Bluetongue virus serotype 8 virus-like particles protect sheep against virulent virus infection as a single or multi-serotype cocktail immunogen

Since 1998, there have been multiple separate outbreaks of Bluetongue disease (BT) in Europe with the largest outbreak ever recorded in Northern Europe caused by Bluetongue virus serotype 8 (BTV-8). Coinciding with the BTV-8 outbreak, a virulent strain of BTV-1 emerged and co-infections of these two serotypes were reported. In response, we generated VLPs for BTV-8 and tested the efficacy of BTV-8 VLPs as a single immunogen and as a component of a multivalent vaccine, with VLPs of BTV-1 and BTV-2, in order to test if there was any interference between serotypes. All pre-Alps sheep vaccinated with BTV-8 VLPs developed a strong neutralising antibody response to BTV-8 and multivalent VLP vaccinated animals also developed neutralising antibodies to BTV-1 and BTV-2. There were no side effects observed due to the vaccination with either the single- or multivalent VLP cocktail. All VLP-vaccinated animals had no clinical manifestation of BT or viraemia after challenge with a virulent BTV-8 isolate. This data indicates that BTV-8 VLPs delivered as a single immunogen or as a component of a multivalent vaccine are highly efficacious. Moreover, there was no interference on the development of a strong protective immune response due to the combination of different phylogenetically unrelated BTV serotypes in the vaccinated animals. This report further highlights that BTV VLPs are safe and efficacious immunogens that are able to afford complete protection against a virulent virus challenge.     

Protective efficacy of Bluetongue virus-like and subvirus-like particles in sheep: presence of the serotype-specific VP2, independent of its geographic lineage, is essential for protection

There have been multiple separate outbreaks of Bluetongue (BT) disease of ruminants in Europe since 1998, often entering via the Mediterranean countries of Italy, Spain and Greece. BT is caused by an orbivirus, Bluetongue virus (BTV), a member of the family Reoviridae. BTV is a non-enveloped double-capsid virus, which encodes 7 structural proteins (VP1-VP7) and several non-structural proteins (NS1, NS2, NS3/3a and NS4) from ten double-stranded RNA segments of the genome. In this report, we have prepared BTV virus-like particles (VLPs, composed of VP2, VP3, VP5 and VP7) and sub-viral, inner core-like particles (CLPs, VP3 and VP7) using a recombinant baculovirus expression system. We compared the protective efficacy of VLPs and CLPs in sheep and investigated the importance of geographical lineages of BTV in the development of vaccines. The Greek crossbred Karagouniko sheep, which display mild to sub-clinical BT, were vaccinated with VLPs or CLPs of BTV-1, derived from western lineage and were challenged with virulent BTV-1 from an eastern lineage. All VLP-vaccinated animals developed a neutralising antibody response to BTV-1 from both lineages prior to challenge. Moreover, post-challenged animals had no clinical manifestation or viraemia and the challenged virus replication was completely inhibited. In contrast, CLP-vaccinated animals did not induce any neutralising antibody response but developed the group specific VP7 antibodies. CLPs also failed to prevent the clinical manifestation and virus replication, but in comparison to controls, the severity of disease manifestation and viraemia was mitigated. The data demonstrated that the outer capsid was essential for complete protection, while the geographical origin of the BTV was not critical for development of a serotype specific vaccine.     

Protective immunity of plant-produced African horse sickness virus serotype 5 chimaeric virus-like particles (VLPs) and viral protein 2 (VP2) vaccines in IFNAR-/- mice

