Modelling African horse sickness emergence and transmission in the South African control area using a deterministic metapopulation approach.

African horse sickness is an equine orbivirus transmitted by Culicoides Latreille biting midges. In the last 80 years, it has caused several devastating outbreaks in the equine population in Europe, the Far and Middle East, North Africa, South-East Asia, and sub-Saharan Africa. The disease is endemic in South Africa; however, a unique control area has been set up in the Western Cape where increased surveillance and control measures have been put in place. A deterministic metapopulation model was developed to explore if an outbreak might occur, and how it might develop, if a latently infected horse was to be imported into the control area, by varying the geographical location and months of import. To do this, a previously published ordinary differential equation model was developed with a metapopulation approach and included a vaccinated horse population. Outbreak length, time to peak infection, number of infected horses at the peak, number of horses overall affected (recovered or dead), re-emergence, and Rv (the basic reproduction number in the presence of vaccination) were recorded and displayed using GIS mapping. The model predictions were compared to previous outbreak data to ensure validity. The warmer months (November to March) had longer outbreaks than the colder months (May to September), took more time to reach the peak, and had a greater total outbreak size with more horses infected at the peak. Rv appeared to be a poor predictor of outbreak dynamics for this simulation. A sensitivity analysis indicated that control measures such as vaccination and vector control are potentially effective to manage the spread of an outbreak, and shortening the vaccination window to July to September may reduce the risk of vaccine-associated outbreaks.

Development of Differentiating Infected from Vaccinated Animals (DIVA) Real-Time PCR for African Horse Sickness Virus Serotype 1.

African horse sickness (AHS) is a highly infectious and often fatal disease caused by 9 serotypes of the orbivirus African horse sickness virus (AHSV). In March 2020, an AHS outbreak was reported in Thailand in which AHSV serotype 1 was identified as the causative agent. Trivalent live attenuated vaccines serotype 1, 3, and 4 were used in a targeted vaccination campaign within a 50-km radius surrounding the infected cases, which promptly controlled the spread of the disease. However, AHS-like symptoms in vaccinated horses required laboratory diagnostic methods to differentiate infected horses from vaccinated horses, especially for postvaccination surveillance. We describe a real-time reverse transcription PCR-based assay for rapid characterization of the affecting field strain. The development and validation of this assay should imbue confidence in differentiating AHS-vaccinated horses from nonvaccinated horses. This method should be applied to determining the epidemiology of AHSV in future outbreaks.

On All Fours: Transient Laborers, the Threat of Movement, and the Aftermath of Disease.

African horse sickness (AHS) plagued the Middle East in 1944 for the first time. It spread into Palestine during a transformative period, as the role of animals as global migrant-laborers was shifting; soon after, automated machines would relieve their burden and transform the relations between farmers, traders, the state and its policing powers, and the global market. By following the movement and management of this outbreak of the disease, along with medical knowledge and tools of prevention and treatment, the article demonstrates that animal health and mobility were substantial matters of concern in British Palestine. It shows, furthermore, that AHS became a catalyst in dismantling the economic, social, and cultural value of animals of burden and their handlers.

African horse sickness: an ancient disease for a current threat / La peste équine : une maladie ancienne pour une menace actuelle

African horse sickness (AHS) is a major arthropod-borne disease that causes significant losses in horses in sub-Saharan Africa. It is caused by the African horse sickness virus (AHSV), which is transmitted during a blood meal by Culicoides biting midges. The distribution of historical African culicoid vectors increases due to global warming. In addition, recent (Thailand, 2020) and earlier (Iberian Peninsula, 1965-66/1987-90) AHS outbreaks outside Africa demonstrate the adaptation of the virus to endogenous species in AHS-free regions, similar to what has been observed for bluetongue disease in recent decades. Therefore, many regions are considered at risk of introduction of AHS which could have important economic consequences for the equine industry. Overall, this prone the European Union to launch research programs to get better diagnostic and prophylactic tools.

