Cytokine mRNA Expression Profile in Target Organs of IFNAR (-/-) Mice Infected with African Horse Sickness Virus.

African horse sickness (AHS) is a highly severe disease caused by a viral etiological agent, African horse sickness virus (AHSV). It is endemic in sub-Saharan Africa, while sporadic outbreaks have occurred in North Africa, Asia, and Europe, with the most recent cases in Thailand. AHSV transmission between equines occurs primarily by biting midges of the genus Culicoides, especially C. imicola, with a wide distribution globally. As research in horses is highly restricted due to a variety of factors, small laboratory animal models that reproduce clinical signs and pathology observed in natural infection of AHSV are highly needed. Here, we investigated the expression profile of several pro-inflammatory cytokines in target organs and serum of IFNAR (-/-) mice, to continue characterizing this established animal model and to go deep into the innate immune responses that are still needed.

An entry risk assessment of African horse sickness virus into the controlled area of South Africa through the legal movement of equids.

South Africa is endemic for African horse sickness (AHS), an important health and trade-sensitive disease of equids. The country is zoned with movement control measures facilitating an AHS-free controlled area in the south-west. Our objective was to quantitatively establish the risk of entry of AHS virus into the AHS controlled area through the legal movement of horses. Outcomes were subcategorised to evaluate movement pathway, temporal, and spatial differences in risk. A 'no-control' scenario allowed for evaluation of the impact of control measures. Using 2019 movement and AHS case data, and country-wide census data, a stochastic model was developed establishing local municipality level entry risk of AHSV at monthly intervals. These were aggregated to annual probability of entry. Sensitivity analysis evaluated model variables on their impact on the conditional means of the probability of entry. The median monthly probability of entry of AHSV into the controlled area of South Africa ranged from 0.75% (June) to 5.73% (February), with the annual median probability of entry estimated at 20.21% (95% CI: 15.89%-28.89%). The annual risk of AHSV entry compared well with the annual probability of introduction of AHS into the controlled area, which is ~10% based on the last 20 years of outbreak data. Direct non-quarantine movements made up most movements and accounted for most of the risk of entry. Spatial analysis showed that, even though reported case totals were zero throughout 2019 in the Western Cape, horses originating from this province still pose a risk that should not be ignored. Control measures decrease risk by a factor of 2.8 on an annual basis. Not only do the outcomes of this study inform domestic control, they can also be used for scientifically justified trade decision making, since in-country movement control forms a key component of export protocols.

Inhibition of Orbivirus Replication by Aurintricarboxylic Acid.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging to the Orbivirus genus, which are transmitted between hosts primarily by biting midges of the genus Culicoides. With recent BTV and AHSV outbreaks causing epidemics and important economy losses, there is a pressing need for efficacious drugs to treat and control the spread of these infections. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antiviral activity. Here, we evaluated ATA as a potential antiviral compound against Orbivirus infections in both mammalian and insect cells. Notably, ATA was able to prevent the replication of BTV and AHSV in both cell types in a time- and concentration-dependent manner. In addition, we evaluated the effect of ATA in vivo using a mouse model of infection. ATA did not protect mice against a lethal challenge with BTV or AHSV, most probably due to the in vivo effect of ATA on immune system regulation. Overall, these results demonstrate that ATA has inhibitory activity against Orbivirus replication in vitro, but further in vivo analysis will be required before considering it as a potential therapy for future clinical evaluation.

African horse sickness virus NS4 is a nucleocytoplasmic protein that localizes to PML nuclear bodies.

