Culicoides species as potential vectors of African horse sickness virus in the southern regions of South Africa.

African horse sickness (AHS), a disease of equids caused by the AHS virus, is of major concern in South Africa. With mortality reaching up to 95% in susceptible horses and the apparent reoccurrence of cases in regions deemed non-endemic, most particularly the Eastern Cape, epidemiological research into factors contributing to the increase in the range of this economically important virus became imperative. The vectors, Culicoides (Diptera: Ceratopogonidae), are considered unable to proliferate during the unfavourable climatic conditions experienced in winter in the province, although the annual occurrence of AHS suggests that the virus has become established and that vector activity continues throughout the year. Surveillance of Culicoides within the province is sparse and little was known of the diversity of vector species or the abundance of known vectors, Culicoides imicola and Culicoides bolitinos. Surveillance was performed using light trapping methods at selected sites with varying equid species over two winter and two outbreak seasons, aiming to determine diversity, abundance and vector epidemiology of Culicoides within the province. The research provided an updated checklist of Culicoides species within the Eastern Cape, contributing to an increase in the knowledge of AHS vector epidemiology, as well as prevention and control in southern Africa.

Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro.

The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family. IMPORTANCE: Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses.

A correlation between capsid protein VP2 and the plaque morphology of African horse sickness virus in cell culture.

The attenuated live virus vaccine that is used in South Africa to protect against African horse sickness infection was developed more than 50 years ago. With the selection of the vaccine strains by cell culture passage, a correlation between the size of plaques formed in monolayer Vero cultures and attenuation of virus virulence in horses was found. The large plaque phenotype was used as an indication of cell culture adaptation and strongly correlated with attenuation of virulence in horses. There was never any investigation into the genetic causes of either the variation in plaque size, or the loss of virulence. An understanding of the underlying mechanisms of attenuation would benefit the production of a safer AHSV vaccine. To this end, the genomes of different strains of two African horse sickness isolates, producing varying plaque sizes, were compared and the differences between them identified. This comparison suggested that proteins VP2, VP3, VP5 and NS3 were most likely involved in the determination of the plaque phenotype. Comparison between genome sequences (obtained from GenBank) of low and high passage strains from two additional serotypes indicated that VP2 was the only protein with amino acid substitutions in all four serotypes. The amino acid substitutions all occurred within the same hydrophilic area, resulting in increased hydrophilicity of VP2 in the large plaque strains.

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.

The effect of alphacypermethrin-treated mesh protection against African horse sickness virus vectors on jet stall microclimate, clinical variables and faecal glucocorticoid metabolites of horses.

BACKGROUND: African horse sickness (AHS) is of importance to health and international trade in horses worldwide. During export from and transit through AHS endemic countries or zones, physical and chemical measures to protect horses from the vectors of AHS virus (AHSV) are recommended by the World Organization for Animal Health. Protection of containerized air transport systems for horses (jet stalls) with alphacypermethrin insecticide-treated high density polyethylene mesh is effective in reducing the Culicoides midge vector attack rate. In order to determine the effect of this mesh on jet stall ventilation and horse welfare under temperate climatic conditions, jet stall microclimate, clinical variables and faecal glucocorticoid metabolite (FGM) levels of 12 horses were monitored during overnight housing in either a treated or untreated stall in two blocks of a 2 × 3 randomized crossover design. RESULTS: Temperature difference between the treated stall and outside was significantly higher than the difference between the untreated stall and outside at 1/15 time points only (P = 0.045, r = 0.70). Relative humidity (RH) difference between the treated stall and outside did not differ from the untreated stall and outside. Temperature and RH in the treated stall were highly and significantly correlated with outside temperature (r = 0.96, P < 0.001) and RH (r = 0.95, P < 0.001), respectively. No significant differences were detected between rectal temperatures, pulse and respiratory rates of horses in the treated stall compared to the untreated stall. Mean FGM concentrations for horses housed in the treated stall peaked earlier (24 h) and at a higher concentration than horses housed in the untreated stall (48 h), but were not significantly different from baseline. No significant difference was detected in FGM concentrations when the treated and untreated stall groups were compared at individual time points up to 72 h after exiting the jet stall. CONCLUSIONS: Alphacypermethrin-treated HDPE mesh could be used under temperate climatic conditions to protect horses in jet stalls against AHSV vectors, without compromising jet stall microclimate and horse welfare.