Next generation vaccines have the capability to contribute to and revolutionise the veterinary vaccine industry. African horse sickness (AHS) is caused by an arbovirus infection and is characterised by respiratory distress and/or cardiovascular failure and is lethal to horses. Mandatory annual vaccination in endemic areas curtails disease occurrence and severity. However, development of a next generation AHSV vaccine, which is both safe and efficacious, has been an objective globally for years. In this study, both AHSV serotype 5 chimaeric virus-like particles (VLPs) and soluble viral protein 2 (VP2) were successfully produced in Nicotiana benthamiana ΔXT/FT plants, partially purified and validated by gel electrophoresis, transmission electron microscopy and liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing before vaccine formulation. IFNAR^-/- mice vaccinated with the adjuvanted VLPs or VP2 antigens in a 10 µg prime-boost regime resulted in high titres of antibodies confirmed by both serum neutralising tests (SNTs) and enzyme-linked immunosorbent assays (ELISA). Although previous studies reported high titres of antibodies in horses when vaccinated with plant-produced AHS homogenous VLPs, this is the first study demonstrating the protective efficacy of both AHSV serotype 5 chimaeric VLPs and soluble AHSV-5 VP2 as vaccine candidates. Complementary to this, coating ELISA plates with the soluble VP2 has the potential to underpin serotype-specific serological assays.     

Application of Bluetongue Disabled Infectious Single Animal (DISA) vaccine for different serotypes by VP2 exchange or incorporation of chimeric VP2

Bluetongue is a disease of ruminants caused by the bluetongue virus (BTV). Bluetongue outbreaks can be controlled by vaccination, however, currently available vaccines have several drawbacks. Further, there are at least 26 BTV serotypes, with low cross protection. A next-generation vaccine based on live-attenuated BTV without expression of non-structural proteins NS3/NS3a, named Disabled Infectious Single Animal (DISA) vaccine, was recently developed for serotype 8 by exchange of the serotype determining outer capsid protein VP2. DISA vaccines are replicating vaccines but do not cause detectable viremia, and induce serotype specific protection. Here, we exchanged VP2 of laboratory strain BTV1 for VP2 of European serotypes 2, 4, 8 and 9 using reverse genetics, without observing large effects on virus growth. Exchange of VP2 from serotype 16 and 25 was however not possible. Therefore, chimeric VP2 proteins of BTV1 containing possible immunogenic regions of these serotypes were studied. BTV1, expressing 1/16 chimeric VP2 proteins was functional in virus replication in vitro and contained neutralizing epitopes of both serotype 1 and 16. For serotype 25 this approach failed. We combined VP2 exchange with the NS3/NS3a negative phenotype in BTV1 as previously described for serotype 8 DISA vaccine. DISA vaccine with 1/16 chimeric VP2 containing amino acid region 249–398 of serotype 16 raised antibodies in sheep neutralizing both BTV1 and BTV16. This suggests that DISA vaccine could be protective for both parental serotypes present in chimeric VP2. We here demonstrate the application of the BT DISA vaccine platform for several serotypes and further extend the application for serotypes that are unsuccessful in single VP2 exchange.     

Strong protection induced by an experimental DIVA subunit vaccine against bluetongue virus serotype 8 in cattle

Bluetongue virus (BTV) infections in ruminants pose a permanent agricultural threat since new serotypes are constantly emerging in new locations. Clinical disease is mainly observed in sheep, but cattle were unusually affected during an outbreak of BTV seroype 8 (BTV-8) in Europe. We previously developed an experimental vaccine based on recombinant viral protein 2 (VP2) of BTV-8 and non-structural proteins 1 (NS1) and NS2 of BTV-2, mixed with an immunostimulating complex (ISCOM)–matrix adjuvant. We demonstrated that bovine immune responses induced by this vaccine were as good or superior to those induced by a classic commercial inactivated vaccine. In this study, we evaluated the protective efficacy of the experimental vaccine in cattle and, based on the detection of VP7 antibodies, assessed its DIVA compliancy following virus challenge. Two groups of BTV-seronegative calves were subcutaneously immunized twice at a 3-week interval with the subunit vaccine (n = 6) or with adjuvant alone (n = 6). Following BTV-8 challenge 3 weeks after second immunization, controls developed viremia and fever associated with other mild clinical signs of bluetongue disease, whereas vaccinated animals were clinically and virologically protected. The vaccine-induced protection was likely mediated by high virus-neutralizing antibody titers directed against VP2 and perhaps by cellular responses to NS1 and NS2. T lymphocyte responses were cross-reactive between BTV-2 and BTV-8, suggesting that NS1 and NS2 may provide the basis of an adaptable vaccine that can be varied by using VP2 of different serotypes. The detection of different levels of VP7 antibodies in vaccinated animals and controls after challenge suggested a compliancy between the vaccine and the DIVA companion test. This BTV subunit vaccine is a promising candidate that should be further evaluated and developed to protect against different serotypes.     

