A Single Immunization with MVA Expressing GnGc Glycoproteins Promotes Epitope-specific CD8+-T Cell Activation and Protects Immune-competent Mice against a Lethal RVFV Infection

Background

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen causing an important disease in ruminants often transmitted to humans after epizootic outbreaks in African and Arabian countries. To help combat the spread of the disease, prophylactic measures need to be developed and/or improved.

Methodology/Principal Findings

In this work, we evaluated the immunogenicity and protective efficacy of recombinant plasmid DNA and modified vaccinia virus Ankara (rMVA) vectored vaccines against Rift Valley fever in mice. These recombinant vaccines encoded either of two components of the Rift Valley fever virus: the viral glycoproteins (Gn/Gc) or the nucleoprotein (N). Following lethal challenge with live RVFV, mice immunized with a single dose of the rMVA-Gn/Gc vaccine showed no viraemia or clinical manifestation of disease, but mounted RVFV neutralizing antibodies and glycoprotein specific CD8+ T-cell responses. Neither DNA-Gn/Gc alone nor a heterologous prime-boost immunization schedule (DNA-Gn/Gc followed by rMVAGn/Gc) was better than the single rMVA-Gn/Gc immunization schedule with regards to protective efficacy. However, the rMVA-Gn/Gc vaccine failed to protect IFNAR^−/− mice upon lethal RVFV challenge suggesting a role for innate responses in protection against RVFV. Despite induction of high titer antibodies against the RVFV nucleoprotein, the rMVA-N vaccine, whether in homologous or heterologous prime-boost schedules with the corresponding recombinant DNA vaccine, only conferred partial protection to RVFV challenge.

Conclusions/Significance

Given the excellent safety profile of rMVA based vaccines in humans and animals, our data supports further development of rMVA-Gn/Gc as a vaccine strategy that can be used for the prevention of Rift Valley fever in both humans and livestock.     

Correlative Gene Expression to Protective Seroconversion in Rift Valley Fever Vaccinates

Rift Valley fever Virus (RVFV), a negative-stranded RNA virus, is the etiological agent of the vector-borne zoonotic disease, Rift Valley fever (RVF). In both humans and livestock, protective immunity can be achieved through vaccination. Earlier and more recent vaccine trials in cattle and sheep demonstrated a strong neutralizing antibody and total IgG response induced by the RVF vaccine, authentic recombinant MP-12 (arMP-12). From previous work, protective immunity in sheep and cattle vaccinates normally occurs from 7 to 21 days after inoculation with arMP-12. While the serology and protective response induced by arMP-12 has been studied, little attention has been paid to the underlying molecular and genetic events occurring prior to the serologic immune response. To address this, we isolated RNA from whole blood of vaccinated calves over a time course of 21 days before and after vaccination with arMP-12. The time course RNAs were sequenced by RNASeq and bioinformatically analyzed. Our results revealed time-dependent activation or repression of numerous gene ontologies and pathways related to the vaccine induced immune response and its regulation. Additional bioinformatic analyses identified a correlative relationship between specific host immune response genes and protective immunity prior to the detection of protective serum neutralizing antibody responses. These results contribute an important proof of concept for identifying molecular and genetic components underlying the immune response to RVF vaccination and protection prior to serologic detection.     

Multiserotype protection elicited by a combinatorial prime-boost vaccination strategy against bluetongue virus

Bluetongue virus (BTV) belongs to the genus Orbivirus within the family Reoviridae. The development of vector-based vaccines expressing conserved protective antigens results in increased immune activation and could reduce the number of multiserotype vaccinations required, therefore providing a cost-effective product. Recent recombinant DNA technology has allowed the development of novel strategies to develop marker and safe vaccines against BTV. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2, VP7 and NS1 proteins from BTV-4. IFNAR((-/-)) mice inoculated with DNA/rMVA-VP2,-VP7-NS1 in an heterologous prime boost vaccination strategy generated significant levels of antibodies specific of VP2, VP7, and NS1, including those with neutralizing activity against BTV-4. In addition, vaccination stimulated specific CD8(+) T cell responses against these three BTV proteins. Importantly, the vaccine combination expressing NS1, VP2 and VP7 proteins of BTV-4, elicited sterile protection against a lethal dose of homologous BTV-4 infection. Remarkably, the vaccine induced cross-protection against lethal doses of heterologous BTV-8 and BTV-1 suggesting that the DNA/rMVA-VP2,-VP7,-NS1 marker vaccine is a promising multiserotype vaccine against BTV.     