Virus uncoating is required for apoptosis induction in cultured mammalian cells infected with African horse sickness virus.

Infection of cultured mammalian cells with African horse sickness virus (AHSV) is known to induce cell death. To date, the trigger(s) of this response, the apoptotic pathways activated during AHSV infection and the functional consequences of apoptosis on the virus replication cycle have yet to be characterized. This study demonstrated that extracellular treatment of BHK-21 cells with both of the AHSV4 outer capsid proteins, VP2 and VP5, was sufficient to trigger apoptosis. Whether steps in AHSV4 replication subsequent to viral attachment were required for AHSV4-induced apoptosis was also investigated. Apoptosis was induced in BHK-21 cells infected with UV-inactivated AHSV4 and in ribavirin-treated cells infected with AHSV4. However, both AHSV4- and VP2/VP5-stimulated apoptotic responses were inhibited in the presence of the endosomal acidification inhibitors ammonium chloride and chloroquine. These results indicated that uncoating of AHSV4 virions, but not viral transcription or subsequent steps in viral replication, was required for AHSV4 to induce apoptosis in BHK-21 cells. Furthermore, this study showed that both the extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways were induced following AHSV4 infection. The inhibition of caspase activity in AHSV4-infected cells did not diminish AHSV4 replication, but reduced the release and dissemination of progeny viral particles. Taken together, the data indicated that uncoating of AHSV virions was required for apoptosis induction, and that apoptosis enhanced virus spread and release.

Circadian activity of Culicoides oxystoma (Diptera: Ceratopogonidae), potential vector of bluetongue and African horse sickness viruses in the Niayes area, Senegal.

Culicoides biting midges (Diptera: Ceratopogonidae) are important vectors of arboviruses in Africa. Culicoides oxystoma has been recently recorded in the Niayes region of Senegal (West Africa) and its high abundance on horses suggests a potential implication in the transmission of the African horse sickness virus in this region. This species is also suspected to transmit bluetongue virus to imported breeds of sheep. Little information is available on the biology and ecology of Culicoides in Africa. Therefore, understanding the circadian host-seeking activity of this putative vector is of primary importance to assess the risk of the transmission of Culicoides-borne pathogens. To achieve this objective, midges were collected using a sheep-baited trap over two consecutive 24-h periods during four seasons in 2012. A total of 441 Culicoides, belonging to nine species including 418 (94.8%) specimens of C. oxystoma, were collected. C. oxystoma presented a bimodal circadian host-seeking activity at sunrise and sunset in July and was active 3 h after sunrise in April. Daily activity appeared mainly related to time periods. Morning activity increased with the increasing temperature up to about 27 °C and then decreased with the decreasing humidity, suggesting thermal limits for C. oxystoma activity. Evening activity increased with the increasing humidity and the decreasing temperature, comprised between 20 and 27 °C according to seasons. Interestingly, males were more abundant in our sampling sessions, with similar activity periods than females, suggesting potential animal host implication in the facilitation of reproduction. Finally, the low number of C. oxystoma collected render practical vector-control recommendations difficult to provide and highlight the lack of knowledge on the bio-ecology of this species of veterinary interest.

Comparative ultrastructural characterization of African horse sickness virus-infected mammalian and insect cells reveals a novel potential virus release mechanism from insect cells.