African horse sickness virus (AHSV) is the causative agent of the often fatal disease African horse sickness in equids. The non-structural protein NS4 is the only AHSV protein that localizes to the nucleus. Here we report that all AHSV reference and representative field strains express one of the two forms of NS4, i.e. NS4-I or NS4-II. Both forms of NS4 are nucleocytoplasmic proteins, but NS4-I has a stronger nuclear presence whilst NS4-II has a proportionally higher cytoplasmic distribution. A subtype of NS4-II containing a nuclear localization signal (NLS), named NLS-NS4-II, displays distinct punctate foci in the nucleus. We showed that NS4 likely enters the nucleus via passive diffusion as a result of its small size. Colocalization analysis with nuclear compartments revealed that NS4 colocalizes with promyelocytic leukaemia nuclear bodies (PML-NBs), suggesting a role in the antiviral response or interferon signalling. Interestingly, we showed that two other AHSV proteins also interact with nuclear components. A small fraction of the NS1 tubules were present in the nucleus and associated with PML-NBs; this was more pronounced for a virus strain lacking NS4. A component of nuclear speckles, serine and arginine rich splicing factor 2 (SRSF2) was recruited to viral inclusion bodies (VIBs) in the cytoplasm of AHSV-infected cells and colocalized with NS2. Nuclear speckles are important sites for cellular mRNA transcript processing and maturation. Collectively, these results provide data on three AHSV non-structural proteins interacting with host cell nuclear components that could contribute to overcoming antiviral responses and creating conditions that will favour viral replication.

Assessing the potential of plains zebra to maintain African horse sickness in the Western Cape Province, South Africa.

African horse sickness (AHS) is a disease of equids that results in a non-tariff barrier to the trade of live equids from affected countries. AHS is endemic in South Africa except for a controlled area in the Western Cape Province (WCP) where sporadic outbreaks have occurred in the past 2 decades. There is potential that the presence of zebra populations, thought to be the natural reservoir hosts for AHS, in the WCP could maintain AHS virus circulation in the area and act as a year-round source of infection for horses. However, it remains unclear whether the epidemiology or the ecological conditions present in the WCP would enable persistent circulation of AHS in the local zebra populations. Here we developed a hybrid deterministic-stochastic vector-host compartmental model of AHS transmission in plains zebra (Equus quagga), where host populations are age- and sex-structured and for which population and AHS transmission dynamics are modulated by rainfall and temperature conditions. Using this model, we showed that populations of plains zebra present in the WCP are not sufficiently large for AHS introduction events to become endemic and that coastal populations of zebra need to be >2500 individuals for AHS to persist >2 years, even if zebras are infectious for more than 50 days. AHS cannot become endemic in the coastal population of the WCP unless the zebra population involves at least 50,000 individuals. Finally, inland populations of plains zebra in the WCP may represent a risk for AHS to persist but would require populations of at least 500 zebras or show unrealistic duration of infectiousness for AHS introduction events to become endemic. Our results provide evidence that the risk of AHS persistence from a single introduction event in a given plains zebra population in the WCP is extremely low and it is unlikely to represent a long-term source of infection for local horses.

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.

African horse sickness virus (AHSV) with a deletion of 77 amino acids in NS3/NS3a protein is not virulent and a safe promising AHS Disabled Infectious Single Animal (DISA) vaccine platform.

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. Currently, nine serotypes have been defined showing limited cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African Horse Sickness (AHS) in equids with a mortality up to 95% in naïve domestic horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are competent vectors of closely related bluetongue virus. AHS outbreaks cause huge economic losses in developing countries. In the developed world, outbreaks will result in losses in the equestrian industry and will have an enormous emotional impact on owners of pet horses. Live-attenuated vaccine viruses (LAVs) have been developed, however, safety of these LAVs are questionable due to residual virulence, reversion to virulence, and risk on virulent variants by reassortment between LAVs or with field AHSV. Research aims vaccines with improved profiles. Reverse genetics has recently being developed for AHSV and has opened endless possibilities including development of AHS vaccine candidates, such as Disabled Infectious Single Animal (DISA) vaccine. Here, virulent AHSV5 was recovered and its high virulence was confirmed by experimental infection of ponies. 'Synthetically derived' virulent AHSV5 with an in-frame deletion of 77 amino acids codons in genome segment 10 encoding NS3/NS3a protein resulted in similar in vitro characteristics as published NS3/NS3a knockout mutants of LAV strain AHSV4LP. In contrast to its highly virulent ancestor virus, this deletion AHSV5 mutant (DISA5) was completely safe for ponies. Two vaccinations with DISA5 as well as two vaccinations with DISA vaccine based on LAV strain AHSV4LP showed protection against lethal homologous AHSV. More research is needed to further improve efficacy, to explore the AHS DISA vaccine platform for all nine serotypes, and to study the vaccine profile in more detail.

