BTV Antibodies Research Articles

Seroprevalence of Bluetongue and Schmallenberg viruses in ruminants in Al-Batinah Governorates, Oman

Bluetongue (BTV) and Schmallenberg viruses (SBV) are the causative agents of Bluetongue and Schmallenberg dis-eases, respectively. Both BTV and SBV are vector-borne viruses that are transmitted mainly by Culicoides (Diptera; Ceratopogo-nidae) and infect do-mestic, wild ruminants, and camelid species. Bluetongue virus be-longs to the genus Orbi-virus, family Reoviridae, whereas Schmallenberg virus belongs to the ge-nus Orthobunyavirus, family Bunyaviridae. In this study, we used en-zyme-linked immuno-sorbent assay (ELISA) to determine the sero-prevalence of both vi-ruses in ruminants in Al-Batinah Gover-norates, Sultanate of Oman. A total of 529 serum samples were randomly collected from 207 sheep, 265 goats, and 57 cattle from five wilayat of Al-Batinah North and Al-Batinah South Governorates. The serum samples were screened for the presence of BTV-specific antibodies against the BTV VP7 protein using ID Screen® Bluetongue Competition (cELISA) and screened for the presence of SBV-specific antibodies against the recombinant SBV nucleoprotein anti-gen using ID Screen® Schmallenberg virus Indirect Mult (iELISA). The overall seropreva-lence of BTV and SBV infections was 69.8% and 44.8%. In cattle, the prevalence of BTV and SBV antibodies was comparatively higher (94.7% and 82.5%) than in goats (83% and 43%) and sheep (45.9% and 36.7%). The high-est BTV seroprevalence was recorded in Nakhal (85.5%), followed by Wadi Al-Maawil (81%), and was lowest in Bar-ka (59.6%). However, the highest seropreva-lence of SBV was ob-served in Wadi Al-Maawil (50%), followed by Barka (48.7%), and lowest in Sohar (34.2%). Overall, the seroprevalence of BTV and SBV in domesticat-ed ruminants was high-er in adults than in young animals. Females showed a higher sero-prevalence of BTV and SBV compared to males. The study pro-vides an update on the epidemiological status of BTV, and to our knowledge, this is the first study on the sero-prevalence of SBV in ruminants in Oman. Future studies are re-quired for the isolation and identification of BTV and SBV along with other potential bio-logical vectors in Oman.

Bluetongue Virus Infections in Cattle Herds of Manabí Province of Ecuador.

Bluetongue (BT) is a viral disease transmitted by Culicoides (Diptera: Ceratopogonidae) to domestic and wild ruminants. Infections in cattle are mainly subclinical, but severe necrotic and hemorrhagic illness and death may occur depending on the strain of the virus and other factors; cattle act as a reservoir for the virus. Although the Ecuadorian coast has climatic conditions that favor the presence of the vector, there are few serologic or virologic BTV studies available. Manabí is a coastal province in which livestock farming is mostly implemented in the northern part. We conducted two studies to assess, for the first time, the presence of active BTV infections in Manabí province. We collected 430 serum samples from 38 randomly selected farms between March and July 2019 to perform BTV competitive ELISA. In addition, six seropositive farms were selected to place eight sentinel BTV-naive calves. All these calves were blood sampled and the presence of BTV RNA and antibodies was tested for by RT-PCR and competitive ELISA, respectively, once a week for 6–8 weeks until seroconversion was evidenced. A high individual seroprevalence (99%) was obtained, and all investigated farms had BTV seropositive animals. All sentinel calves became BTV viremic and seroconverted. The first viremia appeared after 2–5 weeks from arrival at the farm; they seroconverted 1–3 weeks later. We demonstrate for the first time that there is a high level of BTV circulation north of Manabí, with active infections on these farms. Integrated control strategies such as hygienic measures on farms to reduce midge populations would be advisable for the owners as mitigation measures.

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.

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.

