures under laboratory conditions. Considering the continuous spread of this species in the northern part of Europe and its adaptation at cooler temperatures, preventative control measures should be adopted to prevent possible ZIKV epidemics.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

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

Diversity of Transmission Outcomes Following Co-Infection of Sheep with Strains of Bluetongue Virus Serotype 1 and 8.

Bluetongue virus (BTV) causes an economically important disease, bluetongue (BT), in susceptible ruminants and is transmitted primarily by species of Culicoides biting midges (Diptera: Ceratopogonidae). Since 2006, northern Europe has experienced multiple incursions of BTV through a variety of routes of entry, including major outbreaks of strains of BTV serotype 8 (BTV-8) and BTV serotype 1 (BTV-1), which overlapped in distribution within southern Europe. In this paper, we examined the variation in response to coinfection with strains of BTV-1 and BTV-8 using an in vivo transmission model involving Culicoides sonorensis, low passage virus strains, and sheep sourced in the United Kingdom. In the study, four sheep were simultaneously infected using BTV-8 and BTV-1 intrathoracically inoculated C. sonorensis and co-infections of all sheep with both strains were established. However, there were significant variations in both the initiation and peak levels of virus RNA detected throughout the experiment, as well as in the infection rates in the C. sonorensis that were blood-fed on experimentally infected sheep at peak viremia. This is discussed in relation to the potential for reassortment between these strains in the field and the policy implications for detection of BTV strains.

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level.

BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.

Evidence of West Nile Virus (WNV) Circulation in Wild Birds and WNV RNA Negativity in Mosquitoes of the Danube Delta Biosphere Reserve, Romania, 2016.

West Nile virus (WNV) is a zoonotic flavivirus whose transmission cycle in nature includes wild birds as amplifying hosts and ornithophilic mosquito vectors. Bridge vectors can transmit WNV to mammal species potentially causing West Nile Fever. Wild bird migration is a mode of WNV introduction into new areas. The Danube Delta Biosphere Reserve (DDBR) is a major stopover of wild birds migrating between Europe and Africa. The aim of this study was to investigate the presence of WNV in the DDBR during the 2016 transmission season in wild birds and mosquitoes. Blood from 68 wild birds (nine different species) trapped at four different locations was analyzed by competitive ELISA and Virus Neutralization Test (VNT), revealing positive results in 8/68 (11.8%) of the wild birds by ELISA of which six samples (three from juvenile birds) were confirmed seropositive by VNT. Mosquitoes (n = 6523, 5 genera) were trapped with CDC Mini Light traps at two locations and in one location resting mosquitoes were caught. The presence of WNV RNA was tested in 134 pools by reverse transcription quantitative PCR (RT-qPCR). None of the pools was positive for WNV-specific RNA. Based on the obtained results, WNV was circulating in the DDBR during 2016.

Species diversity, host preference and arbovirus detection of Culicoides (Diptera: Ceratopogonidae) in south-eastern Serbia.

BACKGROUND: Culicoides (Diptera: Ceratopogonidae) is a genus of small biting midges (also known as "no-see ums") that currently includes 1368 described species. They are proven or suspected vectors for important pathogens affecting animals such as bluetongue virus (BTV) and Schmallenberg virus (SBV). Currently little information is available on the species of Culicoides present in Serbia. Thus, the aim of this study was to examine species diversity, host preference and the presence of BTV and SBV RNA in Culicoides from the Stara Planina Nature Park in south-eastern Serbia. RESULTS: In total 19,887 individual Culicoides were collected during three nights of trapping at two farm sites and pooled into six groups (Obsoletus group, Pulicaris group, "Others" group and further each group according to the blood-feeding status to freshly engorged and non-engorged). Species identification was done on subsamples of 592 individual Culicoides specimens by morphological and molecular methods (MALDI-TOF mass spectrometry and PCR/sequencing). At least 22 Culicoides species were detected. Four animal species (cow, sheep, goat and common blackbird) as well as humans were identified as hosts of Culicoides biting midges. The screening of 8291 Culicoides specimens in 99 pools for the presence of BTV and SBV RNA by reverse-transcription quantitative PCR were negative. CONCLUSIONS: The biodiversity of Culicoides species in the natural reserve Stara Planina was high with at least 22 species present. The presence of C. imicola Kieffer was not recorded in this area. Culicoides showed opportunistic feeding behaviour as determined by host preference. The absence of SBV and BTV viral RNA correlates with the absence of clinical disease in the field during the time of sampling. These data are the direct outcome of a training programme within the Institutional Partnership Project "AMSAR: Arbovirus monitoring, research and surveillance-capacity building on mosquitoes and biting midges" funded by the programme SCOPES of the Swiss National Science Foundation.

