Bluetongue Virus Serotype Research Articles

Pathogen detection and disease diagnosis in wildlife: challenges and opportunities.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.

Diagnostic applications of molecular and serological assays for bluetongue and African horse sickness.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.

A novel immunohistochemistry slide adhesive 3-(trimethoxysilyl) propyl methacrylate (tpm) for bluetongue virus serotype-1 antigen detection in ovine foetal tissues

The present study describes the use of 3-(trimethoxysilyl) propyl methacrylate (TPM) as a glass slide adhesive for better tissue adherence in immunohistochemistry. TPM is an alkoxysilane, dissolved in acidified water (pH 3.5) with glacial acetic acid. TPM formed a gel cast on the glass slide surface, which enhanced the net positive charge of the slide. Tissues with net negative charge adhered with gel cast adhesive on the slide surface with covalent bonding. Practically, foetal tissues have less tissue adherence capacity due to less net negative charge. Foetal brain, pancreas, lymph node and umbilical cord of sheep were included in the study. Foetal tissues were collected and fixed in Bouin’s fixative and processed as per standard paraffin embedding technique. Immunohistochemistry was performed on foetal tissues and umbilical cord using three different glass slide adhesive such as poly-L-lysin (PLL), aminopropyl triethoxy silane (APES) and TPM under different antigen retrieval system. TPM significantly enhanced the foetal tissue adherence, when compared with commercially available adhesives such as PLL and APES. In the present study, foetal tissues adhered with TPM coating well tolerated the harsh proteinase K antigen retrieval treatment. Comparatively, TPM coated slides revealed mild to zero tissue loss and less to no background staining. In addition, TPM was cost effective as many commercially available slide adhesives were either costlier or needed accessory chemicals for coating which in turn added the cost per slide. The present study showed that TPM was cost-effective, less hazardous, less time consuming, easy-to-use adhesive for manual preparation of coating slides when compared to other protein adhesive systems. The present experiment also emphasized that tissues with less adherence capacity could be coated with TPM adhesive for best IHC results.

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.

Viral Vector Vaccines against Bluetongue Virus.

Bluetongue virus (BTV), the prototype member of the genus Orbivirus (family Reoviridae), is the causative agent of an important livestock disease, bluetongue (BT), which is transmitted via biting midges of the genus Culicoides. To date, up to 29 serotypes of BTV have been described, which are classified as classical (BTV 1–24) or atypical (serotypes 25–27), and its distribution has been expanding since 1998, with important outbreaks in the Mediterranean Basin and devastating incursions in Northern and Western Europe. Classical vaccine approaches, such as live-attenuated and inactivated vaccines, have been used as prophylactic measures to control BT through the years. However, these vaccine approaches fail to address important matters like vaccine safety profile, effectiveness, induction of a cross-protective immune response among serotypes, and implementation of a DIVA (differentiation of infected from vaccinated animals) strategy. In this context, a wide range of recombinant vaccine prototypes against BTV, ranging from subunit vaccines to recombinant viral vector vaccines, have been investigated. This article offers a comprehensive outline of the live viral vectors used against BTV.

The Tip Region on VP2 Protein of Bluetongue Virus Contains Potential IL-4-Inducing Amino Acid Peptide Segments.

