Immune response of horses to inactivated African horse sickness vaccines.

BACKGROUND: African horse sickness (AHS) is a serious viral disease of equids resulting in the deaths of many equids in sub-Saharan Africa that has been recognized for centuries. This has significant economic impact on the horse industry, despite the good husbandry practices. Currently, prevention and control of the disease is based on administration of live attenuated vaccines and control of the arthropod vectors. RESULTS: A total of 29 horses in 2 groups, were vaccinated. Eighteen horses in Group 1 were further divided into 9 subgroups of 2 horses each, were individually immunised with one of 1 to 9 AHS serotypes, respectively. The eleven horses of Group 2 were immunised with all 9 serotypes simultaneously with 2 different vaccinations containing 5 serotypes (1, 4, 7-9) and 4 serotypes (2, 3, 5, 6) respectively. The duration of this study was 12 months. Blood samples were periodically withdrawn for serum antibody tests using ELISA and VNT and for 2 weeks after each vaccination for PCR and virus isolation. After the booster vaccination, these 27 horses seroconverted, however 2 horses responded poorly as measured by ELISA. In Group 1 ELISA and VN antibodies declined between 5 to 7 months post vaccination (pv). Twelve months later, the antibody levels in most of the horses decreased to the seronegative range until the annual booster where all horses again seroconverted strongly. In Group 2, ELISA antibodies were positive after the first booster and VN antibodies started to appear for some serotypes after primary vaccination. After booster vaccination, VN antibodies increased in a different pattern for each serotype. Antibodies remained high for 12 months and increased strongly after the annual booster in 78% of the horses. PCR and virus isolation results remained negative. CONCLUSIONS: Horses vaccinated with single serotypes need a booster after 6 months and simultaneously immunised horses after 12 months. Due to the non-availability of a facility in the UAE, no challenge infection could be carried out.

Plant-produced chimeric virus-like particles - a new generation vaccine against African horse sickness.

BACKGROUND: African horse sickness (AHS) is a severe arthropod-borne viral disease of equids, with a mortality rate of up to 95% in susceptible naïve horses. Due to safety concerns with the current live, attenuated AHS vaccine, alternate safe and effective vaccination strategies such as virus-like particles (VLPs) are being investigated. Transient plant-based expression systems are a rapid and highly scalable means of producing such African horse sickness virus (AHSV) VLPs for vaccine purposes. RESULTS: In this study, we demonstrated that transient co-expression of the four AHSV capsid proteins in agroinfiltrated Nicotiana benthamiana dXT/FT plants not only allowed for the assembly of homogenous AHSV-1 VLPs but also single, double and triple chimeric VLPs, where one capsid protein originated from one AHS serotype and at least one other capsid protein originated from another AHS serotype. Following optimisation of a large scale VLP purification procedure, the safety and immunogenicity of the plant-produced, triple chimeric AHSV-6 VLPs was confirmed in horses, the target species. CONCLUSIONS: We have successfully shown assembly of single and double chimeric AHSV-7 VLPs, as well as triple chimeric AHSV-6 VLPs, in Nicotiana benthamiana dXT/FT plants. Plant produced chimeric AHSV-6 VLPs were found to be safe for administration into 6 month old foals as well as capable of eliciting a weak neutralizing humoral immune response in these target animals against homologous AHSV virus.

Immunogenicity of plant-produced African horse sickness virus-like particles: implications for a novel vaccine.

African horse sickness (AHS) is a debilitating and often fatal viral disease affecting horses in much of Africa, caused by the dsRNA orbivirus African horse sickness virus (AHSV). Vaccination remains the single most effective weapon in combatting AHS, as there is no treatment for the disease apart from good animal husbandry. However, the only commercially available vaccine is a live-attenuated version of the virus (LAV). The threat of outbreaks of the disease outside its endemic region and the fact that the LAV is not licensed for use elsewhere in the world, have spurred attempts to develop an alternative safer, yet cost-effective recombinant vaccine. Here, we report the plant-based production of a virus-like particle (VLP) AHSV serotype five candidate vaccine by Agrobacterium tumefaciens-mediated transient expression of all four capsid proteins in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant expression vector system. The production process is fast and simple, scalable, economically viable, and most importantly, guinea pig antiserum raised against the vaccine was shown to neutralize live virus in cell-based assays. To our knowledge, this is the first report of AHSV VLPs produced in plants, which has important implications for the containment of, and fight against the spread of, this deadly disease.