African horse sickness virus (AHSV) is an arbovirus capable of successfully replicating in both its mammalian host and insect vector. Where mammalian cells show a severe cytopathic effect (CPE) following AHSV infection, insect cells display no CPE. These differences in cell death could be linked to the method of viral release, i.e. lytic or non-lytic, that predominates in a specific cell type. Active release of AHSV, or any related orbivirus, has, however, not yet been documented from insect cells. We applied an integrated microscopy approach to compare the nanomechanical and morphological response of mammalian and insect cells to AHSV infection. Atomic force microscopy revealed plasma membrane destabilization, integrity loss and structural deformation of the entire surface of infected mammalian cells. Infected insect cells, in contrast, showed no morphological differences from mock-infected cells other than an increased incidence of circular cavities present on the cell surface. Transmission electron microscopy imaging identified a novel large vesicle-like compartment within infected insect cells, not present in mammalian cells, containing viral proteins and virus particles. Extracellular clusters of aggregated virus particles were visualized adjacent to infected insect cells with intact plasma membranes. We propose that foreign material is accumulated within these vesicles and that their subsequent fusion with the cell membrane releases entrapped viruses, thereby facilitating a non-lytic virus release mechanism different from the budding previously observed in mammalian cells. This insect cell-specific defence mechanism contributes to the lack of cell damage observed in AHSV-infected insect cells.

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.

Virulent African horse sickness virus serotype 4 interferes with the innate immune response in horse peripheral blood mononuclear cells in vitro.

African horse sickness (AHS) is caused by African horse sickness virus (AHSV), a double stranded RNA (dsRNA) virus of the genus Orbivirus, family Reoviridae. For the development of new generation AHS vaccines or antiviral treatments, it is crucial to understand the host immune response against the virus and the immune evasion strategies the virus employs. To achieve this, the current study used transcriptome analysis of RNA sequences to characterize and compare the innate immune responses activated during the attenuated AHSV serotype 4 (attAHSV4) (in vivo) and the virulent AHSV4 (virAHSV4) (in vitro) primary and secondary immune responses in horse peripheral blood mononuclear cells (PBMC) after 24 h. The pro-inflammatory cytokine and chemokine responses were negatively regulated by anti-inflammatory cytokines, whereas the parallel type I and type III IFN responses were maintained downstream of nucleic acid sensing pattern recognition receptor (PRR) signalling pathways during the attAHSV4 primary and secondary immune responses. It appeared that after translation, virAHSV4 proteins were able to interfere with the C-terminal IRF association domain (IAD)-type 1 (IAD1) containing IRFs, which inhibited the expression of type I and type III IFNs downstream of PRR signalling during the virAHSV4 primary and secondary immune responses. Viral interference resulted in an impaired innate immune response that was not able to eliminate virAHSV4-infected PBMC and gave rise to prolonged expression of pro-inflammatory cytokines and chemokines during the virAHSV4 induced primary immune response. Indicating that virAHSV4 interference with the innate immune response may give rise to an excessive inflammatory response that causes immunopathology, which could be a major contributing factor to the pathogenesis of AHS in a naïve horse. Viral interference was overcome by the fast kinetics and increased effector responses of innate immune cells due to trained innate immunity and memory T cells and B cells during the virAHSV4 secondary immune response.

Genome segment reassortment identifies non-structural protein NS3 as a key protein in African horsesickness virus release and alteration of membrane permeability.

The role of African horsesickness virus (AHSV) nonstructural membrane protein NS3 in determining the effects of AHSV infection on Vero cells was examined. NS3 protein sequences are highly variable and cluster into three phylogenetic groups, alpha, beta, and gamma. Three AHSV strains, with NS3 from alpha, beta, or gamma, were shown to have quantitatively different phenotypes in Vero cells. Reassortants between these strains, in which the S10 genome segment encoding NS3 was exchanged alone or with other segments, were generated and compared to parental strains. Exchange of the NS3 gene resulted in changes in virus release, membrane permeability and total virus yield, indicating an important role for NS3 in these viral properties. Differences in the cytopathicity and the effect on cell viability between the parental strains could not be associated with NS3 alone, and it is likely that a number of viral and host factors play a role.

Spatial distribution modelling of Culicoides (Diptera: Ceratopogonidae) biting midges, potential vectors of African horse sickness and bluetongue viruses in Senegal.