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.

Study of the virulence of serotypes 4 and 9 of African horse sickness virus in IFNAR(-/-), Balb/C and 129 Sv/Ev mice.

African horse sickness virus (AHSV) is a double-stranded RNA virus which belongs to the family Reoviridae, genus Orbivirus. Recent studies have focused on the interferon-α/ß receptor knock-out mice (IFNAR(-/-)) as a small animal laboratory for the development of AHSV vaccines. The aim of this work was to study in vivo the virulence of two strains of AHSV and to compare the outcome of the infection of three mouse strains. To address this, AHSV serotypes 4 (AHSV-4) and 9 (AHSV-9) were inoculated subcutaneously (SC) and intranasally (IN) in two immunocompetent mouse strains (Balb/C and 129 Sv/Ev (129 WT)) as well as IFNAR(-/-) mice (on 129 Sv/Ev genetic background). In IFNAR(-/-) mice, fatality up to 50% was measured and significantly more clinical signs were observed in comparison with SC inoculated immunocompetent mice. The observed clinical signs were significantly more severe after AHSV-4 infection, in particular in immunocompetent mice inoculated by IN route. Considering RNAemia, significantly higher viral loads were measured following AHSV-4 infection. In the organs of 129 WT inoculated by IN route, significantly higher viral loads were detected after AHSV-4 infection. Together the results support a higher virulence for AHSV-4 compared to AHSV-9 and a higher clinical impact following infections in IN inoculated mice, at least in the investigated strains. The study also brought indirect evidences for type I IFN involvement in the control of AHSV infection.

Worldwide niche and future potential distribution of Culicoides imicola, a major vector of bluetongue and African horse sickness viruses.

We modelled the ecoclimatic niche of Culicoides imicola, a major arthropod vector of midge-borne viral pathogens affecting ruminants and equids, at fine scale and on a global extent, so as to provide insight into current and future risks of disease epizootics, and increase current knowledge of the species' ecology. Based on the known distribution and ecology of C. imicola, the species' response to monthly climatic conditions was characterised using CLIMEX with 10' spatial resolution climatic datasets. The species' climatic niche was projected worldwide and under future climatic scenarios. The validated model highlights the role of irrigation in supporting the occurrence of C. imicola in arid regions. In Europe, the modelled potential distribution of C. imicola extended further West than its reported distribution, raising questions regarding ongoing process of colonization and non-climatic habitat factors. The CLIMEX model highlighted similar ecological niches for C. imicola and the Australasian C. brevitarsis raising questions on biogeography and biosecurity. Under the climate change scenarios considered, its' modelled potential distribution could expand northward in the Northern hemisphere, whereas in Africa its range may contract in the future. The biosecurity risks from bluetongue and African horse sickness viruses need to be re-evaluated in regions where the vector's niche is suitable. Under a warmer climate, the risk of vector-borne epizootic pathogens such as bluetongue and African horse sickness viruses are likely to increase as the climate suitability for C. imicola shifts poleward, especially in Western Europe.

Descriptive epidemiology of African horse sickness in Zimbabwe.

A study of the prevalence of African horse sickness in horses was conducted, using records from two private equine practices in Harare for the period 1998-2004. Results indicated a higher prevalence of the disease in horses in Zimbabwe in the late rainy season (March - May). Age of the horse was found to be a significant risk factor, with foals or yearlings appearing to be 1.80 times more likely to contract the disease compared with horses older than two years. The case fatality rate in foals or yearlings was also higher than in older age groups, but this difference was not significant. The vaccination status was an important risk factor, with vaccinated horses 0.12 times less likely to die from the disease compared with unvaccinated horses. Young, unvaccinated horses therefore seem to be the most susceptible to the disease and have greater chances of fatality. This study highlights the importance of adequately protecting horses against African horse sickness by providing immunisation through vaccination and discusses the need to review current vaccination strategies being practiced in Zimbabwe.

