RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells.

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

Bluetongue Virus in the Iberian Lynx (Lynx pardinus), 2010-2022.

Clinical infection and death caused by bluetongue virus infection has been reported in the Eurasian lynx. Bluetongue virus surveillance in the Iberian lynx revealed widespread and repeated exposure to serotypes 1 and 4 in wild and captive populations of this species. This exposure is possibly from a spillover event from sympatric ruminants.

Emergence of Bluetongue Virus Serotype 3, the Netherlands, September 2023.

Since 1998, notifiable bluetongue virus (BTV) serotypes 1-4, 6, 8, 9, 11, and 16 have been reported in Europe. In August 2006, a bluetongue (BT) outbreak caused by BTV serotype 8 began in northwestern Europe. The Netherlands was declared BT-free in February 2012, and annual monitoring continued. On September 3, 2023, typical BT clinical manifestations in sheep were notified to the Netherlands Food and Product Safety Consumer Authority. On September 6, we confirmed BTV infection through laboratory diagnosis; notifications of clinical signs in cattle were also reported. We determined the virus was serotype 3 by whole-genome sequencing. Retrospective analysis did not reveal BTV circulation earlier than September. The virus source and introduction route into the Netherlands remains unknown. Continuous monitoring and molecular diagnostic testing of livestock will be needed to determine virus spread, and new prevention strategies will be required to prevent BTV circulation within the Netherlands and Europe.

Bluetongue Virus Serotype 3 and Schmallenberg Virus in Culicoides Biting Midges, Western Germany, 2023.

In October 2023, bluetongue virus serotype 3 (BTV-3) emerged in Germany, where Schmallenberg virus is enzootic. We detected BTV-3 in 1 pool of Culicoides biting midges collected at the time ruminant infections were reported. Schmallenberg virus was found in many vector pools. Vector trapping and analysis could elucidate viral spread.

The effect of pH on the structure of Bluetongue virus VP5.

The unenveloped Bluetongue virus capsid comprises several structural layers, the inner two comprising a core, which assembles before addition of the outer proteins, VP2 and VP5. Two symmetric trimers of VP5 fit like pegs into two distinct pits on the core and undergo pH conformational changes in the context of the virus, associated with cell entry. Here we show that in isolation VP5 alone undergoes essentially the same changes with pH and confirm a helical transition, indicating that VP5 is a motor during cell entry. In the absence of VP5 the two pits on the core differ from each other, presumably due to the asymmetric underlying structure of VP3, the innermost capsid protein. On insertion of VP5 these pits become closely similar and remain similar at low pH whilst VP5 is present. This natural asymmetry presumably destabilises the attachment of VP5, facilitating ejection upon low pH, membrane penetration and cell entry.

Extensive Schmallenberg virus circulation in Germany, 2023.

Schmallenberg virus (SBV) and bluetongue virus (BTV) are both transmitted by Culicoides biting midges and infect predominantly ruminants. To investigate the extent of virus spread in the 2022 and 2023 vector seasons, we serologically tested wild ruminants from western Germany. While antibodies against BTV were not detected in any animal, regardless of age or sampling time, numerous wild ruminants tested positive for antibodies to SBV. In 2022, a low seroprevalence of 4.92% was measured. In sharp contrast, 40.15% of the animals tested positive in 2023. Of the young animals, about 31.82% were seropositive, clearly indicating large-scale SBV circulation in summer and autumn 2023.

Whole-transcriptome analyses of ovine lung microvascular endothelial cells infected with bluetongue virus.

Bluetongue virus (BTV) infection induces profound and intricate changes in the transcriptional profile of the host to facilitate its survival and replication. However, there have been no whole-transcriptome studies on ovine lung microvascular endothelial cells (OLMECs) infected with BTV. In this study, we comprehensively analysed the whole-transcriptome sequences of BTV-1 serotype-infected and mock-infected OLMECs and subsequently performed bioinformatics differential analysis. Our analysis revealed 1215 differentially expressed mRNA transcripts, 82 differentially expressed long noncoding RNAs (lncRNAs) transcripts, 63 differentially expressed microRNAs (miRNAs) transcripts, and 42 differentially expressed circular RNAs (circRNAs) transcripts. Annotation from Gene Ontology, enrichment from the Kyoto Encyclopedia of Genes and Genomes, and construction of endogenous competing RNA network analysis revealed that the differentially expressed RNAs primarily participated in viral sensing and signal transduction pathways, antiviral and immune responses, inflammation, and extracellular matrix (ECM)-related pathways. Furthermore, protein‒protein interaction network analysis revealed that BTV may regulate the conformation of ECM receptor proteins and change their biological activity through a series of complex mechanisms. Finally, on the basis of real-time fluorescence quantitative polymerase chain reaction results, the expression trends of the differentially expressed RNA were consistent with the whole-transcriptome sequencing data, such as downregulation of the expression of COL4A1, ITGA8, ITGB5, and TNC and upregulation of the expression of CXCL10, RNASEL, IRF3, IRF7, and IFIHI. This study provides a novel perspective for further investigations of the mechanism of the ECM in the BTV-host interactome and the pathogenesis of lung microvascular endothelial cells.