Long-term persistence of neutralising antibodies against bluetongue virus serotype 8 in naturally infected cattle

Neutralising antibodies to bluetongue virus (BTV) in convalescent cattle have been described as persistent. Controlled laboratory studies, however, rarely last longer than a couple of weeks and long-term field data are lacking. This study followed twelve cattle that had been naturally infected with bluetongue virus serotype 8 (BTV-8) in Germany in 2006. Using ELISAs and a serum neutralisation test, we found a strong humoral immune response four to six years after the last exposure to BTV-8; based on data from long-term vaccine studies, it is highly likely that this coincides with immunity to reinfection with the same serotype.     

Recombinant canarypox virus vaccine co-expressing genes encoding the VP2 and VP5 outer capsid proteins of bluetongue virus induces high level protection in sheep

We describe the development and preliminary characterization of a recombinant canarypox virus vectored vaccine for protective immunization of ruminants against bluetongue virus (BTV) infection. Sheep (n=6) immunized with recombinant canarypox virus vector (BTV-CP) co-expressing synthetic genes encoding the two outer capsid proteins (VP2 and VP5) of BTV serotype 17 (BTV-17) developed high titers (40-160) of virus-specific neutralizing antibodies and were resistant to challenge with a field strain of BTV-17. In contrast, sheep (n=5) immunized with a commercial recombinant canarypox virus vector expressing the E and preM genes of West Nile virus were seronegative to BTV and developed pyrexia, lymphopenia, and extended, high-titered viremias following challenge exposure to the field strain of BTV-17. These data confirm that the BTV-CP vaccine may be useful for the protective immunization of ruminants against bluetongue, and it may avoid the problems inherent to live-attenuated (LA) BTV vaccines.     

Bluetongue Disabled Infectious Single Animal (DISA) vaccine: Studies on the optimal route and dose in sheep

Bluetongue (BT) is a disease of ruminants caused by bluetongue virus (BTV) transmitted by biting midges of the Culicoides genus. Outbreaks have been controlled successfully by vaccination, however, currently available BT vaccines have several shortcomings. Recently, we have developed BT Disabled Infectious Single Animal (DISA) vaccines based on live-attenuated BTV without expression of dispensable non-structural NS3/NS3a protein. DISA vaccines are non-pathogenic replicating vaccines, do not cause viremia, enable DIVA and are highly protective. NS3/NS3a protein is involved in virus release, cytopathogenic effect and suppression of Interferon-I induction, suggesting that the vaccination route can be of importance. A standardized dose of DISA vaccine for serotype 8 has successfully been tested by subcutaneous vaccination. We show that 10 and 100 times dilutions of this previously tested dose did not reduce the VP7 humoral response. Further, the vaccination route of DISA vaccine strongly determined the induction of VP7 directed antibodies (Abs). Intravenous vaccination induced high and prolonged humoral response but is not practical in field situations. VP7 seroconversion was stronger by intramuscular vaccination than by subcutaneous vaccination. For both vaccination routes and for two different DISA vaccine backbones, IgM Abs were rapidly induced but declined after 14 days post vaccination (dpv), whereas the IgG response was slower. Interestingly, intramuscular vaccination resulted in an initial peak followed by a decline up to 21 dpv and then increased again. This second increase is a steady and continuous increase of IgG Abs. These results indicate that intramuscular vaccination is the optimal route. The protective dose of DISA vaccine has not been determined yet, but it is expected to be significantly lower than of currently used BT vaccines. Therefore, in addition to the advantages of improved safety and DIVA compatibility, the novel DISA vaccines will be cost–competitive to commercially available live attenuated and inactivated vaccines for Bluetongue.     