BACKGROUND: In Senegal, the last epidemic of African horse sickness (AHS) occurred in 2007. The western part of the country (the Niayes area) concentrates modern farms with exotic horses of high value and was highly affected during the 2007 outbreak that has started in the area. Several studies were initiated in the Niayes area in order to better characterize Culicoides diversity, ecology and the impact of environmental and climatic data on dynamics of proven and suspected vectors. The aims of this study are to better understand the spatial distribution and diversity of Culicoides in Senegal and to map their abundance throughout the country. METHODS: Culicoides data were obtained through a nationwide trapping campaign organized in 2012. Two successive collection nights were carried out in 96 sites in 12 (of 14) regions of Senegal at the end of the rainy season (between September and October) using OVI (Onderstepoort Veterinary Institute) light traps. Three different modeling approaches were compared: the first consists in a spatial interpolation by ordinary kriging of Culicoides abundance data. The two others consist in analyzing the relation between Culicoides abundance and environmental and climatic data to model abundance and investigate the environmental suitability; and were carried out by implementing generalized linear models and random forest models. RESULTS: A total of 1,373,929 specimens of the genus Culicoides belonging to at least 32 different species were collected in 96 sites during the survey. According to the RF (random forest) models which provided better estimates of abundances than Generalized Linear Models (GLM) models, environmental and climatic variables that influence species abundance were identified. Culicoides imicola, C. enderleini and C. miombo were mostly driven by average rainfall and minimum and maximum normalized difference vegetation index. Abundance of C. oxystoma was mostly determined by average rainfall and day temperature. Culicoides bolitinos had a particular trend; the environmental and climatic variables above had a lesser impact on its abundance. RF model prediction maps for the first four species showed high abundance in southern Senegal and in the groundnut basin area, whereas C. bolitinos was present in southern Senegal, but in much lower abundance. CONCLUSIONS: Environmental and climatic variables of importance that influence the spatial distribution of species abundance were identified. It is now crucial to evaluate the vector competence of major species and then combine the vector densities with densities of horses to quantify the risk of transmission of AHS virus across the country.

Protective efficacy of multivalent replication-abortive vaccine strains in horses against African horse sickness virus challenge.

African horse sickness virus (AHSV) is an orbivirus, a member of the Reoviridae family. Nine different serotypes have been described so far. AHSV is vectored by Culicoides spp. to equids, causing high mortality, particularly in horses, with considerable economic impacts. For development of a safe attenuated vaccine, we previously established an efficient reverse genetics (RG) system to generate Entry Competent Replication-Abortive (ECRA) virus strains, for all nine serotypes and demonstrated the vaccine potential of these strains in type I interferon receptor (IFNAR)-knockout mice. Here, we evaluated the protective efficacies of these ECRA viruses in AHSV natural hosts. One monoserotype (ECRA.A4) vaccine and one multivalent cocktail (ECRA.A1/4/6/8) vaccine were tested in ponies and subsequently challenged with a virulent AHSV4. In contrast to control animals, all vaccinated ponies were protected and did not develop severe clinical symptoms of AHS. Furthermore, the multivalent cocktail vaccinated ponies produced neutralizing antibodies against all serotypes present in the cocktail, and a foal born during the trial was healthy and had no viremia. These results validate the suitability of these ECRA strains as a new generation of vaccines for AHSV.

African horse sickness virus infects BSR cells through macropinocytosis.

Cellular pathways involved in cell entry by African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, have not yet been determined. Here, we show that acidic pH is required for productive infection of BSR cells by AHSV-4, suggesting that the virus is likely internalized by an endocytic pathway. We subsequently analyzed the major endocytic routes using specific inhibitors and determined the consequences for AHSV-4 entry into BSR cells. The results indicated that virus entry is dynamin dependent, but clathrin- and lipid raft/caveolae-mediated endocytic pathways were not used by AHSV-4 to enter and infect BSR cells. Instead, binding of AHSV-4 to BSR cells stimulated uptake of a macropinocytosis-specific cargo and inhibition of Na(+)/H(+) exchangers, actin polymerization and cellular GTPases and kinases involved in macropinocytosis significantly inhibited AHSV-4 infection. Altogether, the data suggest that AHSV-4 infects BSR cells by utilizing macropinocytosis as the primary entry pathway.

