Impact of VP2 structure on antigenicity: comparison of BTV1 and the highly virulent BTV8 serotype.

Bluetongue virus (BTV) is an agriculturally and economically significant insect-borne virus that causes serious illness and death in sheep and other domestic and wild ruminants in large areas of the world. Numerous BTV serotypes exist, and distant serotypes exhibit unique neutralizing antibody profiles, which target the outermost capsid protein VP2. The predominant serotype-specific nature of the antibody response to VP2 is a barrier to the development of broad-spectrum prophylactic BTV vaccine candidates. Although VP2 is the main serotype determinant of BTV, the structural basis of serotype specificity has not been investigated. In this study, we utilized the recently available atomic structure of VP2 with a modeled tip domain to carry out in silico structural comparisons between distant serotypes BTV1 and BTV8. These analyses identified structural differences in the tip domain, positioned at the apex of VP2, and informed the design of mutant VP2 constructs. Dissection of tip domain antigenicity demonstrated that the region of structural difference between BTV1 and highly virulent BTV8 was a target of BTV neutralizing antibodies and that mutation of this region resulted in a loss of neutralizing antibody recognition. This study has for the first time provided insights into the structural differences, which underpin the serotype-specific neutralizing antibody response to BTV.IMPORTANCEThe immune system can protect against virus infection by producing antibodies, which bind and inhibit the virus from infecting the susceptible host. These antibodies are termed neutralizing antibodies and generally target the viral receptor binding protein, such as the VP2 of bluetongue virus (BTV). This pressure from the immune system can drive mutation of the viral protein resulting in escape from antibody-mediated neutralization and the evolution of serotypes, as is the case for BTV. Understanding the structural differences, which underpin the different BTV serotypes, could help guide the design of a BTV vaccine that targets multiple serotypes. In this study, we have mapped the VP2 structural differences between distant serotypes, to a region targeted by neutralizing antibodies, and have demonstrated for the first time how VP2 structure is the fundamental basis of serotype specificity.

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.

Climate correlates of bluetongue incidence in southern Portugal.

Model forecasts of the spatiotemporal occurrence dynamics of diseases are necessary and can help understand and thus manage future disease outbreaks. In our study, we used ecological niche modelling to assess the impact of climate on the vector suitability for bluetongue disease, a disease affecting livestock production with important economic consequences. Specifically, we investigated the relationship between the occurrence of bluetongue outbreaks and the environmental suitability of each of the four vector species studied. We found that the main vector for bluetongue disease, Culicoides imicola, a typically tropical and subtropical species, was a strong predictor for disease outbreak occurrence in a region of southern Portugal from 2004 to 2021. The results highlight the importance of understanding the climatic factors that might influence vector presence to help manage infectious disease impacts. When diseases impact economically relevant species, the impacts go beyond mortality and have important economic consequences.

Isolation and comparative analysis of culturable bacterial communities associated with life stages, breeding and rearing substrates of Culicoides oxystoma Kieffer (Diptera: Ceratopogonidae) vector of bluetongue virus.

Culicoides oxystoma Kieffer (Diptera: Ceratopogonidae) has been vectoring several arboviruses, protozoa and nematodes, leading to mortality and morbidity of livestock and wild ruminants in the tropics and subtropics. Insight into the bacterial communities associated with the vector species must be worked out. This work tries to inventorize the bacterial communities associated with this important vector species. Acquisition of gut microbiota may be the parental origin, while some are obtained through feeding during larval stages. Culicoides oxystoma possesses semi-aquatic life cycle strategies for egg-laying and larval survival. The bacteria associated with C. oxystoma were compared throughout (i) life stages: egg, larval instars, pupa, adult: male and female obtained from laboratory colony; (ii) field-collected adult: male and age-graded females; and (iii) natural breeding substrate and artificial rearing substrate. The culture-dependent bacteria were identified by Sanger sequencing of 16S rRNA, and haemolytic bacteria were screened on blood agar. Results show that Firmicutes and Proteobacteria are the predominant Phyla, of which Bacillus spp. was the most abundant across the life stages. Across the life history, Bacillus cereus, Bacillus pumilus, Bacillus tropicus, Lysinibacillus sp. and Paenibacillus sp. were retrieved routinely. Bacillus cereus and Alcaligenes faecalis were detected in the lab-reared specimens and shared between the natural breeding site and rearing medium. From the adults trapped across two locations, B. cereus, Bacillus flexus, A. faecalis, Enterococcus faecium and Pseudomonas sp. were isolated. The bacterial species associated with this vector may influence various physiological traits, such as vectorial capacity, digestion and larval development, which need further investigation.