Single-dose immunization with virus replicon particles confers rapid robust protection against Rift Valley fever virus challenge

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The potential for RVFV introduction outside the area of endemicity highlights the need for fast-acting, safe, and efficacious vaccines. Here, we demonstrate a robust system for the reverse genetics generation of a RVF virus replicon particle (VRP(RVF)) vaccine candidate. Using a mouse model, we show that VRP(RVF) immunization provides the optimal balance of safety and single-dose robust efficacy. VRP(RVF) can actively synthesize viral RNA and proteins but lacks structural glycoprotein genes, preventing spread within immunized individuals and reducing the risk of vaccine-induced pathogenicity. VRP(RVF) proved to be completely safe following intracranial inoculation of suckling mice, a stringent test of vaccine safety. Single-dose subcutaneous immunization with VRP(RVF), although it is highly attenuated, completely protected mice against a virulent RVFV challenge dose which was 100,000-fold greater than the 50% lethal dose (LD(50)). Robust protection from lethal challenge was observed by 24 h postvaccination, with 100% protection induced in as little as 96 h. We show that a single subcutaneous VRP(RVF) immunization initiated a systemic antiviral state followed by an enhanced adaptive response. These data contrast sharply with the much-reduced survivability and immune responses observed among animals immunized with nonreplicating viral particles, indicating that replication, even if confined to the initially infected cells, contributes substantially to protective efficacy at early and late time points postimmunization. These data demonstrate that replicon vaccines successfully bridge the gap between safety and efficacy and provide insights into the kinetics of antiviral protection from RVFV infection.     

Parenteral vaccination of mammalian livestock with Newcastle disease virus-based vector vaccines offers optimal efficacy and safety

Newcastle disease virus (NDV) is an avian virus that is being evaluated as a vaccine vector for the delivery of foreign genes in mammals. The use of NDV as a vaccine vector in these species offers two major advantages. First, NDV is highly attenuated in mammals, rendering its use inherently safe. Second, mammals lack pre-existing NDV immunity, which minimizes the risk of vaccination failure. NDV-vector vaccines are generally administered to mammals via the respiratory route. We recently showed that intramuscular vaccination with NDV-based Rift Valley fever virus (RVFV) vaccines provides complete protection in mice and induces neutralizing antibodies in sheep and cattle, the main target species of RVFV. Here, we discuss the use of NDV as a vaccine vector for applications in mammalian livestock with an emphasis on the vaccination route. We also report the results of novel experiments that underscore our notion that vaccination via a parenteral route is more effective than immunization via the respiratory route.     

Towards a safe, effective vaccine for Rift Valley fever virus

Rift Valley fever virus (RVFV) is an important animal and human threat and leads to longstanding morbidity and mortality in susceptible hosts. Since no therapies currently exist to treat Rift Valley fever, it remains a public and animal health priority to develop safe, effective RVFV vaccines (whether for animals, humans, or both) that provide long-term protective immunity. In the evaluated article, Bhardwaj and colleagues describe the creation and testing of two successful vaccine strategies against RVFV, a DNA plasmid vaccine expressing Gn coupled to C3d, and an alpha-virus replicon vaccine expressing Gn protein. Both vaccines elicited strong neutralizing antibody responses, prevented morbidity and mortality in RVFV-challenged mice, and enabled protection of naive mice via passive antibody transfer from vaccinated mice. Both DNA and replicon RVFV vaccines have previously been shown to protect against RVFV challenge, but these results allow for direct comparison of the two methods and evaluation of a combined prime-boost method. The results also highlight the specific humoral and cell-mediated immune responses to vaccination.     

An equine herpesvirus type 1 (EHV-1) expressing VP2 and VP5 of serotype 8 bluetongue virus (BTV-8) induces protection in a murine infection model

Bluetongue virus (BTV) can infect most species of domestic and wild ruminants causing substantial morbidity and mortality and, consequently, high economic losses. In 2006, an epizootic of BTV serotype 8 (BTV-8) started in northern Europe that caused significant disease in cattle and sheep before comprehensive vaccination was introduced two years later. Here, we evaluate the potential of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus, as a novel vectored DIVA (differentiating infected from vaccinated animals) vaccine expressing VP2 of BTV-8 alone or in combination with VP5. The EHV-1 recombinant viruses stably expressed the transgenes and grew with kinetics that were identical to those of parental virus in vitro. After immunization of mice, a BTV-8-specific neutralizing antibody response was elicited. In a challenge experiment using a lethal dose of BTV-8, 100% of interferon-receptor-deficient (IFNAR(-/-)) mice vaccinated with the recombinant EHV-1 carrying both VP2 and VP5, but not VP2 alone, survived. VP7 was not included in the vectored vaccines and was successfully used as a DIVA marker. In summary, we show that EHV-1 expressing BTV-8 VP2 and VP5 is capable of eliciting a protective immune response that is distinguishable from that after infection and as such may be an alternative for BTV vaccination strategies in which DIVA compatibility is of importance.     