BTV antibody longevity in cattle five to eight years post BTV-8 vaccination

The Bluetongue virus serotype −8 (BTV-8) epizootic in Germany (2006–2008) was successfully eradicated, essentially by the massive application of commercially available inactivated BTV-8 vaccines. While a six-year antibody longevity of BTV antibodies post BTV-8 vaccination in cattle has been described previously, our study investigated the BTV-8-vaccine antibodies in cattle for up to eight years. In total, 157 bovine serum samples were analysed for the presence of group-specific BTV antibodies in both a commercial cELISA, and a BTV-8- specific serum neutralization test. A robust number of cattle were seropositive for group- and serotype-specific neutralising antibodies for five or more years. In selected animals, born and vaccinated in 2009 or later, the presence of BTV antibodies for up to eight years post BTV-8 vaccination could be confirmed. Our data also show, that booster vaccination prolonged the antibody longevity of vaccine-induced antibodies and the number of serologically positive cattle.

Comparative immuno-reactivity of recombinant non-structural protein 2 fragments (N- and C- terminus) to detect bluetongue viral antibodies in small ruminant serum samples

Bluetongue viral antibody detection assays are important for routine disease diagnosis and sero-surveillance in susceptible host’s especially small ruminants. In the present study, a partial NS2 gene encoding for two non-structural protein-2 fragments; N-terminus (1M-A177 aa) and C-terminus (178P-V354 aa) of BTV-23, were cloned separately, expressed and purified as recombinant NS2Nt and NS2Ct fusion proteins (˜39 kDa each) from prokaryotic expression system (Escherichia coli). Subsequent to affinity chromatographic purification under non-denaturing condition, both rNS2Nt and rNS2Ct fusion proteins were obtained in sufficient quantity and quality. Further, both antigens were also found to possess good reactivity in detecting NS2 specific BTV antibodies irrespective of serotypes in ruminant serum samples by indirect-ELISA. Of two fragments, rNS2Nt was found to be more efficient as diagnostic candidate. However, in comparison to structural protein based VP7 c-ELISA and rVP7 i-ELISA, the sensitivity (5.1%, 8.2%) and specificity (2.5%, 3.2%) of rNS2Nt i-ELISA were found to be relatively lower, respectively. The study indicated that N-terminus of NS2 protein could be used either alone or in combination/fusion with other non-structural proteins (NS1/NS3) of BTV for potential utility of NS protein based indirect-ELISA as an alternate assay for routine sero-surveillance of BTV infection in ruminants.

Seroprevalence of bluetongue in dromedaries

Bluetongue (BT) is an infectious, non-contagious, arthropod-borne viral disease, mainly of sheep but many domestic and wild animals are also affected by this disease. A serological study was aimed at the detection of BTV antibodies by Agar Gel Immunodiffusion test (BT-AGID) and competitive Enzyme Linked Immunosorbent Assay (c-ELISA) in dromedaries of North Gujarat and Kachchh regions. Out of 533 serum samples, the BT-AGID test detected antibodies against BTV in 83 cases (15.57%) while c-ELISA test was positive in 136 cases (25.51%).

Production of recombinant non-structural protein-3 hydrophobic domain deletion (NS3ΔHD) protein of bluetongue virus from prokaryotic expression system as an efficient diagnostic reagent