Assembly of Replication-Incompetent African Horse Sickness Virus Particles: Rational Design of Vaccines for All Serotypes.

UNLABELLED: African horse sickness virus (AHSV), an orbivirus in the Reoviridae family with nine different serotypes, causes devastating disease in equids. The virion particle is composed of seven proteins organized in three concentric layers, an outer layer made of VP2 and VP5, a middle layer made of VP7, and inner layer made of VP3 that encloses a replicase complex of VP1, VP4, and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious candidate vaccines against all AHSV serotypes, taking into account not only immunogenic and safety properties but also virus productivity and stability parameters, which are essential criteria for vaccine candidates. To achieve this goal, we first established a highly efficient reverse genetics (RG) system for AHSV serotype 1 (AHSV1) and, subsequently, a VP6-defective AHSV1 strain in combination with in trans complementation of VP6. This was then used to generate defective particles of all nine serotypes, which required the exchange of two to five RNA segments to achieve equivalent titers of particles. All reassortant-defective viruses could be amplified and propagated to high titers in cells complemented with VP6 but were totally incompetent in any other cells. Furthermore, these replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor (IFNAR)-knockout mice. Thus, these defective viruses have the potential to be used for the development of safe and stable vaccine candidates. The RG system also provides a powerful tool for the study of the role of individual AHSV proteins in virus assembly, morphogenesis, and pathogenesis. IMPORTANCE: African horse sickness virus is transmitted by biting midges and causes African horse sickness in equids, with mortality reaching up to 95% in naive horses. Therefore, the development of efficient vaccines is extremely important due to major economic losses in the equine industry. Through the establishment of a highly efficient RG system, replication-deficient viruses of all nine AHSV serotypes were generated. These defective viruses achieved high titers in a cell line complemented with VP6 but failed to propagate in wild-type mammalian or insect cells. Importantly, these candidate vaccine strains showed strong protective efficacy against AHSV infection in an IFNAR(-/-) mouse model.

African Horse Sickness Caused by Genome Reassortment and Reversion to Virulence of Live, Attenuated Vaccine Viruses, South Africa, 2004-2014.

African horse sickness (AHS) is a hemorrhagic viral fever of horses. It is the only equine disease for which the World Organization for Animal Health has introduced specific guidelines for member countries seeking official recognition of disease-free status. Since 1997, South Africa has maintained an AHS controlled area; however, sporadic outbreaks of AHS have occurred in this area. We compared the whole genome sequences of 39 AHS viruses (AHSVs) from field AHS cases to determine the source of 3 such outbreaks. Our analysis confirmed that individual outbreaks were caused by virulent revertants of AHSV type 1 live, attenuated vaccine (LAV) and reassortants with genome segments derived from AHSV types 1, 3, and 4 from a LAV used in South Africa. These findings show that despite effective protection of vaccinated horses, polyvalent LAV may, paradoxically, place susceptible horses at risk for AHS.

VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV.

UNLABELLED: African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate "synthetic" reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replication in vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes. IMPORTANCE: African horse sickness virus is transmitted by species of Culicoides biting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replication in vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since the in vitro release of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.

African horse sickness in The Gambia: circulation of a live-attenuated vaccine-derived strain.

African horse sickness virus serotype 9 (AHSV-9) has been known for some time to be circulating amongst equids in West Africa without causing any clinical disease in indigenous horse populations. Whether this is due to local breeds of horses being resistant to disease or whether the AHSV-9 strains circulating are avirulent is currently unknown. This study shows that the majority (96%) of horses and donkeys sampled across The Gambia were seropositive for AHS, despite most being unvaccinated and having no previous history of showing clinical signs of AHS. Most young horses (<3 years) were seropositive with neutralizing antibodies specific to AHSV-9. Eight young equids (<3 years) were positive for AHSV-9 by serotype-specific RT-PCR and live AHSV-9 was isolated from two of these horses. Sequence analysis revealed the presence of an AHSV-9 strain showing 100% identity to Seg-2 of the AHSV-9 reference strain, indicating that the virus circulating in The Gambia was highly likely to have been derived from a live-attenuated AHSV-9 vaccine strain.

Animal diseases caused by orbiviruses, Algeria.

Antibodies against bluetongue virus were detected in cattle, sheep, goats, and camels in Algeria in 2008. Antibodies against epizootic hemorrhagic disease virus were detected in cattle, but antibodies against African horse sickness virus were not detected in horses and mules. Epizootic hemorrhagic disease in northern Africa poses a major risk for the European Union.