Winter activity of Culicoides (Diptera: Ceratopogonidae) inside and outside stables in Germany.

Culicoides biting midge species (Diptera: Ceratopogonidae) of the Obsoletus Group and the Pulicaris Complex are considered the major vectors of bluetongue and Schmallenberg viruses in Europe. Overwintering strategies of these arboviruses are controversially discussed, with the ongoing activity of vector species and a non-disrupted transmission cycle during winter being a plausible explanation. Although data on Culicoides winter activity are relatively scant, a seasonal vector-free period (SVFP), during which adult Culicoides are not or hardly active, is questionable. To determine winter activity and define SVFPs according to the EU Commission Regulation No 1266/2007, adult Culicoides were trapped weekly with UV-light traps from October to April 2019/2020 and 2020/2021 inside and outside stables on 16 farms throughout Germany. Temperature measurements were taken regularly at each trapping site since the temperature is a known driver of biting midge activity. In 960 indoor and outdoor catches, 32,377 Culicoides were trapped, with 90.9% of them belonging to the Obsoletus Group, 6.1% to the Pulicaris Complex and 3.0% to 'other Culicoides' according to morphological identification. The majority (61.3%) of Culicoides were trapped indoors, with substantial numbers of specimens collected from October to December, in March and in April, and only a few or no specimens in January and February. Obsoletus Group biting midges were active indoors for almost the entire winter. Outdoors, Culicoides numbers decreased from October to December, few or no specimens were caught from January to March, and high numbers were captured in April. Of the collected Culicoides, 2028 were blood-fed, of which 94.6% were trapped in the stables. The indoor SVFP, although calculated for blood-fed instead of parous females, lasted for almost 4 months (late November until mid-March) in winter 2019/2020 and 2 months (January and February) in winter 2020/2021. The outdoor SVFPs covered almost the entire study period in both winters, with slight differences between the onsets and the ends. The Culicoides activity significantly depended on temperature. Specimens of the Obsoletus Group were caught at an average temperature of 7.4°C (minimum 0.3°C) and of the Pulicaris Complex at an average temperature of 10.3°C (minimum 1.2°C). These temperatures were reached inside the stables over more extended periods than outside. The average indoor temperatures were 1.2 K higher than the average outdoor temperatures, although absolute temperature differences of up to 9.0 K were recorded. Based on Culicoides activity, the results of the present study indicate an almost continuous potential for virus transmission in winter within livestock houses.

Quantitative risk assessment for the introduction of bluetongue virus into mainland Europe by long-distance wind dispersal of Culicoides spp.: A case study from Sardinia.

Europe faces regular introductions and reintroductions of bluetongue virus (BTV) serotypes, most recently exemplified by the incursion of serotype 3 in the Netherlands. Although the long-distance wind dispersal of the disease vector, Culicoides spp., is recognized as a virus introduction pathway, it remains understudied in risk assessments. A Quantitative Risk Assessment framework was developed to estimate the risk of BTV-3 incursion into mainland Europe from Sardinia, where the virus has been present since 2018. We used an atmospheric transport model (HYbrid Single-Particle Lagrangian Integrated Trajectory) to infer the probability of airborne dispersion of the insect vector. Epidemiological disease parameters quantified the virus prevalence in vector population in Sardinia and its potential first transmission after introduction in a new area. When assuming a 24h maximal flight duration, the risk of BTV introduction from Sardinia is limited to the Mediterranean Basin, mainly affecting the southwestern area of the Italian Peninsula, Sicily, Malta, and Corsica. The risk extends to the northern and central parts of Italy, Balearic archipelago, and mainland France and Spain, mostly when maximal flight duration is longer than 24h. Additional knowledge on vector flight conditions and Obsoletus complex-specific parameters could improve the robustness of the model. Providing both spatial and temporal insights into BTV introduction risks, our framework is a key tool to guide global surveillance and preparedness against epizootics.

Evaluating Temperature Effects on Bluetongue Virus Serotype 10 and 17 Coinfection in Culicoides sonorensis.