African horse sickness virus induces apoptosis in cultured mammalian cells.

Infection of mammalian cell cultures with African horse sickness virus (AHSV) is known to result in dramatic cytopathic effects (CPE), but no CPE is observed in infected insect cell cultures despite productive virus replication. The basis for this phenomenon has not yet been investigated, but is suggestive of apoptosis being induced following virus infection of the mammalian cells. To investigate whether AHSV can induce apoptosis in infected mammalian cells, Culicoides variipennis (KC) insect cells and BHK-21 mammalian cells were infected with AHSV-9 and analyzed for morphological and biochemical hallmarks of apoptosis. In contrast to KC cells, infection of BHK-21 cells with AHSV-9 resulted in ultrastructural changes and nuclear DNA fragmentation, both of which are associated with the induction of apoptosis. Results also indicated that AHSV-9 infection of BHK-21 cells resulted in activation of caspase-3, a key agent in apoptosis, and in mitochondrial membrane depolarization. Cumulatively, the data indicate that the intrinsic pathway is activated in AHSV-induced apoptosis.

Membrane permeabilization of the African horse sickness virus VP5 protein is mediated by two N-terminal amphipathic α-helices.

The VP5 outer capsid protein of African horse sickness virus (AHSV) is cytotoxic when expressed in Spodoptera frugiperda (Sf-9) cells. Secondary structure analysis of the VP5 amino acid sequence of AHSV-9 identified two N-terminal amphipathic α-helices within the first 43 amino acids. Baculovirus expression of N- and C-terminal truncated VP5 proteins in Sf-9 cells indicated that the N-terminal 43 amino acids correlated with low levels of protein expression and with increased membrane permeabilization and cytotoxicity. Exogenous addition of chemically synthesized VP5 peptides indicated that both N-terminal amphipathic α-helices are required for membrane permeabilization of Sf-9 cells. These findings suggest that AHSV VP5 is a membrane-destabilizing protein.

A comparison of the susceptibility of the biting midge Culicoides imicola to infection with recent and historical isolates of African horse sickness virus.

The susceptibility of Culicoides (Avaritia) imicola Kiefer (Diptera: Ceratopogonidae) to 21 isolates representing all nine known serotypes of African horse sickness virus (AHSV), recovered from clinical cases of the disease in South Africa during 1998-2004, was compared with its susceptibility to approximately 40-year-old isolates stored at the Agricultural Research Council-Onderstepoort Veterinary Institute. Field-collected C. imicola were fed through a chicken skin membrane on sheep blood spiked with one of the virus isolates to a concentration in the range of 5.6-7.5 log (10)TCID(50)/mL. After 10 days incubation at 23.5 degrees C, five of the nine historical serotypes (AHSV-1, -2, -3, -7 and -9) could not be isolated from C. imicola. All nine serotypes were recovered for the 21 recent isolates, for 16 of which the virus recovery rates were higher than for the corresponding historical isolates. These results emphasize the need to assess the oral susceptibility of local Culicoides populations to viruses in circulation during outbreaks in order to estimate their vector potential.

Evaluation of the pathogenicity of African Horsesickness (AHS) isolates in vaccinated animals.

BACKGROUND: The polyvalent African Horsesickness (AHS) attenuated live vaccine (ALV) produced by Onderstepoort Biological Products (OBP) Ltd., South Africa, has been associated with some safety concerns and alleged cases of vaccine failure or vaccine-induced disease. The risk of reassortment and reversion to virulence is a common concern associated with the use of ALVs, and a phenomenon reported for viruses with segmented RNA genomes. The purpose of this study was to determine whether or not reassortment of AHS vaccine strains could result in reassortants and reversion to virulence and therefore cause AHS in susceptible horses. METHODS: Clinical or field isolates of AHS were obtained from horses with AHS symptoms or disease post vaccination. AHS-naïve horses were inoculated with these isolates and monitored for clinical reactions. Laboratory tests were performed at intervals to determine immune responses and viraemia. Viral RNA extraction and complete genome amplification of monovalent AHS-ALV vaccine strains and isolates collected post-vaccination was conducted. cDNA of the genome segments were run on PAGE to determine mobility patterns and genome segments 2, 3, 4, 5 and 6 sequenced for phylogenetic analysis. RESULTS: No clinical symptoms typical of AHS were observed in inoculated horses and all showed a good immune response. A comparison of mobility patterns of the amplified cDNA genome on PAGE allowed the identification and differentiation of reassortants, which were confirmed by sequence and phylogenetic analysis of the nucleotide sequences. CONCLUSION: This study, however, showed no indications that vaccine reassortants were pathogenic or lethal after inoculation in susceptible horses. Assumptions of virulence or reversion to virulence of vaccine reassortants post-vaccination in horses could not be substantiated.