Preliminary investigation reveals novel pathological consequences of bluetongue virus-1 infection in the endocrine glands of pregnant Indian sheep.

Bluetongue virus (BTV), a major peril to the sheep industry, infects a wide range of the cells in the infected animals including mononuclear, dendritic and epithelial cells. However, little is known about its tropism for the secretory epithelial cells of endocrine glands and the pathogenesis it induces. The aim of the study was to assess the BTV load, antigen distribution in the tissue of the pituitary, thyroid as well as adrenal glands and associated histopathological consequences. BTV antigens were localized using immunohistochemistry in the thyroid's epithelial cells, zona fasciculata and zona reticularis cells and the anterior pituitary epithelial cells. The real-time PCR portrayed the high viral load in adrenals at 7th days postinoculation (DPI) and in thyroid and pituitary glands at 15th DPI. Serum examination revealed variation in the T-3 and T-4 of infected animals in comparison to the control group. Caspase-3 immunolocalization revealed BTV-1 induces apoptosis in the affected cells of endocrine gland of infected animals. Further, this study signifies the tropism of BTV in the novel sites (endocrine glands) of the host that might be one of the reasons for the poor performance of infected animals.

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.

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.

Molecular identification of Culicoides oxystoma and Culicoides actoni vectors of bluetongue virus.

Bluetongue is a non-contagious viral disease causing significant economic losses throughout the world. The bluetongue vectors Culicoides oxystoma and Culicoides actoni, which play a significant role in the transmission of various pathogens, are distributed across different geographical realms. Adults are minute in size with wide phenotypic variation, so morphology-based species identification is severely constrained by preparatory time and shortage of taxonomic expertise. To make the identification process rapid and effective, a specific primer was designed for the identification of C. actoni based on the multiple sequence alignment of ITS1 sequences of 11 Culicoides species. Along with this, a refined version of existing C. oxystoma specific primer was proposed. The primer sets distinguished C. oxystoma and C. actoni from a pooled sample consisting of other Culicoides species as well as closely related genera such as Forcipomyia and Alluaudomyia. Our findings suggest that the primers were species specific, sensitive and have potential to discriminate vector species C. oxystoma and C. actoni from pooled samples. To the best of our knowledge, these are the first ITS1 sequences generated and submitted in GenBank for Culicoides innoxius, Culicoides shortti, Culicoides palpifer and Culicoides anophelis and the first for Culicoides peregrinus, Culicoides fulvus and C. actoni from India.

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.

Assessing the export trade risk of bluetongue virus serotypes 4 and 8 in France.

Bluetongue (BT) causes an economic loss of $3 billion every year in the world. After two serious occurrences of BT (bluetongue virus [BTV] occurrence in 2006 and 2015), France has been controlling for decades, but it has not been eradicated. As the largest live cattle export market in the world, France is also one of the major exporters of breeding animals and genetic materials in the world. The biosafety of its exported cattle and products has always been a concern. The scenario tree quantitative model was used to analyze the risk of BTV release from French exported live cattle and bovine semen. The results showed that with the increase in vaccination coverage rates, the risk decreased. If the vaccine coverage is 0%, the areas with the highest average risk probability of BTV-4 and BTV-8 release from exported live cattle were Haute-Savoie and Puy-de-Dôme, and the risk was 2.96 × 10-4 and 4.25 × 10-4 , respectively. When the vaccine coverage was 90%, the risk probability of BTV-4 and BTV-8 release from exported live cattle was 2.96 × 10-5 and 4.24 × 10-5 , respectively. The average probability of BTV-8 release from bovine semen was 1.09 × 10-10 . Sensitivity analysis showed that the probability of false negative polymerase chain reaction (PCR) test and the probability of BT infection in the bull breeding station had an impact on the model. The identification of high-risk areas and the discovery of key control measures provide a reference for decision makers to assess the risk of French exports of live cattle and bovine semen.