Serological diagnostics for bluetongue (BT), which is an infectious, non-contagious and arthropod-borne virus disease of ruminants, are primarily dependent on availability of high quality native or recombinant antigen(s) based on either structural/non-structural proteins in sufficient quantity. Non-structural proteins (NS1-NS4) of BT virus are presumed candidate antigens in development of DIVA diagnostics. In the present study, NS3 fusion gene encoding for NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains (118A to S141 aa and 162S to A182 aa) and intervening variable central domain (142D to K161 aa) of bluetongue virus 23 was constructed, cloned and over-expressed using prokaryotic expression system. The recombinant NS3ΔHD fusion protein (∼38 kDa) including hexa-histidine tag on its both termini was found to be non-cytotoxic to recombinant Escherichia coli cells and purified by affinity chromatography. The purified rNS3ΔHD fusion protein was found to efficiently detect BTV-NS3 specific antibodies in indirect-ELISA format with diagnostic sensitivity (DSn = 94.4%) and specificity (DSp = 93.9%). The study indicated the potential utility of rNS3ΔHD fusion protein as candidate diagnostic reagent in developing an indirect-ELISA for sero-surveillance of animals for BTV antibodies under DIVA strategy, wherever monovalent/polyvalent killed BT vaccine formulations devoid of NS proteins are being practiced for immunization.

HIGH SEROPREVALENCE OF BLUETONGUE IN SHEEP, GOATS AND CATTLE IN ASSIUT GOVERNORATE, EGYPT

A total of 552 serum samples of sheep, goats and cattle from different localities at Assiut governorate, Egypt were screened for qualitative analysis of the BTV antibodies using a commercial competitive ELISA (cELISA) kit. The results showed an overall percentage of BTV positive sheep, goats and cattle serum samples were 41.86%, 24% and 85.42% respectively. A highest percentage of seropositivity was found inArab El- Awamer farm (100%), followed by Abnoub( 63. 64%), Sedfa(11. 43%), veterinary teaching hospital (11.11%), Aboteeg (3.57%) and Bani zeed el akrad (0% ) respectively. From examined sera all over the four years from 2012 to 2015 indicates prevalence of BTV antibodies by ( 62. 69%) during the non hot months and (10%) during the hot months, Female ruminants were more susceptible to the infection (61.29%) than male( 21.67%). The association between sex and prevalence of BT disease among ruminants observed in table (4). The results showed that the prevalence of BT was higher in female (61.29%) than male (21.67%). In conclusion, this study confirms the presence of blue tongue infection in Assiut Governorate, and cELISA were found to be sensitive and effective for screening of BTV group specific antibodies. The disease should be notifying its presence in Egypt since it was free from this disease and more studies should be attempets to estimate the serotype of blue tongue virus

First report of the giant liver fluke (Fascioloides magna) infection in farmed fallow deer (Dama dama) in Poland – pathomorphological changes and molecular identification

Abstract A 3-year-old female fallow deer was subjected to the necropsy and virological testing, due to a suspected infectious disease in the herd of farmed deer in the Southeastern region of Poland. The animal was found negative for the presence of BVDV, BoHV-1, BTV, and EHDV antibodies and BVDV antigen. The toxicological examination did not reveal any coccidiostats, mycotoxins, rodenticides, carbamate pesticides, and organophosphorus pesticides. The flukes found during postmortem examination were first characterised microscopically as Fascioloides magna and later their identity was confirmed by PCR and sequencing. The autopsy revealed lesions characteristic for F. magna infection, including different size cystic spaces in the liver, filled with brownish mucous fluid and flukes, and black pigment covering the surface of parietal and visceral peritoneum with the highest concentrations localised next to the liver. The changes observed in the liver tissue were typical of liver cirrhosis. The results demonstrated that in Poland, where the cervid farming is developing dynamically, the problem of fascioloidosis is present and may probably exert a significantly negative influence on the productivity of such farms if no antiparasitic treatment is performed.

Establishment of evanescent wave fiber-optic immunosensor method for detection bluetongue virus

The evanescent wave fiber immunosensors (EWFI) technique was developed for the real-time rapidly sensitive and specific detection of the monoclonal antibody 3E2 of BTV. The outer-core protein VP7 of BTV was labled on the surface of the exposed fiber-optic core. The monoclonal antibody 3E2 of BTV VP7 were added and then the goat ant-rat IgG conjugated with Cy3 was captured. After the 532nm pulse (excitation source) reached the fiber probe, evanescent wave was generated, which excited the Cy3 bound to the immuno-complex and produced the fluorescent signal, which was changed into electrical signals read through computer. The preliminary results suggested that a detection limit of 10ng/ml was measured for the monoclonal antibody 3E2, which is equal to the sensitivity of ELISA. The 3E2 sample was specifically detected through the EWFI assay in 15min, and the fiber can be recycled at least ten times through TEA solution condition. This developed EWFI was a real-time rapidly sensitive and specific way for the detection of BTV antibodies.