Vector competence of pre-alpine Culicoides (Diptera: Ceratopogonidae) for bluetongue virus serotypes 1, 4 and 8.

BACKGROUND: Bluetongue disease, caused by bluetongue virus serotype 8 (BTV-8), appeared for the first time in the northern part of Europe in 2006, and subsequently rapidly spread causing severe economic losses to the farming industry. The implicated vectors of BTV in Europe are Culicoides species within the subgenus Avaritia (C. chiopterus, C. dewulfi, C. obsoletus and C. scoticus). Epidemiological data from Switzerland have shown that BTV, whose spread was eliminated at an early stage by vaccination campaigns, had not been circulating among livestock at higher altitudes where other species dominate the Culicoides fauna. In this study, we investigated the extent that Culicoides spp. prevailing at higher altitudes (mainly C. grisescens) can act as vectors for BTV. METHODS: Culicoides were collected at farms in the pre-alpine region (two sites at 1550 m above sea level, masl, referred to as pre-alpine I; one site at 2030 masl, pre-alpine II) and, for comparative purposes, from the Swiss Plateau (one site, 650 masl). They were fed on bovine blood/BTV suspensions (BTV-1, 4 or 8) and incubated for eight days under a fluctuating temperature regime (13-25 °C, mean 19 °C), reflecting a mid-summer warm spell in the pre-alpine region. Susceptibility to BTV transmission was assessed from head homogenates by RT-qPCR and virus isolation. RESULTS: Overall, 9196 female Culicoides were exposed to the three BTV strains through an artificial membrane, with feeding rates of 14-27%. Survival rates of blood-engorged Culicoides females at eight days post-infection depended on both virus serotype and altitude of origin. Virus dissemination (Cq ≤ the cut-off value as determined by serial virus dilutions) was confirmed only for BTV-1 in C. scoticus (dissemination efficiency 22.5%; 9/40) and C. obsoletus (5.6%; 1/18) from the Swiss Plateau area. There was no strong evidence of susceptibility to infection for Culicoides from the pre-alpine area when fed with all BTV strains (BTV-1, 4 and 8). CONCLUSIONS: This study confirms the susceptibility of C. scoticus and C. obsoletus to BTV-1 infection, including under cooler temperatures. Culicoides grisescens, which is highly abundant at higher altitudes, cannot be considered a potential vector under these temperature conditions.

The genome of the biting midge Culicoides sonorensis and gene expression analyses of vector competence for bluetongue virus.

BACKGROUND: The new genomic technologies have provided novel insights into the genetics of interactions between vectors, viruses and hosts, which are leading to advances in the control of arboviruses of medical importance. However, the development of tools and resources available for vectors of non-zoonotic arboviruses remains neglected. Biting midges of the genus Culicoides transmit some of the most important arboviruses of wildlife and livestock worldwide, with a global impact on economic productivity, health and welfare. The absence of a suitable reference genome has hindered genomic analyses to date in this important genus of vectors. In the present study, the genome of Culicoides sonorensis, a vector of bluetongue virus (BTV) in the USA, has been sequenced to provide the first reference genome for these vectors. In this study, we also report the use of the reference genome to perform initial transcriptomic analyses of vector competence for BTV. RESULTS: Our analyses reveal that the genome is 189 Mb, assembled in 7974 scaffolds. Its annotation using the transcriptomic data generated in this study and in a previous study has identified 15,612 genes. Gene expression analyses of C. sonorensis females infected with BTV performed in this study revealed 165 genes that were differentially expressed between vector competent and refractory females. Two candidate genes, glutathione S-transferase (gst) and the antiviral helicase ski2, previously recognized as involved in vector competence for BTV in C. sonorensis (gst) and repressing dsRNA virus propagation (ski2), were confirmed in this study. CONCLUSIONS: The reference genome of C. sonorensis has enabled preliminary analyses of the gene expression profiles of vector competent and refractory individuals. The genome and transcriptomes generated in this study provide suitable tools for future research on arbovirus transmission. These provide a valuable resource for these vector lineage, which diverged from other major Dipteran vector families over 200 million years ago. The genome will be a valuable source of comparative data for other important Dipteran vector families including mosquitoes (Culicidae) and sandflies (Psychodidae), and together with the transcriptomic data can yield potential targets for transgenic modification in vector control and functional studies.