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.

Mapping the pH Sensors Critical for Host Cell Entry by a Complex Nonenveloped Virus.

Bluetongue virus (BTV), in the family Reoviridae, is an insect-borne, double-capsid virus causing hemorrhagic disease in livestock around the world. Here, we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5 and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins, and various in vitro and in vivo assays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid nonenveloped virus.IMPORTANCE Virus entry into a susceptible cell is the first step of infection and a significant point at which infection can be prevented. To enter effectively, viruses must sense the cellular environment and, when appropriate, initiate a series of changes that eventually jettison the protective shell and deposit virus genes into the cytoplasm. Many viruses sense pH, but how this happens and the events that follow are often poorly understood. Here, we address this question for a large multilayered bluetongue virus. We show key residues in outer capsid proteins, a pH-sensing histidine of a zinc finger within the receptor-binding VP2 protein, and certain histidine residues in the membrane-penetrating VP5 protein that detect cellular pH, leading to irreversible changes and propel the virus through the cell membrane. Our data reveal a novel mechanism of cell entry for a nonenveloped virus and highlight mechanisms which may also be used by other viruses.

In silico, in ovo and in vitro antiviral efficacy of phosphorylated derivatives of abacavir: an experimental approach.

Outstanding increase of oral absorption, bioavailability, and antiviral efficacy of phosphorylated nucleosides and basic antiviral influence of abacavir is the central idea for the development of new series of phosphorylated abacavir (ABC) derivatives. The designed compounds were primarily screened for antiviral nature against HN protein of NDV and VP7 protein of BTV using the molecular environment approach. Out of all the designed compounds, the compounds which are having higher binding energies against these two viral strains were prompted for the synthesis of the target compounds (5A-K). Among the synthesized title compounds (5A-K), the compounds which have exhibited higher dock scores akin to the rest of the compounds were then selected and screened for the antiviral activity against NDV and BTV infected embryonated eggs and BHK 21 cell lines through the in ovo and in vitro approaches. The results revealed that all the designed compounds have formed higher binding energies against both the targets. Among all, the compounds which are selected based on their dock scores such as 5A, 5F, 5G, 5H, 5I, and 5K against NDV and 5J, 5E, 5I, 5C, 5A, and 5K against BTV have shown significant antiviral activity against HN protein of NDV, VP7 protein of Bluetongue virus in both NDV- and BTV-treated embryonated eggs and BHK 21 cell lines. Hence, it is concluded that, the best lead compounds will stand as the potential antiviral agents and prompted them as virtuous therapeutics against NDV and BTV in future.

VP2 Gene-Based Molecular Evolutionary Patterns of Major Circulating Bluetongue Virus Serotypes Isolated during 2014-2018 from Telangana and Andhra Pradesh States of India.