Bluetongue is an infectious viral hemorrhagic disease of domestic and wild ruminants that has a considerable economic impact on domestic ruminants. There are currently at least 29 serotypes of bluetongue virus (BTV) in the world. Noteworthily, the pathogenesis among BTV serotypes is different, even in the same animal species. In this study, BTV2/KM/2003 and BTV12/PT/2003 were used to investigate the differential immunological effects on bovine peripheral blood mononuclear cells (PBMCs). The BTV viral load and the expression of cytokine messenger RNA (mRNA) in PBMCs were measured by fluorescence-based real-time reverse-transcription PCR (qRT-PCR). The immunofluorescence assay (IFA) was applied to detect BTV signals in monocyte-derived macrophages (MDMs). The SWISS-MODEL and IL-4pred prediction tools were used to predict the interleukin 4 (IL-4)-inducing peptides in BTV-coat protein VP2. Synthetic peptides of VP2 were used to stimulate PBMCs for IL-4-inducing capability. This study demonstrated that the cytokine profiles of BTV-induced PBMCs were significantly different between BTV2/KM/2003 and BTV12/PT/2003. BTV2 preferentially activated the T helper 2 (Th2) pathway, represented by the early induction of IL-4, and likely fed back to inhibit the innate immunity. In contrast, BTV12 preferentially activated the innate immunity, represented by the induction of tumor necrosis factor -α (TNF-α) and interleukin 1 (IL-1), with only minimal subsequent IL-4. The BTV nonstructural protein 3 antibody (anti-BTV-NS3) fluorescent signals demonstrated that monocytes in PBMCs and MDMs were the preferred targets of BTV replication. Bioinformatics analysis revealed that the capability to induce IL-4 was attributed to the tip region of the VP2 protein, wherein a higher number of predicted peptide segments on BTVs were positively correlated with the allergic reaction reported in cattle. Synthetic peptides of BTV2-VP2 induced significant IL-4 within 12–24 h post-infection (hpi) in PBMCs, whereas those of BTV12 did not, consistent with the bioinformatics prediction. Bovine PBMCs and synthetic peptides together seem to serve as a good model for pursuing the BTV-induced IL-4 activity that precedes the development of an allergic reaction, although further optimization of the protocol is warranted.

Bluetongue virus serotype 12 enters Australia - a further incursion of novel western lineage genome segments.

Bluetongue virus (BTV) is an arbovirus (genus: Orbivirus) that occurs worldwide. It infects domestic and wild ruminant species and can cause disease in livestock, producing high economic impact. Recently, it gained extra prominence throughout Europe, with disease occurring in regions traditionally free of BTV. BTV enters Australia from Southeast Asia via wind-borne infected Culicoides spp. The first Australian isolation was 1975 (BTV-20) and further serotypes were isolated between 1979-86 (BTV-1, -3, -9, -15, -16, -21, -23). Despite increased, more sensitive, monitoring, no more were detected in over two decades, implying a stable BTV episystem of eastern ancestry. Isolations of BTV-2, -7 and -5 then occurred between 2007-15, with the latter two possessing genome segments with high sequence identity to western isolates. We report on the first isolation and genomic characterization of BTV-12, which revealed that three more novel western topotype gene segments have entered northern Australia.

Recombinant Rift Valley fever viruses encoding bluetongue virus (BTV) antigens: Immunity and efficacy studies upon a BTV-4 challenge

Many ruminant diseases of viral aetiology can be effectively prevented using appropriate vaccination measures. For diseases such as Rift Valley fever (RVF) the long inter-epizootic periods make routine vaccination programs unfeasible. Coupling RVF prophylaxis with seasonal vaccination programmes by means of multivalent vaccine platforms would help to reduce the risk of new RVF outbreaks. In this work we generated recombinant attenuated Rift Valley fever viruses (RVFVs) encoding in place of the virulence factor NSs either the VP2 capsid protein or a truncated form of the non-structural NS1 protein of bluetongue virus serotype 4 (BTV-4). The recombinant viruses were able to carry and express the heterologous BTV genes upon consecutive passages in cell cultures. In murine models, a single immunization was sufficient to protect mice upon RVFV challenge and to elicit a specific immune response against BTV-4 antigens that was fully protective after a BTV-4 boost. In sheep, a natural host for RVFV and BTV, both vaccines proved immunogenic although conferred only partial protection after a virulent BTV-4 reassortant Morocco strain challenge. Though additional optimization will be needed to improve the efficacy data against BTV in sheep, our findings warrant further developments of attenuated RVFV as a dual vaccine platform carrying heterologous immune relevant antigens for ruminant diseases in RVF risk areas.

Re-emergence of BTV serotype 4 in North Macedonia, July 2020.

Bluetongue virus serotype 4 (BTV-4) was confirmed in sheep in North Macedonia in July 2020. The full genome of this BTV-4 strain (MKD2020/06) was shown to be most closely related (99.74% nt identity) to the Greek GRE2014/08 and the Hungarian HUN1014 strains, indicating the re-emergence of this BTV serotype in the Balkan region since it was last reported in 2017.