Experimental evaluation of infection, dissemination, and transmission rates for two West Nile virus strains in European Aedes japonicus under a fluctuating temperature regime.

West Nile virus (WNV) is continuously spreading in Eastern and Southern Europe. However, the extent of vector competence of Aedes japonicus (Theobald, 1901) is controversial. In this work, we elucidated the dynamics of virus growth in this invasive mosquito species. Females of Ae. japonicus were reared from eggs collected in the field in Switzerland and fed on bovine blood spiked with two WNV lineage 1 strains (FIN, Italy; NY99, USA). Fully engorged females were incubated for 14 days under a fluctuating temperature regime of 24 ± 7 °C (average 24 °C), 45-90% relative humidity, which is realistic for a Central European mid-summer day. Infection, dissemination, and transmission rates were assessed from individual mosquitoes by analyzing the abdomen, legs and wings, and saliva for the presence of viral RNA. Saliva was also investigated for the presence of infectious virus particles. Overall, 302 females were exposed to WNV strain FIN and 293 to strain NY99. A higher infection rate was observed for NY99 (57.4%) compared to FIN (30.4%) (p = 0.003). There was no statistical evidence that the dissemination rate (viral RNA in legs and wings) was different between females infected with FIN (57.1%) compared to NY99 (35.5%) (p = 0.16). Viral RNA load of FIN compared to NY99 was significantly higher in the hemocoel (p = 0.031) of exposed females but not at other sites (legs and wings, saliva). This is the first study describing the vector competence parameters for two WNV strains in a European population of Ae. japonicus. The high dissemination and transmission rates for WNV under a realistic temperature regime in Ae. japonicus together with recent findings on its opportunistic feeding behavior (mammals and birds) indicate its potential role in WNV transmission in Central Europe where it is highly abundant.

Blood-feeding, susceptibility to infection with Schmallenberg virus and phylogenetics of Culicoides (Diptera: Ceratopogonidae) from the United Kingdom.

BACKGROUND: Culicoides biting midges (Diptera: Ceratopogonidae) are responsible for the biological transmission of internationally important arboviruses of livestock. In 2011, a novel Orthobunyavirus was discovered in northern Europe causing congenital malformations and abortions in ruminants. From field studies, Culicoides were implicated in the transmission of this virus which was subsequently named Schmallenberg virus (SBV), but to date no assessment of susceptibility to infection of field populations under standardised laboratory conditions has been carried out. We assessed the influence of membrane type (chick skin, collagen, Parafilm M®) when offered in conjunction with an artificial blood-feeding system (Hemotek, UK) on field-collected Culicoides blood-feeding rates. Susceptibility to infection with SBV following blood-feeding on an SBV-blood suspension provided via either (i) the Hemotek system or via (ii) a saturated cotton wool pledglet was then compared. Schmallenberg virus susceptibility was defined by RT-qPCR of RNA extractions of head homogenates and related to Culicoides species and haplotype identifications based on the DNA barcode region of the mitochondrial cytochrome c oxidase 1 (cox1) gene. RESULTS: Culicoides blood-feeding rates were low across all membrane types tested (7.5% chick skin, 0.0% for collagen, 4.4% Parafilm M®, with 6029 female Culicoides being offered a blood meal in total). Susceptibility to infection with SBV through membrane blood-feeding (8 of 109 individuals tested) and pledglet blood-feeding (1 of 94 individuals tested) was demonstrated for the Obsoletus complex, with both C. obsoletus (Meigen) and C. scoticus Downes & Kettle susceptible to infection with SBV through oral feeding. Potential evidence of cryptic species within UK populations was found for the Obsoletus complex in phylogenetic analyses of cox1 DNA barcodes of 74 individuals assessed from a single field-site. CONCLUSIONS: Methods described in this study provide the means to blood-feed Palaearctic Culicoides for vector competence studies and colonisation attempts. Susceptibility to SBV infection was 7.3% for membrane-fed members of the subgenus Avaritia and 1.1% for pledglet-fed. Both C. obsoletus and C. scoticus were confirmed as being susceptible to infection with SBV, with potential evidence of cryptic species within UK Obsoletus complex specimens, however the implications of cryptic diversity in the Obsoletus complex on arbovirus transmission remains unknown.