La peste équine est une arbovirose majeure qui entraîne des pertes importantes chez les chevaux en Afrique subsaharienne. Elle est provoquée par le virus de la peste équine (African horse sickness virus, AHSV) dont la transmission s'effectue au cours d'un repas sanguin par des petits moucherons hématophages appartenant au genre Culicoides. En outre, les espèces vectrices historiques de culicoïdes présentes en Afrique voient leur aire de répartition s'étendre en lien avec le réchauffement climatique à l'échelle mondiale. Par ailleurs, des épisodes épizootiques récents (Thaïlande, 2020) ou un peu plus anciens (péninsule ibérique, 1965-66/1987-90) en dehors du continent africain soulignent la capacité d'adaptation du virus à des espèces vectrices autochtones, à l'instar de ce qui a été observé pour la fièvre catarrhale ovine ces dernières décennies. Ces facteurs laissent craindre à tout moment une introduction de la peste équine dans des régions indemnes. L'urgence est donc donnée actuellement par l'Union européenne pour se doter de meilleurs outils diagnostiques et prophylactiques afin de prévenir des conséquences économiques brutales pour l'industrie équine.

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.

Safety and immunogenicity of plant-produced African horse sickness virus-like particles in horses.

African horse sickness (AHS) is caused by multiple serotypes of the dsRNA AHSV and is a major scourge of domestic equids in Africa. While there are well established commercial live attenuated vaccines produced in South Africa, risks associated with these have encouraged attempts to develop new and safer recombinant vaccines. Previously, we reported on the immunogenicity of a plant-produced AHS serotype 5 virus-like particle (VLP) vaccine, which stimulated high titres of AHS serotype 5-specific neutralizing antibodies in guinea pigs. Here, we report a similar response to the vaccine in horses. This is the first report demonstrating the safety and immunogenicity of plant-produced AHS VLPs in horses.

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.

Outdoor and indoor monitoring of livestock-associated Culicoides spp. to assess vector-free periods and disease risks.

BACKGROUND: Within the last few decades Culicoides spp. (Diptera: Ceratopogonidae) emerged Europe-wide as a major vector for epizootic viral diseases e.g. caused by Bluetongue (BT) or Schmallenberg virus. In accordance with the EU regulation 1266/2007, veterinary authorities are requested to determine vector-free periods for loosing trade and movement restrictions of susceptible livestock. Additionally, the widely used basic reproduction number [Formula: see text] is optionally applied for risk assessment of vector-borne diseases. Values of R0 < 1 indicate periods with no disease transmission risk. For the determination of vector-free period and R0 a continuously operating daily Culicoides spp. monitoring in Vienna (Austria) was established. It covered the period 2009-2013 and depicts the seasonal vector abundance indoor and outdoor. Future BT and African horse sickness (AHS) outbreak risks were estimated by projecting R0 to climate change scenarios. Therefore, temperature-dependent vector parameters were applied. RESULTS: The vector-free period lasted about 100 days inside stables, while less than five Culicoides were trapped outdoors on 150 days per season, i.e. winter half year. Additionally, the potential outbreak risk was assessed for BT and AHS. For BT, a basic reproduction number of R0 > 1 was found each year between June and August. The periods without transmission risk, i.e. R0 < 1, were notably higher (200 days). Contrary, values of R0 < 1 were estimated for AHS during the whole period. Finally, the basic reproduction numbers were projected to the future by using temperature forecasts for the period 2014-2100. While the mean summer peak values for BT increase from of R0 = 2.3 to R0 = 3.4 until 2100 (1.1/100 years), no risk for AHS was estimated even under climate warming assumptions. CONCLUSIONS: Restrictions to trade and movement are always associated with an economic impact during epidemic diseases. To minimize these impacts, risk assessments based on the vector-free period or the basic reproduction number R0 can essentially support veterinary authorities to improve protection and control measurements.

African horse sickness.