INTRODUCTION: Bluetongue disease is an economically important viral disease of livestock caused by bluetongue virus (BTV) having multiple serotypes. It belongs to the genus Orbivirus of family Reoviridae and subfamily Sedoreovirinae. The genome of BTV is 10 segmented dsRNA that codes for 7 structural and 4 nonstructural proteins, of which VP2 was reported to be serotype-specific and a major antigenic determinant. OBJECTIVE: It is important to know the circulating serotypes in a particular geographical location for effective control of the disease. The present study unravels the molecular evolution of the circulating BTV serotypes during 2014-2018 in Telangana and Andhra Pradesh states of India. METHODS: Multiple sequence alignment with available BTV serotypes in GenBank and phylogenetic analysis were performed for the partial VP2 sequences of major circulating BTV serotypes during the study period. RESULTS: The multiple sequence alignment of circulating serotypes with respective reference isolates revealed variations in antigenic VP2. The phylogenetic analysis revealed that the major circulating serotypes were grouped into eastern topotypes (BTV-1, BTV-2, BTV-4, and BTV-16) and Western topotypes (BTV-5, BTV-12, and BTV-24). CONCLUSION: Our study strengthens the need for development of an effective vaccine, which can induce the immune response for a range of serotypes within and in between topotypes.

Epizootic Hemorrhagic Disease in White-Tailed Deer, Canada.

Epizootic hemorrhagic disease affects wild and domestic ruminants and has recently spread northward within the United States. In September 2017, we detected epizootic hemorrhagic disease virus in wild white-tailed deer, Odocoileus virginianus, in east-central Canada. Culicoides spp. midges of the subgenus Avaritia were the most common potential vectors identified on site.

Interaction between a Unique Minor Protein and a Major Capsid Protein of Bluetongue Virus Controls Virus Infectivity.

Among the Reoviridae family of double-stranded RNA viruses, only members of the Orbivirus genus possess a unique structural protein, termed VP6, within their particles. Bluetongue virus (BTV), an important livestock pathogen, is the prototype Orbivirus BTV VP6 is an ATP-dependent RNA helicase, and it is indispensable for virus replication. In the study described in this report, we investigated how VP6 might be recruited to the virus capsid and whether the BTV structural protein VP3, which forms the internal layer of the virus capsid core, is involved in VP6 recruitment. We first demonstrated that VP6 interacts with VP3 and colocalizes with VP3 during capsid assembly. A series of VP6 mutants was then generated, and in combination with immunoprecipitation and size exclusion chromatographic analyses, we demonstrated that VP6 directly interacts with VP3 via a specific region of the C-terminal portion of VP6. Finally, using our reverse genetics system, mutant VP6 proteins were introduced into the BTV genome and interactions between VP6 and VP3 were shown in a live cell system. We demonstrate that BTV strains possessing a mutant VP6 are replication deficient in wild-type BSR cells and fail to recruit the viral replicase complex into the virus particle core. Taken together, these data suggest that the interaction between VP3 and VP6 could be important in the packaging of the viral genome and early stages of particle formation.IMPORTANCE The orbivirus bluetongue virus (BTV) is the causative agent of bluetongue disease of livestock, often causing significant economic and agricultural impacts in the livestock industry. In the study described in this report, we identified the essential region and residues of the unique orbivirus capsid protein VP6 which are responsible for its interaction with other BTV proteins and its subsequent recruitment into the virus particle. The nature and mechanism of these interactions suggest that VP6 has a key role in packaging of the BTV genome into the virus particle. As such, this is a highly significant finding, as this new understanding of BTV assembly could be exploited to design novel vaccines and antivirals against bluetongue disease.

Biological tumour volumes of gliomas in early and standard 20-40 min 18F-FET PET images differ according to IDH mutation status.