Prevalence of antibodies to bluetongue virus in large ruminants of Marathwada region of Maharashtra state

Aim: Seroprevalence study of BT antibodies in large ruminants of Marathwada region of Maharashtra state. Materials and Methods: A total of 246 serum samples of Cattle and Buffalo were screened for qualitative analysis of the BTV antibodies using a commercial competitive ELISA (cELISA) kit. Results: The results showed an overall percentage of BTV positive cattle and buffalo serum samples were 89.80% and 80% respectively. The results based upon the serum samples showing optical density values more than 50 per cent of the mean of negative control were taken as positive for presence of BTV antibodies. A high percentage of cattle showed BTV antibodies in places Parbhani (92.53%), Nanded (86%) and Aurangabad (81.81%). The percentage of BTV antibodies in buffaloes was 70%, 100%, 100% and 87.5% respectively in Parbhani, Nanded, Aurangabad and Hingoli. Conclusion: BTV antibodies were widely prevalent in cattle & buffaloes of Marathwada region and cELISA were found to be sensitive and effective for screening of BTV group specific antibodies.

Functionalizing gold nanoparticles with bluetongue virus multiple peptide antigens utilizing gold–thiol interaction: A novel approach to develop pen side test

Bluetongue is an economically important viral disease of small ruminants. The present/current diagnostic kits and methods to diagnose BTV are laborious, time consuming and expensive. In the present study, we have attempted to develop a novel approach to detect BTV antibodies in sera that in future can be harnessed for developing a pen side diagnostic test. Briefly, we identified the immunodominant regions of the VP7 protein of BTV and synthesized them in the multiple antigenic peptide (MAP) format with cysteine at C-terminal of the lysine mosaic, which elicited highly ordered conformation as well as ELISA reactivity. Finally, we coated the MAP peptides on the gold nanoparticles that can be used to detect BTV specific antibodies in the sera using a spot test.

High seroprevalence of bluetongue virus antibodies in Sheep, Goats, Cattle and Camel in different districts of Saudi Arabia

Aim: To estimate the prevalence and distribution of serum antibodies to BTV in different domesticated animals in different localities of Saudi Arabia. Materials and Methods: A total of 4845 field sera collected from different animal species within 10 districts in the Kingdom of Saudi Arabia were screened for the presence of group-specific BTV antibodies by competitive ELISA (c ELISA). Results: The overall BTV antibody prevalence was 54.1%, 53.3%, 44.8% and 25.7% in sheep, goat, cattle and camel respectively (at 95% confidence level). The Jizan and Eastern Province districts were the regions with the highest prevalence resulting 65.8% of sheep, 68.2% of goats, 49.3% of cattle, 44% of camel in Jizan and 65.8% of sheep, 62.5% of goats, 53.4% of cattle, 28.5% of camel in Eastern Province positive to c-ELISA. The second highest rate was in Najran district where the seropositivity for Bluetongue was found to be 60% of sheep, 57.9% of goats, 47.2% of cattle and 29.3% of camel. Our results recorded positive animals in all examined districts which indicate serological evidence of exposure to infection was widely distributed all over the country. Conclusions: These results demonstrate the high occurrence of the BTV that emphasize the necessity to a well-defined control strategy for preventing and controlling the BTV in Saudi Arabia.