Protective duration of immunity of an inactivated bluetongue (BTV) serotype 2 vaccine against a virulent BTV serotype 2 challenge in sheep

The protective properties of an inactivated bluetongue virus serotype 2 (BTV-2) vaccine were evaluated in sheep. Sheep (two groups of seven), vaccinated with either one or two doses of the vaccine, were monitored for antibody response over one year. All sheep developed high titres of neutralizing antibodies by 35 days after first vaccination and titres were maintained over one year. Control sheep (n = 7) remained seronegative until challenge. One year after vaccination, all sheep were inoculated with a virulent BTV-2. All controls developed pyrexia, clinical signs and viraemia. In contrast, the sheep vaccinated with one or two doses of inactivated BTV-2 vaccine were protected from clinical disease and viraemia was completely prevented. These data show that a single dose of the BTV-2 vaccine given to sheep induces a strong immunity which confers protection for at least one year.     

Development of a competitive ELISA for NS3 antibodies as DIVA test accompanying the novel Disabled Infectious Single Animal (DISA) vaccine for Bluetongue

Recently, we have developed a novel vaccine for Bluetongue named BT Disabled Infectious Single Animal (DISA) vaccine. Due to the lack of non-essential NS3/NS3a protein, BT DISA vaccine is a replicating vaccine, but without the inherent risks of live-attenuated vaccines, such as residual virulence or reversion to virulence by mutations, reassortment with field virus, horizontal spread by vectors and vertical transmission. The immune response induced by BT DISA vaccines is rapidly induced, highly protective and serotype specific which is dependent on the immunodominant and serotype determining VP2 protein. The BT DISA vaccine platform provides the replacement of exclusively VP2 from different serotypes in order to safely formulate multivalent cocktail vaccines. The lack of NS3/NS3a directed antibodies by BT DISA vaccination enables differentiation of infected from vaccinated animals (DIVA principle). A highly conserved immunogenic site corresponding to the late domain was mapped in the N-terminal region of NS3. We here established an NS3-specific competitive ELISA (NS3 cELISA) as serological DIVA test accompanying BT DISA vaccines. To this end, NS3 protein missing putative transmembrane regions was produced in large amounts in bacteria and used as antigen in the NS3 cELISA which was investigated with a variety of sera. The NS3 cELISA displayed a high sensitivity and specificity similar to the commercially available VP7-specific cELISA. Results of previously performed vaccination-challenge trials with BT DISA vaccines clearly demonstrate the DIVA system based on the NS3 cELISA and BT vaccine free of NS3 protein.     

Evaluation of humoral response and protective efficacy of two inactivated vaccines against bluetongue virus after vaccination of goats

Bluetongue serotype 8 has become a major animal health issue in the European Union and the European member States have agreed on a vaccination strategy, which involves only inactivated vaccines. In this study, the efficacy of two inactivated vaccines against bluetongue virus serotype 8 (BTV-8) used in Europe since 2008, BTVPUR ALSAP^® 8 (MERIAL) and BOVILIS^® BTV8 (Intervet/SP-AH), was evaluated in goats immunized and challenged with BTV-8 field isolates under experimental conditions. Serological, virological and clinical examinations were conducted before and after challenge. Three groups of 10 goats each (groups A, B and C) were randomly constituted and 2 groups (A and C) were subcutaneously vaccinated twice with one dose of the two commercial vaccines BTVPUR ALSAP 8 (group A) or BOVILIS BTV8 (group C) respectively. Animals of the groups A, C and B (B: controls) were challenged with a virulent inoculum containing BTV-8. During the experiment, it was found out that the BTV-8 challenge inoculum was contaminated with another BTV serotype. However, results demonstrated that vaccination of goats with two injections of BTVPUR ALSAP 8 or BOVILIS BTV8 provided a significant clinical protection against a BTV-8 challenge and completely prevented BTV-8 viraemia in all vaccinated animals. Qualitative data showed no difference in the kinetics and levels of the humoral response induced by these two inactivated vaccines.     