Rift Valley fever virus vaccine lacking the NSs and NSm genes is safe, nonteratogenic, and confers protection from viremia, pyrexia, and abortion following challenge in adult and pregnant sheep

Rift Valley fever virus (RVFV) is a mosquito-borne human and veterinary pathogen causing large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Safe and effective vaccines are critically needed, especially those that can be used in a targeted one-health approach to prevent both livestock and human disease. We report here on the safety, immunogenicity, and efficacy of the ΔNSs-ΔNSm recombinant RVFV (rRVFV) vaccine (which lacks the NSs and NSm virulence factors) in a total of 41 sheep, including 29 timed-pregnant ewes. This vaccine was proven safe and immunogenic for adult animals at doses ranging from 1.0 × 10(3) to 1.0 × 10(5) PFU administered subcutaneously (s.c.). Pregnant animals were vaccinated with 1.0 × 10(4) PFU s.c. at day 42 of gestation, when fetal sensitivity to RVFV vaccine-induced teratogenesis is highest. No febrile reactions, clinical illness, or pregnancy loss was observed following vaccination. Vaccination resulted in a rapid increase in anti-RVFV IgM (day 4) and IgG (day 7) titers. No seroconversion occurred in cohoused control animals. A subset of 20 ewes progressed to full-term delivery after vaccination. All lambs were born without musculoskeletal, neurological, or histological birth defects. Vaccine efficacy was assessed in 9 pregnant animals challenged at day 122 of gestation with virulent RVFV (1.0 × 10(6) PFU intravenously). Following challenge, 100% (9/9) of the animals were protected, progressed to full term, and delivered healthy lambs. As expected, all 3 sham-vaccinated controls experienced viremia, fetal death, and abortion postchallenge. These results demonstrate that the ΔNSs-ΔNSm rRVFV vaccine is safe and nonteratogenic and confers high-level protection in sheep.     

Factors Associated with Severe Human Rift Valley Fever in Sangailu,Garissa County,Kenya

BackgroundMosquito-borne Rift Valley fever virus (RVFV) causes acute, often severe, disease in livestock and humans. To determine the exposure factors and range of symptoms associated with human RVF, we performed a population-based cross-sectional survey in six villages across a 40 km transect in northeastern Kenya.Conclusions/SignificanceOur results demonstrate that a significant proportion of the population in northeastern Kenya has been infected with RVFV. Village and certain animal husbandry activities were associated with more severe disease. Older age, male gender, herder occupation, killing and butchering livestock, and poor visual acuity were useful markers for increased RVFV infection. Formal vision testing may therefore prove to be a helpful, low-technology tool for RVF screening during epidemics in high-risk rural settings.     

Seroprevalence of Sheep and Goat Pox,Peste Des Petits Ruminants and Rift Valley Fever in Saudi Arabia

Sheep and goat pox, peste des petits ruminants and Rift Valley fever are important diseases of small ruminant livestock. Sheep and goat pox, along with peste des petits ruminants, are endemic throughout most of Africa, Asia and the Middle East. Whereas Rift Valley fever is endemic in Africa, outbreaks in the Middle East have been reported over the past decade, including the Arabian Peninsula. Saudi Arabia is a major importer of livestock, and understanding the prevalence of these viral infections would be useful for disease control. In this study, sera from sheep and goats were collected from 3 regions in Saudi Arabia. They were evaluated for antibodies specific to sheep and goat pox, peste des petits ruminants and Rift Valley fever by virus neutralization assays. To the best of our knowledge, this is the first study to evaluate the seroprevalence of these viruses in sheep and goats.     

A Single Vaccination with an Improved Nonspreading Rift Valley Fever Virus Vaccine Provides Sterile Immunity in Lambs

Rift Valley fever virus (RVFV) is an important pathogen that affects ruminants and humans. Recently we developed a vaccine based on nonspreading RVFV (NSR) and showed that a single vaccination with this vaccine protects lambs from viremia and clinical signs. However, low levels of viral RNA were detected in the blood of vaccinated lambs shortly after challenge infection. These low levels of virus, when present in a pregnant ewe, could potentially infect the highly susceptible fetus. We therefore aimed to further improve the efficacy of the NSR vaccine. Here we report the expression of Gn, the major immunogenic protein of the virus, from the NSR genome. The resulting NSR-Gn vaccine was shown to elicit superior CD8 and CD4-restricted memory responses and improved virus neutralization titers in mice. A dose titration study in lambs revealed that the highest vaccination dose of 10^6.3 TCID₅₀/ml protected all lambs from clinical signs and viremia. The lambs developed neutralizing antibodies within three weeks after vaccination and no anamnestic responses were observed following challenge. The combined results suggest that sterile immunity was achieved by a single vaccination with the NSR-Gn vaccine.     