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an understanding of the effect of temperature on reassortment is lacking. The objectives of this study were to compare how temperature affected Culicoides survival, virogenesis, and reassortment in Culicoides sonorensis coinfected with two BTV serotypes. Midges were fed blood meals containing BTV-10, BTV-17, or BTV serotype 10 and 17 and maintained at 20 °C, 25 °C, or 30 °C. Midge survival was assessed, and pools of midges were collected every other day to evaluate virogenesis of BTV via qRT-PCR. Additional pools of coinfected midges were collected for BTV plaque isolation. The genotypes of plaques were determined using next-generation sequencing. Warmer temperatures impacted traits related to vector competence in offsetting ways: BTV replicated faster in midges at warmer temperatures, but midges did not survive as long. Overall, plaques with BTV-17 genotype dominated, but BTV-10 was detected in some plaques, suggesting parental strain fitness may play a role in reassortment outcomes. Temperature adds an important dimension to host-pathogen interactions with implications for transmission and evolution.

Visualisation of Bluetongue Virus in the Salivary Apparatus of Culicoides Biting Midges Highlights the Accessory Glands as a Primary Arboviral Infection Site.

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.

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.

Sialic Acid Binding Sites in VP2 of Bluetongue Virus and Their Use during Virus Entry.

Bluetongue virus (BTV), a member of the Orbivirus genus, is transmitted by biting midges (gnats, Culicoides sp.) and is one of the most widespread animal pathogens, causing serious outbreaks in domestic animals, particularly in sheep, with high economic impact. The non-enveloped BTV particle is a double-capsid structure of seven proteins and a genome of 10 double-stranded RNA segments. Although the outermost spike-like VP2 acts as the attachment protein during BTV entry, no specific host receptor has been identified for BTV. Recent high-resolution cryo-electron (cryoEM) structures and biological data have suggested that VP2 may interact with sialic acids (SAs). To confirm this, we have generated protein-based nanoparticles displaying multivalent VP2 and used them to probe glycan arrays. The data show that VP2 binds α2,3-linked SA with high affinity but also binds α2,6-linked SA. Further, Maackia amurensis lectin II (MAL II) and Sambucus nigra lectin (SNA), which specifically bind α2,3-linked and α2,6-linked SAs, respectively, inhibited BTV infection and virus growth in susceptible sheep cells while SNA alone inhibited virus growth in Culicoides-derived cells. A combination of hydrogen deuterium exchange mass spectrometry and site-directed mutagenesis allowed the identification of the specific SA binding residues of VP2. This study provides direct evidence that sialic acids act as key receptor for BTV and that the outer capsid protein VP2 specifically binds SA during BTV entry in both mammalian and insect cells. IMPORTANCE To date no receptor has been assigned for non-enveloped bluetongue virus. To determine if the outermost spike-like VP2 protein is responsible for host cell attachment via interaction with sialic acids, we first generated a protein-based VP2-nanoparticle, for the multivalent presentation of recombinant VP2 protein. Using nanoparticles displaying VP2 to probe a glycan array, we identified that VP2 binds both α2,3-linked and α2,6-linked sialic acids. Lectin inhibitors targeting both linkages of sialic acids showed strong inhibition to BTV infection and progeny virus production in mammalian cells; however the inhibition was only seen with the lectin targeting α2,6-linked sialic acid in insect vector cells. In addition, we identified the VP2 sialic acid binding sites in the exposed tip domain. Our data provides direct evidence that sialic acids act as key receptors for BTV attachment and entry in to both mammalian and insect cells.

The Combined Expression of the Nonstructural Protein NS1 and the N-Terminal Half of NS2 (NS21-180) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge.

Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.

Characterization of two novel reassortant bluetongue virus serotype 1 strains isolated from farmed white-tailed deer (Odocoileus virginianus) in Florida, USA.

Hemorrhagic diseases caused by epizootic hemorrhagic disease virus or by bluetongue virus (BTV) are the most important orbivirus diseases affecting ruminants, including white-tailed deer (WTD). Bluetongue virus is of particular concern for farmed WTD in Florida, given its lethality and its wide distribution throughout the state. This study reports the clinical findings, ancillary diagnostics, and genomic characterization of two BTV serotype 1 strains isolated from two farmed WTD, from two different farms in Florida in 2019 and 2022. Phylogenetic and genetic analyses indicated that these two novel BTV-1 strains were reassortants. In addition, our analyses reveal that most genome segments of these strains were acquired from BTVs previously detected in ruminants in Florida, substantiating their endemism in the Southeastern U.S. Our findings underscore the need for additional research to determine the genetic diversity of BTV strains in Florida, their prevalence, and the potential risk of new BTV strains to WTD and other ruminants.

A serological survey of pathogens associated with the respiratory and digestive system in the Polish European bison (Bison bonasus) population in 2017-2022.