Clinical disease in sheep caused by bluetongue virus serotype 8, and prevention by an inactivated vaccine

The ability to reduce clinical signs, induce neutralizing antibodies, and perhaps most importantly, to prevent or reduce viraemia (and therefore virus-transmission), represent primary criteria for assessment of bluetongue virus (BTV) vaccine efficacy. Identification of BTV challenge-strains that reliably induce viraemia and clinical signs comparable to those in naturally infected animals, is therefore important for vaccine evaluation. Texel cross-breed and Dorset Poll sheep vaccinated with inactivated BTV-8 vaccine (‘Bovilis^® BTV8’ from MSD Animal Health), were challenged with low-passage BTV-8 (Northern European strain) grown in either insect (Culicoides) or mammalian cell-cultures. The severity of clinical signs was recorded (using a modified numerical scoring-system, which is described) along with viraemia and serum neutralizing (SN) antibody levels. Low level SN-antibodies were detected at the time of challenge (three weeks after vaccination). All unvaccinated control animals became infected after challenge, developing high SN-antibody titres by 21 days post challenge (dpc). Vaccinees showed faster increases in SN-antibody titres (‘booster’ response), with significantly higher titres at 6 dpc than unvaccinated controls. Although only limited clinical-signs could be attributed to BTV in younger animals infected with the mammalian-cell-culture derived virus, both BTV-8 challenge preparations induced severe clinical signs comparable to ‘bluetongue’ observed during natural outbreaks in older unvaccinated animals. Challenge with BTV-8 grown in Culicoides cell-cultures seemed to induce greater severity of clinical-scores and ‘post-mortem lesions’ than the mammalian-derived BTV-8 strain. Vaccination reduced clinical signs, fever, and viraemia equally well after challenge with either virus preparation.     

Presence of bluetongue and epizootic hemorrhagic disease viruses in Egypt in 2016 and 2017

BTV and EHDV are closely-related orbiviruses that are transmitted between domestic and wild ruminants via the bites of hematophagous midges. Previous studies have reported seropositivity against BTV antibodies in sheep and goats in two Egyptian governorates (Beni Suef and Menoufia). However, no recent data are available on the BTV serotype(s) circulating in Egypt and the likely presence of EHDV has never been explored. This study investigated the presence of BTV and EHDV among cattle which had been found BTV-seropositive by ELISA method. These cattle living in proximity to sheep and goats previously found BTV-seropositive. These cattle displayed no clinical signs of BT but reproductive problems had been reported in herds. A total of 227 cattle blood samples were therefore collected in 2016 and 2017. Ninety-four of the 227 animals tested by a BTV ELISA were positive for BTV antibodies (41.4%). Of these 94 ELISA-positive cattle, only 83 EDTA-blood samples were available and therefore tested for BTV and EHDV genome detection by RT-PCR and sequencing.Of the cattle sampled in 2016, results revealed that two were RT-PCR-positive for BTV and seven for EHDV. Sequencing showed the presence of EHDV-1 and BTV-3 genome sequences. EHDV-1 S2 shared 99.5% homology with an EHDV-1 S2 from a strain isolated in 2016 in Israel. BTV-3 S2 and S8 sequences shared >99.8% nucleotide similarity with the BTV-3 Zarzis S2 and S8 sequences (Tunisian BTV, also detected in 2016). Of the 66 blood samples tested following their collection in 2017, they were all EHDV-negative by RT-qPCR while five were BTV- positive by RT-qPCR. However, attempts to identify the BTV serotype of these five samples were unsuccessful. Only part of BTV S8 was sequenced and it showed 79% nucleotide similarity with S8 of atypical BTV serotypes (particularly with BTV-26 and another BTV serotype strain isolated from a sheep pox vaccine). Overall, these findings demonstrate that both BTV and EHDV were circulating in Egypt in 2016 and 2017.     