Implications of a conserved region of bluetongue virus protein VP2 in cross-neutralisation of bluetongue virus serotypes.

Bluetongue (BT) is a vector-borne disease of ruminants caused by Bluetongue virus (BTV). Twenty-nine different serotypes of BTV are currently reported throughout the world. The main objective of this study is the development of a subunit vaccine model that could potentially be adapted to provide broad spectrum protection against multiple BTV serotypes, which the conventional vaccines fail to address. To this end, three different BTV proteins (conserved region of viral protein [VP]2, VP5 and NS1) were expressed and purified in an Escherichia coli expression system. The immunogenicity of these proteins was tested in murine models using the MontanideTM ISA 201 VG adjuvant. BALB/c mice were immunised thrice (with individual proteins and a mixture of three proteins) at two-week intervals and were monitored until Day 40 post-infection/vaccination. Protein-specific antibodies directed against the recombinant proteins were detected by indirect enzyme-linked immunosorbent assay. Neutralising antibody (Nab) titres and cross-neutralisation against a range of BTV serotypes (BTV-1, -2, -4, -5, -9, -10, -12, -16, -21, -23 and -24) were determined by serum neutralisation test. The recombinant proteins elicited higher Nab titres compared with the inactivated vaccine group, except for BTV-1, where the inactivated vaccine group elicited higher Nab titres. Additive effect of the three proteins was not observed as the Nab titres generated with a combination of conserved VP2, VP5 and NS1 was similar to those of the individual protein groups. Whilst BTV-12 could only be neutralised by serum raised against the inactivated vaccine group, BTV-5 and -24 could not be neutralised by any of the groups tested. Our cumulative data suggest that the conserved regions of VP2 (cVP2), VP5 and NS1 could play an important part in the novel vaccine design against multiple BTV serotypes. Importantly, given that VP2 was already known to elicit a serotype-specific immune response against BT, we report, for the first time, that the conserved region of VP2 has the ability to induce cross-protective immune response.

Isolation and Cultivation of a New Isolate of BTV-25 and Presumptive Evidence for a Potential Persistent Infection in Healthy Goats.

Recently, several so-called “atypical” Bluetongue virus (BTV) serotypes were discovered, including BTV-25 (Toggenburg virus), in Switzerland. Most “atypical” BTV were identified in small ruminants without clinical signs. In 2018, two goats from a holding in Germany tested positive for BTV-25 genome by RT-qPCR prior to export. After experimental inoculation of the two goats with the BTV-25 positive field blood samples for generation of reference materials, viremia could be observed in one animal. For the first time, the BTV-25-related virus was isolated in cell culture from EDTA-blood and the full genome of isolate “BTV-25-GER2018” could be generated. BTV-25-GER2018 was only incompletely neutralized by ELISA-positive sera. We could monitor the BTV-25 occurrence in the respective affected goat flock of approximately 120 goats over several years. EDTA blood samples were screened with RT-qPCR using a newly developed BTV-25 specific assay. For serological surveillance, serum samples were screened using a commercial cELISA. BTV-25-GER2018 was detected over 4.5 years in the goat flock with intermittent PCR-positivity in some animals, and with or without concomitantly detected antibodies since 2015. We could demonstrate the viral persistence of BTV-25-GER2018 in goats for up to 4.5 years, and the first BTV-25 isolate is now available for further characterization.

Co-Circulation of Multiple Serotypes of Bluetongue Virus in Zambia.