BACKGROUND: The European bison (Bison bonasus) is a near threatened species and requires health monitoring. The aim of the present study was to determine the prevalence of antibodies to pathogens known to cause respiratory and digestive illness in ruminants. RESULTS: In the studied 328 European bison, the highest seroprevalence was observed for Bovine herpesvirus-1 (BoHV-1) (50.27%), Bovine Coronavirus (BCoV) (26.36%), and Bluetongue Virus (BTV) (12.83%). For Mycoplasma bovis strains and Bovine Viral Diarrhea Virus (BVDV), positive results were rare. Interestingly, a higher prevalence of BTV antibodies was noted in the northeastern populations and older animals. CONCLUSIONS: Our findings indicate that the Polish European bison population appears to have considerable contact with BoHV-1; however, this does not appear to be of great significance, as clinical symptoms and post-mortem lesions are rarely noted in Polish European bison population. The high seroprevalence of BTV in the north-east of Poland is an ongoing trend, also noted in previous studies. It is possible that European bison may perpetuate the virus in this region. This is the first report of antibodies for BCoV in European bison.

Experimental infection of Aedes (Stegomyia) albopictus and Culex pipiens mosquitoes with Bluetongue virus.

Bluetongue disease (BT), caused by Bluetongue virus (BTV), infects wild and domestic ruminants, causing severe economic damage in the cattle and sheep industry. Proven vectors of BTV are biting midges belonging to the Culicoides genus, but other arthropods are considered potential vectors, such as ticks, mosquitoes, wingless flies, and sand flies. The present study represents the first attempt to evaluate the vectorial capacity of Culex pipiens and Aedes albopictus for BTV. Mosquitoes were artificially fed with blood containing BTV serotype 1. Infection, dissemination and transmission rates were evaluated at 0, 3, 7, 14 and 21 days after an infected blood meal. Viral RNA was only detected up to 3 days post infection in the bodies of both species. This study indicates that the two Italian populations of Cx. pipiens and Ae. albopictus are not susceptible to BTV infection.

Development of real-time RT-PCR systems for detection and quantitation of bovine enteric viral pathogens.

The enteric viruses in animals are responsible for severe and devastating losses to the livestock owners with a profound negative impact on animal, health, welfare, and productivity. These viruses are usually transmitted via the feco-oral route and primarily infect the digestive tract of the humans, bovines and different mammals as well as birds. Some of the important enteric viruses in ruminants are: Rotavirus A (RVA), Peste des petits virus (PPRV), Norovirus (NV), Bovine corona virus (BoCV) and Bluetongue virus (BTV). In the present study, sensitive, specific and reliable TaqMan probe-based RT-qPCRs were developed and standardized for the rapid detection and quantification of enteric viruses from fecal samples. The assays result in efficient amplification of the RVA, BTV and BoCV RNA with a limit of detection (LoD) of 5, 5 and 4 copies, respectively, which is 1000 times more sensitive than the traditional gel-based RT-PCR. The reproducibility of each assay was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. In conclusion, real time PCR developed for these viruses are highly specific and sensitive technique for the detection of diarrheic viral pathogens of cattle and buffalo.

Identification of Orbivirus Non-Structural Protein 5 (NS5), Its Role and Interaction with RNA/DNA in Infected Cells.

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids. Confocal microscopy shows that although NS5 accumulates in the nucleus, particularly in the nucleolus, which becomes disrupted, it also appears in the cell cytoplasm, co-localising with mitochondria. NS5 helps to prevent the degradation of ribosomal RNAs during infection and reduces host-cell protein synthesis However, it helps to extend cell viability by supporting viral protein synthesis and virus replication. Pulldown studies showed that NS5 binds to ssRNAs and supercoiled DNAs and demonstrates interactions with ZBP1, suggesting that it modulates host-cell responses.

Blood meal analysis of Culicoides species associated with livestock in West Bengal, India.

Knowledge gaps exist on the feeding pattern and host range of bluetongue virus vectors, Culicoides species, associated with livestock in India. Adult midges were trapped with ultraviolet light traps at 13 household farms adjacent to human biotope. Host DNA was isolated from individual females (n = 101; blood engorged-82, gravid-4 and parous-15) and subjected to PCR amplification targeting CytB and 16S rRNA gene fragments followed by sequencing of amplified DNA samples. However, DNA sequences from only 71 individuals (70.3%) comprising of 10 Culicoides species were obtained. Blood meal analysis revealed at least 10 species that fed on five mammalian hosts including humans, but surprisingly none tested positive for birds. Results revealed that Culicoides innoxius tested positive for four not previously recognized species indicating a potential role as a vector species. Likewise, Culicoides shortti and Culicoides hegneri preferred goat and cattle respectively as hosts, whereas Culicoides palpifer preferred cattle along with buffalo as hosts, which is being reported for the first time. This is the first document on DNA-based blood meal identification and feeding preference of Culicoides midges associated with livestock in India.