Bluetongue virus serotypes 1 and 4 in Sardinia during autumn 2012: New incursions or re-infection with old strains?

Since 2000 several bluetongue virus (BTV) incursions have occurred in Sardinia (Italy) involving serotypes 1, 2, 4, 8 and 16. In October 2012, new BT outbreaks caused by BTV-1 and BTV-4 were reported. Nearly 500 flocks were infected and 9238 sheep died because of the infection. When sequenced, Seg-10 of both strains shared 99% similarity at nucleotide level with BTV strains that have circulated in the Mediterranean basin in the last few years. Similarly, Seg-5 sequences of BTV-1 and BTV-4 newly isolated Sardinian strains are identical and cluster together with recent BTV-1 circulating in the Mediterranean Basin and the BTV-4 strains isolated in Tunisia in 2007 and 2009. These BTV-4 strains differ from the ones that circulated in Europe from 2003 to 2005 and appear to be reassortant strains.     

VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges. Vaccination is the most effective measure to control vector borne diseases; however, there are 26 known BTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment 2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are available for a limited number of serotypes, but each of these have their specific disadvantages, including the inability to differentiate infected from vaccinated animals (DIVA).BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but are important for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro. Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep is strongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 without NS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is a promising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine.Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment 2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, by exchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccines with respect to reassortment between vaccine viruses.Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAs are therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, we developed an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibodies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTV challenge.     

Effect of vaccination with an inactivated vaccine on transplacental transmission of BTV-8 in mid term pregnant ewes and heifers

The effect of vaccination with a commercial inactivated Bluetongue virus serotype 8 (BTV-8) vaccine on the ability of BTV-8 to cross the ruminant placenta was investigated in two experiments. Ten pregnant ewes (Experiment 1) or heifers (Experiment 2) were vaccinated according to the manufacturer's instructions. Three weeks after the completion of the vaccination schedule, all vaccinated animals were infected with BTV-8 together with ten non-vaccinated pregnant animals that served as challenged controls. Four additional pregnant animals received a mock challenge at the same time point. Three weeks after the challenge, the foetuses were collected. In the sheep experiment, the lambs of the vaccinated ewes and the mock infected ewes were negative in the virus isolation, whereas BTV-8 could be isolated from 11/23 lambs of 6/10 ewes in the BTV-8 challenged control group. The incidence and severity of BTV associated lesions, such as haemorrhages, meningitis/encephalitis and necrosis in the placentomes was significantly higher in the BTV-8 challenged control group. The rate of transplacental transmission was less in the cattle experiment: BTV-8 could be detected in 2/10 calves in the BTV-8 challenged control group. All other calves were negative.Vaccination clearly reduced transplacental transmission of BTV-8 in the sheep experiment, whereas in the cattle experiment, the incidence of transmission was too low to demonstrate a significant reduction of transmission by vaccination. However, the vaccine very effectively blocked viraemia, which suggests that the vaccine might prevent transmission in cattle as well.Transplacental transmission of BTV has serious economical consequences, due to the loss of progeny to the livestock industry. Vaccination can be an important aid in the reduction of these economic losses.     

Bluetongue epidemiology in the European Union

Bluetongue (BT) is a reportable disease of considerable socioeconomic concern and of major importance in the international trade of animals and animal products. Before 1998, BT was considered an exotic disease in Europe. From 1998 through 2005, at least 6 BT virus strains belonging to 5 serotypes (BTV-1, BTV-2, BTV-4, BTV-9, and BTV-16) were continuously present in the Mediterranean Basin. Since August 2006, BTV-8 has caused a severe epizootic of BT in northern Europe. The widespread recrudescence and extension of BTV-8 infections in northern Europe during 2007 suggest that requirements for BTV establishment may now be fulfilled in this area. In addition, the radial extension of BTV-8 across Europe increases the risk for an encounter between this serotype and others, particularly those that occur in the Mediterranean Basin, where vector activity continues for more of the year. This condition increases the risk for reassortment of individual BTV gene segments.     

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 log₁₀ TCID₅₀/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.