Tissue tropism of African horsesickness virus in the chicken embryo demonstrated with the avidin-biotin complex immunoperoxidase method.

In horses, African horsesickness virus (AHSV) exhibits marked tropism for certain microvascular endothelia and components of the mononuclear phagocyte system. In this study, the tropism of a field isolate of AHSV serotype 5 was studied in 24 chicken embryos. Histopathology on embryonic tissues harvested with 12 hour intervals revealed progressive changes associated with endothelial damage. Immunolabeling demonstrated viral antigens in the microvascular endothelium of the spleen, lungs, and the mesenchymal connective tissue at the base of the neck, from 24 hours post inoculation. Subsequently, specific immunolabeling increased steadily in endothelia of these and other tissues such as skeletal and cardiac muscle, gastrointestinal smooth muscle, mesonephric glomeruli, liver, subcutis and feathers. Positive immunolabeling was also occasionally observed in circulating mononuclear cells and in Kupffer cells in the liver. It was concluded, that this isolate of AHSV displayed similar tissue tropism in the chicken embryo as in the horse.

Serological survey of African horse sickness in selected districts of Jimma zone, Southwestern Ethiopia.

A cross-sectional serological survey was undertaken in selected districts of different agro-ecology of Jimma zone (Dedo, Yebu, Seka, Serbo, and Jimma town) from November 2009 to February 2010 to determine the seroprevalence of African horse sickness virus and associated risk factors of the disease. Two hundred seventy-four equids (189 horses, 43 mules, and 47 donkeys) with a history of non-vaccination for at least 2 years were selected randomly from the above areas. Sera samples were collected and assayed for the presence of specific antibody against African horse sickness virus using blocking ELISA. An overall seroprevalence of 89 (32.5%) was found and it was 24 (51.1%) for donkeys, 13 (30.2%) for mules, and 52(28.3%) for horses. Seroprevalence was significantly (X(2) = 11.05, P < 0.05) different among the different species of equids. Seroprevalence was also significantly (X(2) = 11.43, P < 0.05) different among the different agro-ecological areas being higher in highlands 47 (40.5%) followed by midland 30 (34.5%) and lowland 12 (16.9%). Age and sex were not significantly (X(2) = 3.15, P > 0.05 and X(2) = 3.38, P > 0.05, respectively) associated with seroprevalence of AHSV. The present study showed that African horse sickness (AHS) is highly prevalent disease for the horses followed by mules and then donkeys in Jimma zone explained by lower seroconversion rate. Therefore, control strategy against AHS should target at high risk species of all age and sex in their locality in the initial stage for better containment of the disease.

Reverse genetics approaches: a novel strategy for African horse sickness virus vaccine design.

African horse sickness (AHS) is a devastating disease caused by African horse sickness virus (AHSV) and transmitted by arthropods between its equine hosts. AHSV is endemic in sub-Saharan Africa, where polyvalent live attenuated vaccine is in use even though it is associated with safety risks. This review article summarizes and compares new strategies to generate safe and effective AHSV vaccines based on protein, virus like particles, viral vectors and reverse genetics technology. Manipulating the AHSV genome to generate synthetic viruses by means of reverse genetic systems has led to the generation of potential safe vaccine candidates that are under investigation.

Cross-protective immune responses against African horse sickness virus after vaccination with protein NS1 delivered by avian reovirus muNS microspheres and modified vaccinia virus Ankara.

African horse sickness virus (AHSV) is an insect-borne pathogen that causes acute disease in horses and other equids. In an effort to improve the safety of currently available vaccines and to acquire new knowledge about the determinants of AHSV immunogenicity, new generation vaccines are being developed. In this work we have generated and tested a novel immunization approach comprised of nonstructural protein 1 (NS1) of AHSV serotype 4 (AHSV-4) incorporated into avian reovirus muNS protein microspheres (MS-NS1) and/or expressed using recombinant modified vaccinia virus Ankara vector (MVA-NS1). The protection conferred against AHSV by a homologous MS-NS1 or heterologous MS-NS1 and MVA-NS1 prime/boost was evaluated in IFNAR (-/-) mice. Our results indicate that immunization based on MS-NS1 and MVA-NS1 afforded complete protection against the infection with homologous AHSV-4. Moreover, priming with MS-NS1 and boost vaccination with MVA-NS1 (MS-MVA-NS1) triggered NS1 specific cytotoxic CD8 + T cells and prevented AHSV disease in IFNAR (-/-) mice after challenge with heterologous serotype AHSV-9. Cross-protective immune responses are highly important since AHS can be caused by nine different serotypes, which means that a universal polyvalent vaccination would need to induce protective immunity against all serotypes.