Bluetongue (BT) is an arthropod-borne viral disease of ruminants with serious trade and socio-economic implications. Although the disease has been reported in a number of countries in sub-Saharan Africa, there is currently no information on circulating serotypes and disease distribution in Zambia. Following surveillance for BT in domestic and wild ruminants in Zambia, BT virus (BTV) nucleic acid and antibodies were detected in eight of the 10 provinces of the country. About 40% (87/215) of pooled blood samples from cattle and goats were positive for BTV nucleic acid, while one hartebeest pool (1/43) was positive among wildlife samples. Sequence analysis of segment 2 revealed presence of serotypes 3, 5, 7, 12 and 15, with five nucleotypes (B, E, F, G and J) being identified. Segment 10 phylogeny showed Zambian BTV sequences clustering with Western topotype strains from South Africa, intimating likely transboundary spread of BTV in Southern Africa. Interestingly, two Zambian viruses and one isolate from Israel formed a novel clade, which we designated as Western topotype 4. The high seroprevalence (96.2%) in cattle from Lusaka and Central provinces and co-circulation of multiple serotypes showed that BT is widespread, underscoring the need for prevention and control strategies.

Evaluation of Bluetongue Virus (BTV) Antibodies for the Immunohistochemical Detection of BTV and Other Orbiviruses.

The detection of bluetongue virus (BTV) antigens in formalin-fixed tissues has been challenging; therefore, only a limited number of studies on suitable immunohistochemical approaches have been reported. This study details the successful application of antibodies for the immunohistochemical detection of BTV in BSR variant baby hamster kidney cells (BHK-BSR) and infected sheep lungs that were formalin-fixed and paraffin-embedded (FFPE). BTV reactive antibodies raised against non-structural (NS) proteins 1, 2, and 3/3a and viral structural protein 7 (VP7) were first evaluated on FFPE BTV-infected cell pellets for their ability to detect BTV serotype 1 (BTV-1). Antibodies that were successful in immunolabelling BTV-1 infected cell pellets were further tested, using similar methods, to determine their broader immunoreactivity against a diverse range of BTV and other orbiviruses. Antibodies specific for NS1, NS2, and NS3/3a were able to detect all BTV isolates tested, and the VP7 antibody cross-reacted with all BTV isolates, except BTV-15. The NS1 antibodies were BTV serogroup-specific, while the NS2, NS3/3a, and VP7 antibodies demonstrated immunologic cross-reactivity to related orbiviruses. These antibodies also detected viral antigens in BTV-3 infected sheep lung. This study demonstrates the utility of FFPE-infected cell pellets for the development and validation of BTV immunohistochemistry.

Re-emergence of bluetongue virus serotype 4 in Iberian ibex (Capra pyrenaica) and sympatric livestock in Spain, 2018-2019.

Between early October and mid-December 2018, mortalities were detected in Iberian ibex (Capra pyrenaica) populations in southern Spain. In the same region and period, bluetongue virus (BTV) circulation was also reported in sentinel and clinically affected domestic ruminant herds. Molecular analyses confirmed BTV serotype 4 (BTV-4) infection in eight Iberian ibexes from six hunting areas, and in 46 domestic ruminants from seven herds in close proximity to affected hunting estates. Histopathological analyses revealed vascular changes in several organs, pneumonia, lymphoid depletion, inflammatory mononuclear cell infiltrate and fibrosis as the most frequently observed lesions in the affected Iberian ibexes. Epidemiological and laboratory results indicate that BTV-4 was the main aetiological agent involved in outbreaks detected in Iberian ibex populations during the study period. Sequence analyses indicated that the BTV-4 strain detected in Iberian ibex had high homology (99.4%-100%) with strains isolated in livestock during the same period, and with previous isolates (≥98.9%) from Spain and Mediterranean Basin countries. Further studies are warranted to determine the impact of BTV-4 on the health status of Iberian ibex populations after the outbreaks. The inclusion of this species in the surveillance programme may be useful for early detection of BTV, especially in epidemiological scenarios at the wildlife-livestock interface.

Heterologous Combination of ChAdOx1 and MVA Vectors Expressing Protein NS1 as Vaccination Strategy to Induce Durable and Cross-Protective CD8+ T Cell Immunity to Bluetongue Virus.