PURPOSE: For the clinical evaluation of O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) PET images, the use of standard summation images obtained 20-40 min after injection is recommended. However, early summation images obtained 5-15 min after injection have been reported to allow better differentiation between low-grade glioma (LGG) and high-grade glioma (HGG) by capturing the early 18F-FET uptake peak specific for HGG. We compared early and standard summation images with regard to delineation of the PET-derived biological tumour volume (BTV) in correlation with the molecular genetic profile according the updated 2016 WHO classification. METHODS: The analysis included 245 patients with newly diagnosed, histologically verified glioma and a positive 18F-FET PET scan prior to any further treatment. BTVs were delineated during the early 5-15 min and standard 20-40 min time frames using a threshold of 1.6 × background activity and were compared intraindividually. Volume differences between early and late summation images of >20% were considered significant and were correlated with WHO grade and the molecular genetic profile (IDH mutation and 1p/19q codeletion status). RESULTS: In 52.2% of the patients (128/245), a significant difference in BTV of >20% between early and standard summation images was found. While 44.3% of WHO grade II gliomas (31 of 70) showed a significantly smaller BTV in the early summation images, 35.0% of WHO grade III gliomas (28/80) and 37.9% of WHO grade IV gliomas (36/95) had a significantly larger BTVs. Among IDH-wildtype gliomas, an even higher portion (44.4%, 67/151) showed significantly larger BTVs in the early summation images, which was observed in 5.3% (5/94) of IDH-mutant gliomas only: most of the latter had significantly smaller BTVs in the early summation images, i.e. 51.2% of IDH-mutant gliomas without 1p/19q codeletion (21/41) and 39.6% with 1p/19q codeletion (21/53). CONCLUSION: BTVs delineated in early and standard summation images differed significantly in more than half of gliomas. While the standard summation images seem appropriate for delineation of LGG as well as IDH-mutant gliomas, a remarkably high percentage of HGG and, particularly, IDH-wildtype gliomas were depicted with significantly larger volumes in early summation images. This finding might be of interest for optimization of treatment planning (e.g. radiotherapy) in accordance with the individual IDH mutation status.

Revealing potential bridge vectors for BTV and SBV: a study on Culicoides blood feeding preferences in natural ecosystems in Spain.

Several species of Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens, such as the bluetongue (BTV) and Schmallenberg (SBV) viruses, which cause important diseases in domestic and wild ruminants. As wild ruminants can contribute to overwintering and epizootics of both diseases, knowledge of the host-feeding behaviour of Culicoides in natural ecosystems is important to better understand their epidemiology. Blood-engorged Culicoides females trapped in natural areas inhabited by different wild ruminant species were genetically analysed to identify host species. The origin of bloodmeals was identified in 114 females of 14 species of Culicoides. A total of 104 (91.1%) Culicoides fed on mammals and 10 (8.9%) on birds. The most abundant host identified was red deer (66.7%), followed by humans (13%) and fallow deer (6.1%). Eleven of the 14 species of Culicoides fed exclusively on mammalian hosts. Among them, five are mammalophilic species considered to be important BTV and/or SBV vectors. The results of the present study confirm that Culicoides imicola, Culicoides obsoletus, Culicoides scoticus, Culicoides pulicaris and Culicoides punctatus fed on wild ruminants, and therefore support the hypothesis that these species can act as bridge vectors by facilitating the circulation of pathogens between wild and domestic ruminant communities.

Proteomic analysis of sheep primary testicular cells infected with bluetongue virus.

Bluetongue virus (BTV) causes a non-contagious, arthropod-transmitted disease in wild and domestic ruminants, such as sheep. In this study, we used iTRAQ labeling coupled with LC-MS/MS for quantitative identification of differentially expressed proteins in BTV-infected sheep testicular (ST) cells. Relative quantitative data were obtained for 4455 proteins in BTV- and mock-infected ST cells, among which 101 and 479 proteins were differentially expressed at 24 and 48 h post-infection, respectively, indicating further proteomic changes during the later stages of infection. Ten corresponding genes of differentially expressed proteins were validated via real-time RT-PCR. Expression levels of three representative proteins, eIF4a1, STAT1 and HSP27, were further confirmed via western blot analysis. Bioinformatics analysis disclosed that the differentially expressed proteins are primarily involved in biological processes related to innate immune response, signal transduction, nucleocytoplasmic transport, transcription and apoptosis. Several upregulated proteins were associated with the RIG-I-like receptor signaling pathway and endocytosis. To our knowledge, this study represents the first attempt to investigate proteome-wide dysregulation in BTV-infected cells with the aid of quantitative proteomics. Our collective results not only enhance understanding of the host response to BTV infection but also highlight multiple potential targets for the development of antiviral agents.