Rift Valley Fever Risk Map Model and Seroprevalence in Selected Wild Ungulates and Camels from Kenya

Since the first isolation of Rift Valley fever virus (RVFV) in the 1930s, there have been multiple epizootics and epidemics in animals and humans in sub-Saharan Africa. Prospective climate-based models have recently been developed that flag areas at risk of RVFV transmission in endemic regions based on key environmental indicators that precede Rift Valley fever (RVF) epizootics and epidemics. Although the timing and locations of human case data from the 2006–2007 RVF outbreak in Kenya have been compared to risk zones flagged by the model, seroprevalence of RVF antibodies in wildlife has not yet been analyzed in light of temporal and spatial predictions of RVF activity. Primarily wild ungulate serum samples from periods before, during, and after the 2006–2007 RVF epizootic were analyzed for the presence of RVFV IgM and/or IgG antibody. Results show an increase in RVF seropositivity from samples collected in 2007 (31.8%), compared to antibody prevalence observed from 2000–2006 (3.3%). After the epizootic, average RVF seropositivity diminished to 5% in samples collected from 2008–2009. Overlaying maps of modeled RVF risk assessments with sampling locations indicated positive RVF serology in several species of wild ungulate in or near areas flagged as being at risk for RVF. Our results establish the need to continue and expand sero-surveillance of wildlife species Kenya and elsewhere in the Horn of Africa to further calibrate and improve the RVF risk model, and better understand the dynamics of RVFV transmission.     

Rift Valley fever virus and European mosquitoes: vector competence of Culex pipiens and Stegomyia albopicta (= Aedes albopictus)

Rift Valley fever (RVF) is a mosquito‐borne disease caused by the Rift Valley fever virus (RVFV). Rift Valley fever affects a large number of species, including human, and has severe impact on public health and the economy, especially in African countries. The present study examined the vector competence of three different European mosquito species, Culex pipiens (Linnaeus, 1758) form molestus (Diptera: Culicidae), Culex pipiens hybrid form and Stegomyia albopicta (= Aedes albopictus) (Skuse, 1894) (Diptera: Culicidae). Mosquitoes were artificially fed with blood containing RVFV. Infection, disseminated infection and transmission efficiency were evaluated. This is the first study to assess the transmission efficiency of European mosquito species using a virulent RVFV strain. The virus disseminated in Cx. pipiens hybrid form and in S. albopicta. Moreover, infectious viral particles were isolated from saliva of both species, showing their RVFV transmission capacity. The presence of competent Cx. pipiens and S. albopicta in Spain indicates that an autochthonous outbreak of RVF may occur if the virus is introduced. These findings provide information that will help health authorities to set up efficient entomological surveillance and RVFV vector control programmes.     

Characterization of protection afforded by a bivalent virus-like particle vaccine against bluetongue virus serotypes 1 and 4 in sheep

Background

Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines.

Methodology/Principal Findings

Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals'' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died.

Conclusions

There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if immunodominance of particular serotypes could interfere with vaccine efficacy.     

Culex pipiens, an experimental efficient vector of West Nile and Rift Valley fever viruses in the Maghreb region

West Nile fever (WNF) and Rift Valley fever (RVF) are emerging diseases causing epidemics outside their natural range of distribution. West Nile virus (WNV) circulates widely and harmlessly in the old world among birds as amplifying hosts, and horses and humans as accidental dead-end hosts. Rift Valley fever virus (RVFV) re-emerges periodically in Africa causing massive outbreaks. In the Maghreb, eco-climatic and entomologic conditions are favourable for WNV and RVFV emergence. Both viruses are transmitted by mosquitoes belonging to the Culex pipiens complex. We evaluated the ability of different populations of Cx. pipiens from North Africa to transmit WNV and the avirulent RVFV Clone 13 strain. Mosquitoes collected in Algeria, Morocco, and Tunisia during the summer 2010 were experimentally infected with WNV and RVFV Clone 13 strain at titers of 10(7.8) and 10(8.5) plaque forming units/mL, respectively. Disseminated infection and transmission rates were estimated 14-21 days following the exposure to the infectious blood-meal. We show that 14 days after exposure to WNV, all mosquito st developed a high disseminated infection and were able to excrete infectious saliva. However, only 69.2% of mosquito strains developed a disseminated infection with RVFV Clone 13 strain, and among them, 77.8% were able to deliver virus through saliva. Thus, Cx. pipiens from the Maghreb are efficient experimental vectors to transmit WNV and to a lesser extent, RVFV Clone 13 strain. The epidemiologic importance of our findings should be considered in the light of other parameters related to mosquito ecology and biology.     