A Serologic Survey of Viral Infections in Captive Ungulates in Turkish Zoos

Zoos and zoologic gardens make optimal environments for interspecies transmission of viral infections. There are seven zoos and several small zoologic collections in Turkey. This study aimed to determine the current status of viral infections in captive ungulates living in these environments. Blood samples were taken from 163 captive animals from two zoos. There were 39 Cameroon sheep (Ovis ammon f aries), 11 Barbary sheep (Ammotragus lervia), 57 pygmy goats (Capra hircus), 9 Angora goats (Capra hircus), 21 mountain goats (Capra aegagrus-aegagrus), 7 llamas (Lama glama), 8 Persian goitred gazelle (Gazella subgutturosa subgutturosa), 7 Caspian red deer (Cervus elaphus maral), 2 fallow deer (Dama dama), and 2 camels (Camelus dromedarius). Antibodies against bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV-1), bovine adenoviruses (BAV-1 and -3), parainfluenzavirus 3 (PI-3), and bluetongue viruses (BTV-4 and -9) were investigated using the virus neutralization test, and malignant catarrhal fever (MCF) antibodies were screened by ELISA. All animals were negative for BVDV and BHV-1 antibodies. Seroprevalence of BAV-1, BAV-3, PI-3, BRSV, BT-4, BT-9, and MCF were detected as follows: 46.6%, 60.1%, 0.6%, 7.3%, 1.8%, 1.2%, and 51.6%, respectively. Seroprevalence of BAVs and MCF were more common than all other viruses (P < 0.0001). Ten sheep (37.0%), 48 goats (84.2), and 1 Ilama (14.2%) were the only species positive for MCF antibodies. Prevalence of BRSV and MCF antibodies were found to be significantly higher in goats than in sheep. BTV antibodies were detected both in Cameroon sheep and mountain goats and suggest that zoo animals are at risk for BTV in endemic regions.

155 EMBRYONIC APOPTOSIS AFTER BTV-8 INFECTION IN BOVINE HATCHED IN VITRO PRODUCED BLASTOCYSTS

The recent blue tongue virus serotype-8 (BTV-8) epidemic in central Western Europe has been associated with field fertility problems (De Clercq et al. 2008 Transbound. Emerg. Dis. 55, 352–359). Previous research clearly showed that in vitro produced bovine hatched blastocysts are susceptible for BTV-8 infection (Vandaele et al. 2010 Reprod. Fertil. Dev. 22, 254 abst.). The aim of the present study was to investigate the effect of a BTV-8 infection on the occurrence of apoptosis in embryos in order to gain a clear insight into the role BTV-8 might play in early embryonic death. Immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes were denuded 24 h post-insemination and cultured in 50-μL droplets of modified SOF medium with 10% fetal calf serum (tested negative for BTV antibodies) at 38.5°C in 5% CO2, 5% O2, and 90% N2. At 7 days post-insemination (dpi), blastocysts were grouped to enhance hatching. At 8.5 dpi, 4 to 8 hatched embryos were placed in 800 μL of minimum essential medium (MEM), containing 103.8 to 104.9 50% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/3, VAR, Brussels, Belgium) and incubated for 1 h at 38.5°C in an atmosphere of 5% CO2 in air. Two groups of hatched control embryos were incubated under the same circumstances in 800 μL of SOF and 800 μL of MEM, respectively. After inoculation, embryos were washed according to IETS guidelines with the exception that they were not ZP-I and cultured in new SOF. At 48 and 72 h post-inoculation (hpi), half of the embryos of each group were fixed in 4% paraformaldehyde for 12 to 24 h and subsequently stained for BTV-8 and apoptosis with a double immunofluorescent staining using a BTV-8 monoclonal antibody (8A3B.6, ID-Vet, Montpellier, France) in combination with TUNEL (Roche Diagnostics, Mannheim, Germany). All mock-inoculated embryos were negative for BTV-8 virus antigen. At 48 and 72 hpi, respectively, 45 and 38% of the embryos were BTV-8 positive in all embryonic cells, and all remaining embryos had at least some BTV-8 blastomeres. The overall apoptotic cell ratio in infected embryos (17.9 ± 1.14% at 48 hpi and 15.3 ± 1.16% at 72 hpi) was significantly higher than in noninfected, mock-inoculated embryos (10.4 ± 0.57% at 48 hpi and 4.3 ± 0.30% at 72 hpi) (P &lt; 0.01). Furthermore, total cell number was substantially lower in infected embryos (103 ± 14.2 at 48 hpi and 120 ± 17.5 at 72 hpi) compared with noninfected, mock-inoculated embryos (258 ± 47.4 at 48 hpi and 348 ± 64.1 at 72 hpi) (P &lt; 0.05). This study indicates that embryonic apoptosis after BTV-8 infection results in embryonic arrest and early embryonic death and thus might be involved in herd fertility problems during a BTV epidemic. The first author is supported by Research Foundation Flanders (Grant number G.0210.09).