Standardization and validation of an immunoperoxidase assay for the detection of African horse sickness virus in formalin-fixed, paraffin-embedded tissues.

An immunoperoxidase assay for the detection of African horse sickness virus (AHSV) in formalin-fixed tissues is a valuable tool in the study of the pathogenesis of the disease, as well as a useful addition to existing diagnostic tests when only preserved tissues are available. An assay that uses Hamblin antiserum in a basic avidin-biotin complex detection system was standardized and validated in accordance with the guidelines of the American Association of Veterinary Laboratory Diagnosticians Subcommittee on Standardization of Immunohistochemistry. Using 128 positive cases of African horse sickness confirmed by viral isolation and serotyping and 119 negative cases from countries where the disease has never occurred, diagnostic sensitivity and diagnostic specificity were 100% in the prime target tissues of heart and lung. There was no variation in the ability of the assay to detect all 9 serotypes of AHSV, and there was no cross-reactivity with other orbiviruses in formalin-fixed tissues. The only cross-reactivity observed was in the lungs of 2 negative cases infected with Rhodococcus equi. The assay gave good results on tissues that had been fixed in formalin for up to 365 days. Nonspecific staining was minimal provided that the standard procedures for processing and staining tissues were followed. Good immunohistochemical results were also obtained on samples fixed as long as 24 hr after death. The assay, therefore, provides a robust diagnostic tool for detection of AHSV in formalin-fixed tissues, provided the analysis is done by an experienced pathologist.

African Horse Sickness: A Review of Current Understanding and Vaccine Development.

African horse sickness is a devastating disease that causes great suffering and many fatalities amongst horses in sub-Saharan Africa. It is caused by nine different serotypes of the orbivirus African horse sickness virus (AHSV) and it is spread by Culicoid midges. The disease has significant economic consequences for the equine industry both in southern Africa and increasingly further afield as the geographic distribution of the midge vector broadens with global warming and climate change. Live attenuated vaccines (LAV) have been used with relative success for many decades but carry the risk of reversion to virulence and/or genetic re-assortment between outbreak and vaccine strains. Furthermore, the vaccines lack DIVA capacity, the ability to distinguish between vaccine-induced immunity and that induced by natural infection. These concerns have motivated interest in the development of new, more favourable recombinant vaccines that utilize viral vectors or are based on reverse genetics or virus-like particle technologies. This review summarizes the current understanding of AHSV structure and the viral replication cycle and also evaluates existing and potential vaccine strategies that may be applied to prevent or control the disease.

A field investigation of an African horse sickness outbreak in the controlled area of South Africa in 2016.

An outbreak of African horse sickness (AHS) caused by AHS virus type 1 occurred within the South African AHS surveillance zone during April and May 2016. The index case was detected by a private veterinarian through passive surveillance. There were 21 cases in total, which is relatively low compared to case totals during prior AHS outbreaks in the same region (and of the same AHS virus type) in 2004, 2011 and 2014. The affected proportion of horses on affected properties was 0.07 (95% CI 0.04, 0.11). Weather conditions were conducive to high midge activity immediately prior to the outbreak but midge numbers decreased rapidly with the advent of winter. The outbreak was localized, with 18 of the 21 cases occurring within 8 km of the index property and the three remaining cases on two properties within 21 km of the index property, with direction of spread consistent with wind-borne dispersion of infected midges. Control measures included implementation of a containment zone with movement restrictions on equids. The outbreak was attributed to a reversion to virulence of a live attenuated vaccine used extensively in South Africa. Outbreaks in the AHS control zones have a major detrimental impact on the direct export of horses from South Africa, notably to the European Union.

Production of monoclonal antibodies binding to the VP7 protein of African horse sickness virus.