African horse sickness (AHS) is a devastating disease of equids caused by an arthropod-borne virus belonging to the Reoviridae family, genus Orbivirus. It is considered a major health threat for horses in endemic areas in sub-Saharan Africa. African horse sickness virus (AHSV) repeatedly caused large epizootics in the Mediterranean region (North Africa and southern Europe in particular) as a result of trade in infected equids. The unexpected emergence of a closely related virus, the bluetongue virus, in northern Europe in 2006 has raised fears about AHSV introduction into Europe, and more specifically into AHSV-free regions that have reported the presence of AHSV vectors, e.g. Culicoides midges. North African and European countries should be prepared to face AHSV incursions in the future, especially since two AHSV serotypes (serotypes 2 and 7) have recently spread northwards to western (e.g. Senegal, Nigeria, Gambia) and eastern Africa (Ethiopia), where historically only serotype 9 had been isolated. The authors review key elements of AHS epidemiology, surveillance and prophylaxis.

Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications.

The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.

Immunogenic profile of a plant-produced nonavalent African horse sickness viral protein 2 (VP2) vaccine in IFNAR-/- mice.

A safe, highly immunogenic multivalent vaccine to protect against all nine serotypes of African horse sickness virus (AHSV), will revolutionise the AHS vaccine industry in endemic countries and beyond. Plant-produced AHS virus-like particles (VLPs) and soluble viral protein 2 (VP2) vaccine candidates were developed that have the potential to protect against all nine serotypes but can equally well be formulated as mono- and bi-valent formulations for localised outbreaks of specific serotypes. In the first interferon α/ß receptor knock-out (IFNAR-/-) mice trial conducted, a nine-serotype (nonavalent) vaccine administered as two pentavalent (5 µg per serotype) vaccines (VLP/VP2 combination or exclusively VP2), were directly compared to the commercially available AHS live attenuated vaccine. In a follow up trial, mice were vaccinated with an adjuvanted nine-serotype multivalent VP2 vaccine in a prime boost strategy and resulted in the desired neutralising antibody titres of 1:320, previously demonstrated to confer protective immunity in IFNAR-/- mice. In addition, the plant-produced VP2 vaccine performed favourably when compared to the commercial vaccine. Here we provide compelling data for a nonavalent VP2-based vaccine candidate, with the VP2 from each serotype being antigenically distinguishable based on LC-MS/MS and ELISA data. This is the first preclinical trial demonstrating the ability of an adjuvanted nonavalent cocktail of soluble, plant-expressed AHS VP2 proteins administered in a prime-boost strategy eliciting high antibody titres against all 9 AHSV serotypes. Furthermore, elevated T helper cells 2 (Th2) and Th1, indicative of humoral and cell-mediated memory T cell immune responses, respectively, were detected in mouse serum collected 14 days after the multivalent prime-boost vaccination. Both Th2 and Th1 may play a role to confer protective immunity. These preclinical immunogenicity studies paved the way to test the safety and protective efficacy of the plant-produced nonavalent VP2 vaccine candidate in the target animals, horses.

Clinical, Virological and Immunological Responses after Experimental Infection with African Horse Sickness Virus Serotype 9 in Immunologically Naïve and Vaccinated Horses.

This study described the clinical, virological, and serological responses of immunologically naïve and vaccinated horses to African horse sickness virus (AHSV) serotype 9. Naïve horses developed a clinical picture resembling the cardiac form of African horse sickness. This was characterized by inappetence, reduced activity, and hyperthermia leading to lethargy and immobility-recumbency by days 9-10 post-infection, an end-point criteria for euthanasia. After challenge, unvaccinated horses were viremic from days 3 or 4 post-infection till euthanasia, as detected by serogroup-specific (GS) real time RT-PCR (rRT-PCR) and virus isolation. Virus isolation, antigen ELISA, and GS-rRT-PCR also demonstrated high sensitivity in the post-mortem detection of the pathogen. After infection, serogroup-specific VP7 antibodies were undetectable by blocking ELISA (b-ELISA) in 2 out of 3 unvaccinated horses during the course of the disease (9-10 dpi). Vaccinated horses did not show significant side effects post-vaccination and were largely asymptomatic after the AHSV-9 challenge. VP7-specific antibodies could not be detected by the b-ELISA until day 21 and day 30 post-inoculation, respectively. Virus neutralizing antibody titres were low or even undetectable for specific serotypes in the vaccinated horses. Virus isolation and GS-rRT-PCR detected the presence of AHSV vaccine strains genomes and infectious vaccine virus after vaccination and challenge. This study established an experimental infection model of AHSV-9 in horses and characterized the main clinical, virological, and immunological parameters in both immunologically naïve and vaccinated horses using standardized bio-assays.