Characterization of the nucleic acid binding activity of inner core protein VP6 of African horse sickness virus.

Minor structural protein VP6 is the putative helicase of African horse sickness virus (AHSV), of the genus Orbivirus in the Reoviridae family. We investigated how the protein interacts with double-stranded (ds) RNA and other nucleic acids. Binding was assayed using an electrophoretic migration retardation assay and a nucleic acid overlay protein blot assay. VP6 bound double and single stranded RNA and DNA in a NaCl concentration sensitive reaction. Of six truncated VP6 peptides investigated, two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. The distinction between the peptides appeared to be the pI which ranged from more than 8.0 to just above 6.0. Changing the pH of the binding buffer modified the binding activity. Regardless of assay conditions, only peptides with a specific region of amino acids in common, showed evidence of binding activity. No sequence homology was identified with other binding domains, however, the presence of charged amino acids are assumed to be important for binding activity. The results suggested dsRNA binding in the blot assay was strongly affected by the net charge on the peptide.

African horse sickness.

African horse sickness virus (AHSV) causes a non-contagious, infectious insect-borne disease of equids and is endemic in many areas of sub-Saharan Africa and possibly Yemen in the Arabian Peninsula. However, periodically the virus makes excursions beyond its endemic areas and has at times extended as far as India and Pakistan in the east and Spain and Portugal in the west. The vectors are certain species of Culicoides biting midge the most important of which is the Afro-Asiatic species C. imicola. This paper describes the effects that AHSV has on its equid hosts, aspects of its epidemiology, and present and future prospects for control. The distribution of AHSV seems to be governed by a number of factors including the efficiency of control measures, the presence or absence of a long term vertebrate reservoir and, most importantly, the prevalence and seasonal incidence of the major vector which is controlled by climate. However, with the advent of climate-change the major vector, C. imicola, has now significantly extended its range northwards to include much of Portugal, Spain, Italy and Greece and has even been recorded from southern Switzerland. Furthermore, in many of these new locations the insect is present and active throughout the entire year. With the related bluetongue virus, which utilises the same vector species of Culicoides this has, since 1998, precipitated the worst outbreaks of bluetongue disease ever recorded with the virus extending further north in Europe than ever before and apparently becoming endemic in that continent. The prospects for similar changes in the epidemiology and distribution of AHSV are discussed.

Membrane association of African horsesickness virus nonstructural protein NS3 determines its cytotoxicity.

The smallest RNA genome segment of African horsesickness virus (AHSV) encodes the nonstructural protein NS3 (24K). NS3 localizes in areas of plasma membrane disruption and is associated with events of viral release. Conserved features in all AHSV NS3 proteins include the synthesis of a truncated NS3A protein from the same open reading frame as that of NS3, a proline-rich region, a region of strict sequence conservation and two hydrophobic domains. To investigate whether these features are associated with the cytotoxicity of NS3 or altered membrane permeability, a series of mutants were constructed and expressed in the BAC-TO-BAC baculovirus-expression system. Our results indicate that mutations in either of the two hydrophobic domains do not prevent membrane targeting of the mutant proteins but abolish their membrane anchoring. This prevents their localization to the cell surface and obviates their cytotoxic effect. The cytotoxicity of NS3 is therefore dependent on its membrane topography and thus involves both hydrophobic domains. NS3 has many of the characteristics of lytic viral proteins that play a central role in viral pathogenesis through modifying membrane permeability.