Monoclonal antibodies (MAbs) against AHSV were produced by immunising BALB/c mice with AHSV serotype 9 and six clones able to recognize specifically the VP7-AHSV with a strong reactivity were selected. The specificity of the MAbs was assessed in i-ELISA against a commercial VP7-AHSV and in immunoblot against a home-made VP7-AHSV, expressed by a Baculovirus expression system; potential cross-reactions with related orbiviruses (Bluetongue virus and Epizootic Haemorrhagic Disease virus) were investigated as well. One of the six MAbs selected, MAb 7F11E14, was tested in direct immunofluorescence and reacted with all nine AHSV serotypes, but didn't cross-react with BTV and EHDV. MAb 7F11E14 was also used to develop a competitive ELISA and was able to detect AHSV antibodies in the sera of AHS infected animals.

A serological survey of African horse sickness in Botswana.

A retrospective serological survey of African horse sickness (AHS) in Botswana covering a 10-year period (1995-2004) is reported. The survey involved horses showing clinical symptoms of the disease; the horses had not been vaccinated against AHS. Over the period surveyed, serological evidence suggestive of infection with AHS virus (AHSV) was found in 99 clinical cases out of which 41.4% (41/99) cases were found during the 1st half (1995-1999) and 58.6 % (58/99) cases were found in the 2nd half of the survey period (2000-2004). These serological findings are discussed in relation to AHSV serotypes isolated from diseased horses in Botswana before and during the period of this serological survey.

[Preparation and identification of monoclonal antibodies against African horse sickness virus VP7].

Objective To prepare the monoclonal antibody (mAb) against the African horsefever virus (AHSV) VP7 protein and to identify it. Methods mAbs were prepared by using baculovirus expressed VP7 protein in BALB/c mice, and the effect of mAb was detected by ELISA, indirect immunofluorescence assay (IFA), and AHSV positive serum blockade. Results Four mAb strains were selected, including 20A8, 28B3, 30G8 and 47E6, among which 47E6 had the best blocking effect. Conclusion mAbs were successfully prepared against VP7 protein.

Evidence of African horse sickness virus infection of Equus zebra hartmannae in the south-western Khomas Region, Namibia.

Equine mortalities suspected to be due to African horse sickness (AHS) were reported from the arid Khomas Region, Namibia, in 2008. The area was previously considered a localized AHS-free area. Hartmann's mountain zebra (Equus zebra hartmannae), a potential but unconfirmed reservoir host of African horse sickness virus (AHSV), occurs in the region. Between 2009 and 2010 serum, blood and tissue samples from 31 culled E. z. hartmannae were analysed by reverse transcription-polymerase chain reaction (RT-PCR) (n = 31) and enzyme-linked immunosorbent assay (ELISA) (n = 18) to determine the presence of AHSV and/or antibodies against AHSV. The presence of antibodies against AHSV was demonstrated in all 18 samples assayed, and AHSV double stranded RNA was detected in 26% of the animals. This is evidence that E. z. hartmannae can become infected with AHSV.

Diagnostic DIVA tests accompanying the Disabled Infectious Single Animal (DISA) vaccine platform for African horse sickness.

African Horse Sickness Virus (AHSV) (Orbivirus genus, Reoviridae family) causes high mortality in naïve domestic horses with enormous economic and socio-emotional impact. There are nine AHSV serotypes showing limited cross neutralization. AHSV is transmitted by competent species of Culicoides biting midges. AHS is a serious threat beyond the African continent as endemic Culicoides species in moderate climates transmit the closely related prototype bluetongue virus. There is a desperate need for safe and efficacious vaccines, while DIVA (Differentiating Infected from Vaccinated) vaccines would accelerate control of AHS. Previously, we have shown that highly virulent AHSV with an in-frame deletion of 77 amino acids (aa) in NS3/NS3a is completely safe, does not cause viremia and shows protective capacity. This deletion mutant is a promising DISA (Disabled Infectious Single Animal) vaccine platform, since exchange of serotype specific virus proteins has been shown for all nine serotypes. Here, we show that a prototype NS3 competitive ELISA is DIVA compliant to AHS DISA vaccine platforms. Epitope mapping of NS3/NS3a shows that more research is needed to evaluate this prototype serological DIVA assay regarding sensitivity and specificity, in particular for AHSVs expressing antigenically different NS3/NS3a proteins. Further, an experimental panAHSV PCR test targeting genome segment 10 is developed that detects reference AHSV strains, whereas AHS DISA vaccine platforms were not detected. This DIVA PCR test completely guarantees genetic DIVA based on in silico and in vitro validation, although test validation regarding diagnostic sensitivity and specificity has not been performed yet. In conclusion, the prototype NS3 cELISA and the PCR test described here enable serological and genetic DIVA accompanying AHS DISA vaccine platforms.