The sequence of non-structural protein NS1 of bluetongue virus (BTV), which contains immunodominant CD8+ T cell epitopes, is highly conserved among BTV serotypes, and has therefore become a major tool in the development of a universal BTV vaccine. In this work, we have engineered multiserotype BTV vaccine candidates based on recombinant chimpanzee adenovirus (ChAdOx1) and modified vaccinia virus Ankara (MVA) vectors expressing the NS1 protein of BTV-4 or its truncated form NS1-Nt. A single dose of ChAdOx1-NS1 or ChAdOx1-NS1-Nt induced a moderate CD8+ T cell response and protected IFNAR(-/-) mice against a lethal dose of BTV-4/MOR09, a reassortant strain between BTV-1 and BTV-4, although the animals showed low viremia after infection. Furthermore, IFNAR(-/-) mice immunized with a single dose of ChAdOx1-NS1 were protected after challenge with a lethal dose of BTV-8 in absence of viremia nor clinical signs. Additionally, the heterologous prime-boost ChAdOx1/MVA expressing NS1 or NS1-Nt elicited a robust NS1 specific CD8+ T cell response and protected the animals against BTV-4/MOR09 even 16 weeks after immunization, with undetectable levels of viremia at any time after challenge. Subsequently, the best immunization strategy based on ChAdOx1/MVA-NS1 was assayed in sheep. Non-immunized animals presented fever and viremia levels up to 104 PFU/mL after infection. In contrast, although viremia was detected in immunized sheep, the level of virus in blood was 100 times lower than in non-immunized animals in absence of clinical signs.

BTV-14 Infection in Sheep Elicits Viraemia with Mild Clinical Symptoms.

In 2011, Bluetongue virus serotype 14 (BTV-14) was detected in Russia during routine surveillance, and was subsequently found in a number of European countries. The strain had high sequence similarity to a BTV-14 vaccine strain. We aimed to determine the risk of this BTV-14 strain causing disease in a UK sheep breed. Four Poll Dorset sheep were infected with a Polish isolate of BTV-14 and infection kinetics were monitored over 28 days. BTV RNA was detected in EDTA blood by 4 days post-infection (dpi) and remained detectable at 28 days post-infection (dpi). Peak viraemia occurred at 6 and 7 dpi with Ct values ranging between 24.6 and 27.3 in all infected animals. BTV antibodies were detected by 10 dpi using a commercial ELISA and neutralising antibodies were detected from 10 dpi. BTV was isolated between 6 and 12 dpi. All infected sheep developed mild clinical signs such as reddening of conjunctiva and mucosal membranes, with one sheep demonstrating more overt clinical signs. Two uninoculated control animals remained clinically healthy and did not have detectable BTV RNA or antibodies. The overall mild clinical symptoms caused by this BTV-14 in this highly susceptible sheep breed were in accordance with the asymptomatic infections observed in the affected countries.

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.

Continuous Cell Lines from the European Biting Midge Culicoides nubeculosus (Meigen, 1830).

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase 1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens.

A model for the assessment of bluetongue virus serotype 1 persistence in Spain.

Bluetongue virus (BTV) is an arbovirus of ruminants that has been circulating in Europe continuously for more than two decades and has become endemic in some countries such as Spain. Spain is ideal for BTV epidemiological studies since BTV outbreaks from different sources and serotypes have occurred continuously there since 2000; BTV-1 has been reported there from 2007 to 2017. Here we develop a model for BTV-1 endemic scenario to estimate the risk of an area becoming endemic, as well as to identify the most influential factors for BTV-1 persistence. We created abundance maps at 1-km2 spatial resolution for the main vectors in Spain, Culicoides imicola and Obsoletus and Pulicaris complexes, by combining environmental satellite data with occurrence models and a random forest machine learning algorithm. The endemic model included vector abundance and host-related variables (farm density). The three most relevant variables in the endemic model were the abundance of C. imicola and Obsoletus complex and density of goat farms (AUC 0.86); this model suggests that BTV-1 is more likely to become endemic in central and southwestern regions of Spain. It only requires host- and vector-related variables to identify areas at greater risk of becoming endemic for bluetongue. Our results highlight the importance of suitable Culicoides spp. prediction maps for bluetongue epidemiological studies and decision-making about control and eradication measures.

"Frozen evolution" of an RNA virus suggests accidental release as a potential cause of arbovirus re-emergence.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.