Eva Veronesi

Complete Genome Characterisation of a Novel 26th Bluetongue Virus Serotype from Kuwait

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.

Implicating Culicoides Biting Midges as Vectors of Schmallenberg Virus Using Semi-Quantitative RT-PCR

Background 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 Cq values used to infer the presence of viral RNA in samples. Methodology/Principal Findings 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. Conclusions/Significance 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.

Temperature Dependence of the Extrinsic Incubation Period of Orbiviruses in Culicoides Biting Midges

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Viraemia and clinical disease in Dorset Poll sheep following vaccination with live attenuated bluetongue virus vaccines serotypes 16 and 4

The spread of bluetongue virus (BTV) is most successfully controlled by vaccination of susceptible ruminant populations. Currently two different types of BTV vaccines are used for this purpose; inactivated, mostly monovalent vaccine formulations and modified live virus vaccines (MLVs). Clinical signs and viraemia in Dorset Poll sheep vaccinated with BTV-4 and BTV-16 MLVs or inoculated with homogenates of midges (C. sonorensis and C. nubeculosus) previously infected with BTV-4 MLV are presented. All sheep vaccinated with the two MLVs mounted an infectious viraemia lasting for a minimum of 9 up to 23 days post vaccination and developed a range of clinical signs associated with BTV infection. Peak viraemia titres recorded in individual sheep ranged from 3.5 to 6.83 log(10)TCID(50)/ml indicating a high potential for infection of vector insects and onward transmission. The implications of these results are discussed with reference to the current outbreaks of BTV occurring in northern Europe and in relation to the future development of vaccines for this virus.

Transplacental transmission of bluetongue virus 8 in cattle, UK.

To determine whether transplacental transmission could explain overwintering of bluetongue virus in the United Kingdom, we studied calves born to dams naturally infected during pregnancy in 2007–08. Approximately 33% were infected transplacentally; some had compromised health. In all infected calves, viral load decreased after birth; no evidence of persistent infection was found.

Evidence for Transmission of Bluetongue Virus Serotype 26 through Direct Contact

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.

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture. Methodology/Principal Findings A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (Cq values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination. Conclusions/Significance Cq values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.

Drosophila melanogaster as a Model Organism for Bluetongue Virus Replication and Tropism

ABSTRACT Bluetongue virus (BTV) is the etiological agent of bluetongue (BT), a hemorrhagic disease of ruminants that can cause high levels of morbidity and mortality. BTV is an arbovirus transmitted between its ruminant hosts by Culicoides biting midges (Diptera: Ceratopogonidae). Recently, Europe has experienced some of the largest BT outbreaks ever recorded, including areas with no known history of the disease, leading to unprecedented economic and animal welfare issues. The current lack of genomic resources and genetic tools for Culicoides restricts any detailed study of the mechanisms involved in the virus-insect interactions. In contrast, the genome of the fruit fly (Drosophila melanogaster) has been successfully sequenced, and it is used extensively as a model of molecular pathways due to the existence of powerful genetic technology. In this study, D. melanogaster is investigated as a model for the replication and tropism of BTV. Using reverse genetics, a modified BTV-1 that expresses the fluorescent mCherry protein fused to the viral nonstructural protein NS3 (BTV-1/NS3mCherry) was generated. We demonstrate that BTV-1/NS3mCherry is not only replication competent as it retains many characteristics of the wild-type virus but also replicates efficiently in D. melanogaster after removal of the bacterial endosymbiont Wolbachia pipientis by antibiotic treatment. Furthermore, confocal microscopy shows that the tissue tropism of BTV-1/NS3mCherry in D. melanogaster resembles that described previously for BTV in Culicoides. Overall, the data presented in this study demonstrate the feasibility of using D. melanogaster as a genetic model to investigate BTV-insect interactions that cannot be otherwise addressed in vector species.

Quantifying bluetongue virus in adult Culicoides biting midges (Diptera: Ceratopogonidae).

Abstract A TissueLyser system (QIAGEN) was used to rapidly and accurately estimate bluetongue virus “loads” in individual adult Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae). The optimized homogenization program that was developed, involved shaking insects for 1 min at 25 Hz with 2- or 3-mm stainless steel ball bearings. This program was used to measure the quantities of bluetongue virus present in insects that had either been inoculated or had ingested a viremic bloodmeal through an artificial membrane. The virus titers obtained using either infection technique and the optimized program did not differ significantly from those obtained using a polypropylene motor-driven pestle, a method that is currently in common use for studies of vector competence). The advantages of the new method, as a rapid means of detecting fully disseminated infections in individual field-caught flies, are discussed. Its use is compared with the processing of pools of Culicoides by conventional methods, where the extent of dissemination of the virus is unknown and could wrongly implicate species that are of low importance in transmission.

Testing of UK Populations of Culex pipiens L. for Schmallenberg Virus Vector Competence and Their Colonization

Background Schmallenberg virus (SBV), an arboviral pathogen of ruminants, emerged in northern Europe during 2011 and has subsequently spread across a vast geographic area. While Culicoides biting midges (Diptera: Ceratopogonidae) have been identified as a biological transmission agent of SBV, the role of mosquitoes (Diptera: Culicidae) as potential vectors has not been defined beyond small-scale field collections in affected areas. Culex pipiens L. are one of the most widespread mosquitoes in northern Europe; they are present on farms across the region and have previously been implicated as vectors of several other arboviruses. We assessed the ability of three colony lines of Cx. pipiens, originating from geographically diverse field populations, to become fully infected by SBV using semi-quantitative real-time RT-PCR (sqPCR). Findings Two colony lines of Cx. pipiens were created in the UK (‘Brookwood’ and ‘Caldbeck’) from field collections of larvae and pupae and characterised using genetic markers. A third strain of Cx. pipiens from CVI Wageningen, The Netherlands, was also screened during experiments. Intrathoracic inoculation of the Brookwood line resulted in infections after 14 days that were characterised by high levels of RNA throughout individuals, but which demonstrated indirect evidence of salivary gland barriers. Feeding of 322 individuals across the three colony lines on a membrane based infection system resulted in no evidence of full dissemination of SBV, although infections did occur in a small proportion of Cx. pipiens from each line. Conclusions/Significance This study established two novel lines of Cx. pipiens mosquitoes of UK origin in the laboratory and subsequently tested their competence for SBV. Schmallenberg virus replication and dissemination was restricted, demonstrating that Cx. pipiens is unlikely to be an epidemiologically important vector of the virus in northern Europe.