Four poll Dorset sheep and four Holstein‐Friesian cattle were infected with the northern European strain of bluetongue virus ( btv ), btv‐8 , to assess its pathogenicity in uk breeds. The time course of infection was monitored in both species by using real‐time reverse transcriptase‐ pcr ( rt‐pcr ), conventional rt‐pcr and serology. Two of the sheep developed severe clinical signs that would have been fatal in the field; the other two were moderately and mildly ill, respectively. The cattle were clinically unaffected, but had high levels of viral rna in their bloodstream. Real‐time rt‐pcr detected viral rna as early as one day after infection in the cattle and three days after infection in the sheep. Antibodies against btv were detected by six days after infection in the sheep and eight days after infection in the cattle. Postmortem examinations revealed pathology in the cattle that was more severe than suggested by the mild clinical signs, but the pathological and clinical findings in the sheep were more consistent.

Bluetongue virus is the “type” species of the genus Orbivirus, family Reoviridae. Twenty four distinct bluetongue virus (BTV) serotypes have been recognized for decades, any of which is thought to be capable of causing “bluetongue” (BT), an insect-borne disease of ruminants. However, two further BTV serotypes, BTV-25 (Toggenburg orbivirus, from Switzerland) and BTV-26 (from Kuwait) have recently been identified in goats and sheep, respectively. The BTV genome is composed of ten segments of linear dsRNA, encoding 7 virus-structural proteins (VP1 to VP7) and four distinct non-structural (NS) proteins (NS1 to NS4). We report the entire BTV-26 genome sequence (isolate KUW2010/02) and comparisons to other orbiviruses. Highest identity levels were consistently detected with other BTV strains, identifying KUW2010/02 as BTV. The outer-core protein and major BTV serogroup-specific antigen “VP7” showed 98% aa sequence identity with BTV-25, indicating a common ancestry. However, higher level of variation in the nucleotide sequence of Seg-7 (81.2% identity) suggests strong conservation pressures on the protein of these two strains, and that they diverged a long time ago. Comparisons of Seg-2, encoding major outer-capsid component and cell-attachment protein “VP2” identified KUW2010/02 as 26th BTV, within a 12th Seg-2 nucleotype [nucleotype L]. Comparisons of Seg-6, encoding the smaller outer capsid protein VP5, also showed levels of nt/aa variation consistent with identification of KUW2010/02 as BTV-26 (within a 9th Seg-6 nucleotype - nucleotype I). Sequence data for Seg-2 of KUW2010/02 were used to design four sets of oligonucleotide primers for use in BTV-26, type-specific RT-PCR assays. Analyses of other more conserved genome segments placed KUW2010/02 and BTV-25/SWI2008/01 closer to each other than to other “eastern” or “western” BTV strains, but as representatives of two novel and distinct geographic groups (topotypes). Our analyses indicate that all of the BTV genome segments have evolved under strong purifying selection.

The rate at which viruses replicate and disseminate in competent arthropod vectors is limited by the temperature of their environment, and this can be an important determinant of geographical and seasonal limits to their transmission by arthropods in temperate regions. Here, we present a novel statistical methodology for estimating the relationship between temperature and the extrinsic incubation period (EIP) and apply it to both published and novel data on virus replication for three internationally important orbiviruses (African horse sickness virus (AHSV), bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV)) in their Culicoides vectors. Our analyses show that there can be differences in vector competence for different orbiviruses in the same vector species and for the same orbivirus in different vector species. Both the rate of virus replication (approximately 0.017-0.021 per degree-day) and the minimum temperature required for replication (11-13°C), however, were generally consistent for different orbiviruses and across different Culicoides vector species. The estimates obtained in the present study suggest that previous publications have underestimated the replication rate and threshold temperature because the statistical methods they used included an implicit assumption that all negative vectors were infected. Robust estimates of the temperature dependence of arbovirus replication are essential for building accurate models of transmission and for informing policy decisions about seasonal relaxations to movement restrictions. The methodology developed in this study provides the required robustness and is superior to methods used previously. Importantly, the methods are generic and can readily be applied to other arbovirus-vector systems, as long as the assumptions described in the text are valid.

The aim of this study was to assess the mechanisms of transmission of bluetongue virus serotype 26 (BTV-26) in goats. A previous study, which investigated the pathogenicity and infection kinetics of BTV-26 in goats, unexpectedly revealed that one control goat may have been infected through a direct contact transmission route. To investigate the transmission mechanisms of BTV-26 in more detail an experimental infection study was carried out in which three goats were infected with BTV-26, three goats were kept uninfected, but were housed in direct contact with the infected goats, and an additional four goats were kept in indirect contact separated from infected goats by metal gates. This barrier allowed the goats to have occasional face-to-face contact in the same airspace, but feeding, watering, sampling and environmental cleaning was carried out separately. The three experimentally infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from their blood. At 21 dpi viral RNA was detected in, and virus was isolated from the blood of the three direct contact goats, which also seroconverted. The four indirect barrier contact goats remained uninfected throughout the duration of the experiment. In order to assess replication in a laboratory model species of Culicoides biting midge, more than 300 Culicoides sonorensis were fed a BTV-26 spiked blood meal and incubated for 7 days. The dissemination of BTV-26 in individual C. sonorensis was inferred from the quantity of virus RNA and indicated that none of the insects processed at day 7 possessed transmissible infections. This study shows that BTV-26 is easily transmitted through direct contact transmission between goats, and the strain does not seem to replicate in C. sonorensis midges using standard incubation conditions.

The recent unprecedented emergence of arboviruses transmitted by Culicoides biting midges in northern Europe has necessitated the development of techniques to differentiate competent vector species. At present these techniques are entirely reliant upon interpretation of semi-quantitative RT-PCR (sqPCR) data in the form of C_q values used to infer the presence of viral RNA in samples. This study investigates the advantages and limitations of sqPCR in this role by comparing infection and dissemination rates of Schmallenberg virus (SBV) in two colony lines of Culicoides. Through the use of these behaviorally malleable lines we provide tools for demarcating arbovirus infection and dissemination rates in Culicoides which to date have prevented clear implication of primary vector species in northern Europe. The study demonstrates biological transmission of SBV in an arthropod vector, supporting the conclusions from field-caught Culicoides and provides a general framework for future assessment of vector competence of Culicoides for arboviruses using sqPCR. When adopting novel diagnostic technologies, correctly implicating vectors of arboviral pathogens requires a coherent laboratory framework to fully understand the implications of results produced in the field. This study illustrates these difficulties and provides a full examination of sqPCR in this role for the Culicoides-arbovirus system.