Inference for a spatio-temporal model with partial spatial data: African horse sickness virus in Morocco.

African horse sickness virus (AHSV) is a vector-borne virus spread by midges (Culicoides spp.). The virus causes African horse sickness (AHS) disease in some species of equid. AHS is endemic in parts of Africa, previously emerged in Europe and in 2020 caused outbreaks for the first time in parts of Eastern Asia. Here we analyse a unique historic dataset from the 1989-1991 emergence of AHS in Morocco in a naïve population of equids. Sequential Monte Carlo and Markov chain Monte Carlo techniques are used to estimate parameters for a spatial-temporal model using a transmission kernel. These parameters allow us to observe how the transmissibility of AHSV changes according to the distance between premises. We observe how the spatial specificity of the dataset giving the locations of premises on which any infected equids were reported affects parameter estimates. Estimations of transmissibility were similar at the scales of village (location to the nearest 1.3 km) and region (median area 99 km2), but not province (median area 3000 km2). This data-driven result could help inform decisions by policy makers on collecting data during future equine disease outbreaks, as well as policies for AHS control.

Economic assessment of African horse sickness vaccine impact.

BACKGROUND: African horse sickness (AHS) is endemic in sub-Saharan Africa posing a threat to equine populations in non-endemic regions. Available vaccine technologies have limitations, creating barriers to horse movement, AHS control and, in non-endemic areas or countries, rapid elimination of virus after incursion. The literature lacks an economic assessment of the benefits of bringing a new, more effective AHS vaccine to market. OBJECTIVES: The study assesses the economic impact of AHS and tests the hypothesis that investment in a safer, more effective AHS vaccine would give an economic return. STUDY DESIGN: Cost-benefit analysis. METHODS: Primary and secondary data were collected to populate the cost-benefit analysis model. A literature review was followed by a questionnaire survey and interviews to gather primary data. At-risk populations were defined and qualitative assessment completed to narrow the target populations for quantitative assessment. A deterministic cost-benefit model was developed in Excel and different scenarios tested. Break-even and sensitivity analysis were conducted on key parameters. RESULTS: The economic impact of AHS was estimated to be US$95 million per annum, and this was mainly in endemic regions with domestic equine industries and involved in international trade. Investment required to bring a new AHS vaccine to market was estimated to be up to US$3.5 million, which was very small relative to the benefits estimated in this study. The economic return on investment in bringing a new AHS vaccine to market was predicted to be positive and the analysis demonstrates this result was robust. MAIN LIMITATIONS: Data for the analysis were scarce, requiring expert opinion and extrapolation by the authors. Sensitivity analysis with the deterministic modelling structure indicated there was no justification for stochastic modelling, given the robustness of the return on investment. CONCLUSIONS: The analysis predicts a strong and robust economic return on the investment in bringing a new AHS vaccine to market. Main economic beneficiaries would be the high value horse sectors, specifically the equine industries in Republic of South Africa (RSA) and in non-endemic countries. In addition, major benefits would be captured in poor communities in sub-Saharan Africa where working equids are of high economic and social importance.

Entry-competent-replication-abortive African horse sickness virus strains elicit robust immunity in ponies against all serotypes.

African horse sickness virus (AHSV) is an Orbivirus within the Reoviridae family, spread by Culicoides species of midges, which infects equids with high mortality, particularly in horses and has a considerable impact on the equine industry. In order to control the disease, we previously described Entry Competent Replication Abortive (ECRA) virus strains for each of the nine distinct AHSV serotypes and demonstrated their potential as vaccines, first in type I interferon receptor (IFNAR-/-) knockout mice, and then in ponies. In this report we have investigated whether or not a combination ECRA vaccine comprising nine vaccine strains as two different cocktails is as efficient in ponies and the duration of the immunity triggered by ECRA vaccines. In one study, a group of ponies were vaccinated with a cocktail of 4 vaccine strains, followed by a vaccination of the remaining 5 vaccine strains, mimicking the current live attenuated vaccine regimen. In the second study, ponies were vaccinated with a single ECRA-AHSV strain and monitored for 6 months. The first group of ponies developed neutralising antibody responses against all 9 serotypes, indicating that no cross-serotype interference occurred, while the second group developed robust neutralising antibody responses against the single serotype that were sustained at the same level throughout a 6-month study. The results support our previous data and further validate ECRA vaccines as a safe and efficacious replacement of current live vaccines.