A Modeling Approach to Investigate Epizootic Outbreaks and Enzootic Maintenance of Rift Valley Fever Virus

We propose a mathematical model to investigate the transmission dynamics of Rift Valley fever (RVF) virus among ruminants. Our findings indicate that in endemic areas RVF virus maintains at a very low level among ruminants after outbreaks and subsequent outbreaks may occur when new susceptible ruminants are recruited into endemic areas or abundant numbers of mosquitoes emerge when herd immunity decreases. Many factors have been shown to have impacts on the severity of RVF outbreaks; a higher probability of death due to RVF among ruminants, a higher mosquito:ruminant ratio, or a shorter lifespan of animals can amplify the magnitude of the outbreaks; vaccination helps to reduce the magnitude of RVF outbreaks and the loss of animals efficiently, and the maximum vaccination effort (a high vaccination rate and a larger number of vaccinated animals) is recommended before the commencement of an outbreak but can be reduced later during the enzootic.     

Vaccination with DNA Plasmids Expressing Gn Coupled to C3d or Alphavirus Replicons Expressing Gn Protects Mice against Rift Valley Fever Virus

Background

Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent.

Methodology/Principal Findings

We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12.

Conclusion/Significance

These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use.     

Generation of replication-defective virus-based vaccines that confer full protection in sheep against virulent bluetongue virus challenge

The reverse genetics technology for bluetongue virus (BTV) has been used in combination with complementing cell lines to recover defective BTV-1 mutants. To generate a potential disabled infectious single cycle (DISC) vaccine strain, we used a reverse genetics system to rescue defective virus strains with large deletions in an essential BTV gene that encodes the VP6 protein (segment S9) of the internal core. Four VP6-deficient BTV-1 mutants were generated by using a complementing cell line that provided the VP6 protein in trans. Characterization of the growth properties of mutant viruses showed that each mutant has the necessary characteristics for a potential vaccine strain: (i) viral protein expression in noncomplementing mammalian cells, (ii) no infectious virus generated in noncomplementing cells, and (iii) efficient replication in the complementing VP6 cell line. Further, a defective BTV-8 strain was made by reassorting the two RNA segments that encode the two outer capsid proteins (VP2 and VP5) of a highly pathogenic BTV-8 with the remaining eight RNA segments of one of the BTV-1 DISC viruses. The protective capabilities of BTV-1 and BTV-8 DISC viruses were assessed in sheep by challenge with specific virulent strains using several assay systems. The data obtained from these studies demonstrated that the DISC viruses are highly protective and could offer a promising alternative to the currently available attenuated and killed virus vaccines and are also compliant as DIVA (differentiating infected from vaccinated animals) vaccines.     

Protection of Spanish Ibex (Capra pyrenaica) against Bluetongue virus serotypes 1 and 8 in a subclinical experimental infection

Many wild ruminants such as Spanish ibex (Capra pyrenaica) are susceptible to Bluetongue virus (BTV) infection, which causes disease mainly in domestic sheep and cattle. Outbreaks involving either BTV serotypes 1 (BTV-1) and 8 (BTV-8) are currently challenging Europe. Inclusion of wildlife vaccination among BTV control measures should be considered in certain species. In the present study, four out of fifteen seronegative Spanish ibexes were immunized with a single dose of inactivated vaccine against BTV-1, four against BTV-8 and seven ibexes were non vaccinated controls. Seven ibexes (four vaccinated and three controls) were inoculated with each BTV serotype. Antibody and IFN-gamma responses were evaluated until 28 days after inoculation (dpi). The vaccinated ibexes showed significant (P<0.05) neutralizing antibody levels after vaccination compared to non vaccinated ibexes. The non vaccinated ibexes remained seronegative until challenge and showed neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of non vaccinated ibexes from 2 to the end of the study (28 dpi) and in target tissue samples obtained at necropsy (8 and 28 dpi). BTV-1 was successfully isolated on cell culture from blood and target tissues of non vaccinated ibexes. Clinical signs were unapparent and no gross lesions were found at necropsy. Our results show for the first time that Spanish ibex is susceptible and asymptomatic to BTV infection and also that a single dose of vaccine prevents viraemia against BTV-1 and BTV-8 replication.