Culicoides-virus interactions: infection barriers and possible factors underlying vector competence.

In the United States, Culicoides midges vector arboviruses of economic importance such as Bluetongue Virus and Epizootic Hemorrhagic Disease Virus. A limited number of studies have demonstrated the complexities of midge-virus interactions, including dynamic changes in virus titer and prevalence over the infection time course. These dynamics are, in part, dictated by mesenteron infection and escape barriers. This review summarizes the overarching trends in viral titer and prevalence throughout the course of infection. Essential barriers to infection and dissemination in the midge are highlighted, along with heritable and extrinsic factors that likely contribute to these barriers. Next generation molecular tools and techniques, now available for Culicoides midges, give researchers the opportunity to test how these factors contribute to vector competence.

Advancing vector biology research: a community survey for future directions, research applications and infrastructure requirements.

Vector-borne pathogens impact public health, animal production, and animal welfare. Research on arthropod vectors such as mosquitoes, ticks, sandflies, and midges which transmit pathogens to humans and economically important animals is crucial for development of new control measures that target transmission by the vector. While insecticides are an important part of this arsenal, appearance of resistance mechanisms is increasingly common. Novel tools for genetic manipulation of vectors, use of Wolbachia endosymbiotic bacteria, and other biological control mechanisms to prevent pathogen transmission have led to promising new intervention strategies, adding to strong interest in vector biology and genetics as well as vector-pathogen interactions. Vector research is therefore at a crucial juncture, and strategic decisions on future research directions and research infrastructure investment should be informed by the research community. A survey initiated by the European Horizon 2020 INFRAVEC-2 consortium set out to canvass priorities in the vector biology research community and to determine key activities that are needed for researchers to efficiently study vectors, vector-pathogen interactions, as well as access the structures and services that allow such activities to be carried out. We summarize the most important findings of the survey which in particular reflect the priorities of researchers in European countries, and which will be of use to stakeholders that include researchers, government, and research organizations.

Using shared needles for subcutaneous inoculation can transmit bluetongue virus mechanically between ruminant hosts.

Bluetongue virus (BTV) is an economically important arbovirus of ruminants that is transmitted by Culicoides spp. biting midges. BTV infection of ruminants results in a high viraemia, suggesting that repeated sharing of needles between animals could result in its iatrogenic transmission. Studies defining the risk of iatrogenic transmission of blood-borne pathogens by less invasive routes, such as subcutaneous or intradermal inoculations are rare, even though the sharing of needles is common practice for these inoculation routes in the veterinary sector. Here we demonstrate that BTV can be transmitted by needle sharing during subcutaneous inoculation, despite the absence of visible blood contamination of the needles. The incubation period, measured from sharing of needles, to detection of BTV in the recipient sheep or cattle, was substantially longer than has previously been reported after experimental infection of ruminants by either direct inoculation of virus, or through blood feeding by infected Culicoides. Although such mechanical transmission is most likely rare under field condition, these results are likely to influence future advice given in relation to sharing needles during veterinary vaccination campaigns and will also be of interest for the public health sector considering the risk of pathogen transmission during subcutaneous inoculations with re-used needles.