Humoral and cell-mediated immune responses to plant-produced African horse sickness virus VP7 quasi-crystals.

African horse sickness (AHS) is a devastating viral disease affecting equines and has resulted in many disastrous epizootics. To date, no successful therapeutic treatment exists for AHS, and commercially used live-attenuated vaccines have various undesirable side effects. Previous studies have shown that mice inoculated with insoluble African horse sickness virus (AHSV) VP7 crystals are protected from live challenge with a lethal dose of AHSV. This study investigates the humoral and cell-mediated immune responses in guinea-pigs to a safer monovalent vaccine alternative based on AHSV-5 VP7 quasi-crystals produced in plants. Guinea-pigs received prime- and boost-inoculations of between 10 and 50 µg of purified plant-produced AHSV VP7. Western immunoblot analysis of the humoral response showed stimulation of high titres of anti-VP7 antibodies 28 days after the boost-inoculation in sera from three of the five experimental animals. In addition, RNA-seq transcriptome profiling of guinea-pig spleen-derived RNA highlighted thirty significantly (q ≤ 0.05) differentially expressed genes involved in innate and adaptive immunity. Differential expression of genes involved in Th1, Th2 and Th17 cell differentiation suggest a cell-mediated immune response to AHSV-5 VP7. Upregulation of several important cytokines and cytokine receptors were noted, including TNFSF14, CX3CR1, IFNLR1 and IL17RA. Upregulation of IL17RA suggests a Th17 response which has been reported as a key component in AHSV immunity. While further investigation is needed to validate these findings, these results suggest that AHSV-5 VP7 quasi-crystals produced in N. benthamiana are immunogenic and induce both humoral and cell-mediated responses.

Humoral antibody response of 10 horses after vaccination against African horse sickness with an inactivated vaccine containing all 9 serotypes in one injection.

BACKGROUND: African horse sickness (AHS) is a devastating viral disease of equids that was first recorded in 1327. Currently, prevention and control of the disease are based on attenuated vaccines and midge control. It has been shown that attenuated Orbivirus vaccines are not always safe as they may reverse to virulence. OBJECTIVES: In the Emirate of Dubai, a vaccination experiment was carried out with an inactivated AHS vaccine produced at the Central Veterinary Research Laboratory (CVRL), Dubai, UAE to investigate the humoral antibody response of AHS-naïve horses to this vaccine. Our vaccination experiment was performed to establish an AHS vaccine bank in the UAE to protect horses from the disease in case of an outbreak. Therefore, CVRL established an inactivated AHS vaccine containing all nine serotypes which induce high neutralising antibodies. STUDY DESIGN: A total of 10 horses kept in a desert isolation area were subcutaneously and intramuscularly vaccinated with an inactivated vaccine containing all nine AHS serotypes previously isolated from Kenyan horse fatalities. Primary immunisation was followed by two booster immunisations 4 weeks and 6 months apart. After 13 months, an annual booster was administered. METHODS: Blood samples were regularly withdrawn for ELISA and virus neutralisation testing. Additionally, EDTA blood was tested every second day for 14 days post each vaccination for the presence of AHS virus or its RNA. RESULTS: Results show that ELISA and virus neutralising antibodies appeared after the first booster, declined after 4-6 months and therefore three vaccinations and an annual vaccination are necessary to achieve high protective virus neutralising antibodies. MAIN LIMITATIONS: No challenge infection was carried out due to the lack of a safe facility in the UAE. CONCLUSION: Before more advanced AHS vaccines become a reality, inactivated vaccines containing all nine serotypes should be used as they produce high ELISA and neutralising antibodies.