192 SUSCEPTIBILITY OF BOVINE-HATCHED BLASTOCYSTS TO BLUETONGUE VIRUS SEROTYPE 8 INFECTION

In 2006 and 2007, Bluetongue virus serotype 8 (BTV-8) caused devastating outbreaks in Northern Europe; the outbreaks were controlled in 2008 and 2009 by an international vaccination policy. Remarkably, BTV-8 differs from other serotypes in that it spread transplacentally (De Clercq K et al. 2008 Transboundary and Emerging Diseases 55, 352-359). Apart from the transplacental spreading, a significant increase in the incidence of abortions was reported in Belgium (Meroc E et al. 2009 Transboundary and Emerging Diseases 56, 39-48). The aim of the present study was to investigate the susceptibility of bovine-hatched, in vitro-produced blastocysts to BTV-8. A total of 1390 immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes (n = 1148) were denuded 24 h post-insemination and cultured in 50-μL droplets of modified synthetic oviduct fluid (SOF) medium with 10% fetal calf serum (tested negative for BTV antibodies) at 39.0°C in 5% CO2, 5% O2, and 90% N2. At 7 days post-insemination (dpi), blastocysts were grouped to enhance hatching. For virus incubation, BTV-8 Bel 2006/2 from Veterinary and Agrochemical Research Centre (VAR, Brussels, Belgium) was used. At 8.5 dpi, hatched embryos were placed in 800μL of minimum essential medium (MEM) containing 103.8 50% tissue culture infectious doses (TCID50) of BTV-8 and incubated for 1 h at 39°C in an atmosphere of 5% CO2 in air. At the same time, 2 groups of hatched control embryos were incubated under the same circumstances in 800 μL of SOF and 800 μL of MEM, respectively. After infection, all embryos were washed according to IETS guidelines with the exception that they were not zona pellucida intact and cultured in new SOF. At 48, 60, 72, and 96 h post-infection (hpi), one-fourth of the embryos of each group were fixed in 4% paraformaldehyde for 12 to 24 h and subsequently stained for BTV-8 with double immunofluorescent staining using a BTV-8 monoclonal antibody (8A3B.6, ID-Vet, Montpellier, France). All control embryos (CTRL and MEM) were negative for BTV-8 virus antigen at all time points. At 48 hpi, only 1 out of 7 infected embryos was positive for virus antigen (in all its cells). At 60 hpi, all remaining embryos (n = 6) were negative, whereas at 72 hpi and 96 hpi all embryos had 25% to 100% BTV-8-positive cells (n = 6 at 72 hpi and n = 7 at 96 hpi). Furthermore, 1 embryo at 72 hpi and 2 embryos at 96 hpi showed morphological signs of degeneration. This study has showed for the first time that hatched in vitro-produced blastocysts are susceptible for BTV-8 virus infection and replication in vitro. The relatively long time between virus infection and detection of viral antigen is in accordance with the slow replication cycle of the virus. Further research is ongoing to investigate the importance of BTV-8 infection in early embryonic death. The first author is supported by Research Foundation-Flanders.