Culicoides (Diptera: Ceratopogonidae) midges, the vectors of African horse sickness virus--a host/vector contact study in the Niayes area of Senegal.

BACKGROUND: African horse sickness (AHS) is an equine disease endemic to Senegal. The African horse sickness virus (AHSV) is transmitted to the mammalian hosts by midges of the Culicoides Latreille genus. During the last epizootic outbreak of AHS in Senegal in 2007, 1,169 horses died from this disease entailing an estimated cost of 1.4 million euros. In spite of the serious animal health and economic implications of AHS, very little is known about determinants involved in transmission such as contact between horses and the Culicoides species suspected of being its vectors. METHODS: The monthly variation in host/vector contact was determined in the Niayes area, Senegal, an area which was severely affected by the 2007 outbreak of AHS. A horse-baited trap and two suction light traps (OVI type) were set up at each of five sites for three consecutive nights every month for one year. RESULTS: Of 254,338 Culicoides midges collected 209,543 (82.4%) were female and 44,795 (17.6%) male. Nineteen of the 41 species collected were new distribution records for Senegal. This increased the number of described Culicoides species found in Senegal to 53. Only 19 species, of the 41 species found in light trap, were collected in the horse-baited trap (23,669 specimens) largely dominated by Culicoides oxystoma (22,300 specimens, i.e. 94.2%) followed by Culicoides imicola (482 specimens, i.e. 2.0%) and Culicoides kingi (446 specimens, i.e. 1.9%). CONCLUSIONS: Culicoides oxystoma should be considered as a potential vector of AHSV in the Niayes area of Senegal due to its abundance on horses and its role in the transmission of other Culicoides-borne viruses.

Can insecticide-treated netting provide protection for Equids from Culicoides biting midges in the United Kingdom?

BACKGROUND: Biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) cause a significant biting nuisance to equines and are responsible for the biological transmission of African horse sickness virus (AHSV). While currently restricted in distribution to sub-Saharan Africa, AHSV has a history of emergence into southern Europe and causes one of the most lethal diseases of horses and other species of Equidae. In the event of an outbreak of AHSV, the use of insecticide treated nets (ITNs) to screen equine accomodation is recommended by competent authorities including the Office International des Épizooties (OIE) in order to reduce vector-host contact. METHODS: Seven commercially avaliable pyrethroid insecticides and three repellent compounds, all of which are licensed for amateur use, were assessed in modified World Health Organization (WHO) cone bioassay trials in the laboratory using a colony line of Culicoides nubeculosus (Meigen), 1830. Two field trials were subsequently conducted to test the efficiency of treated net screens in preventing entry of Culicoides. RESULTS: A formulation of cypermethrin (0.15 % w/w) and pyrethrins (0.2 % w/w) (Tri-Tec 14®, LS Sales (Farnham) Ltd, Bloxham, UK) applied to black polyvinyl-coated polyester insect screen (1.6 mm aperture; 1.6 mm thickness) inflicted 100 % mortality on batches of C. nubeculosus following a three minute exposure in the WHO cone bioassays at 1, 7 and 14 days post-treatment. Tri-Tec 14® outperformed all other treatments tested and was subsequently selected for use in field trials. The first trial demonstrated that treated screens placed around an ultraviolet light-suction trap entirely prevented Culicoides being collected, despite their collection in identical traps with untreated screening or no screening. The second field trial examined entry of Culicoides into stables containing horses and found that while the insecticide treated screens reduced entry substantially, there was still a small risk of exposure to biting. CONCLUSIONS: Screened stables can be utilised as part of an integrated control program in the event of an AHSV outbreak in order to reduce vector-host contact and may also be applicable to protection of horses from Culicoides during transport.

Protection of horses against Culicoides biting midges in different housing systems in Switzerland.

Species belonging to the Culicoides complexes (Diptera, Ceratopogonidae), obsoletus and pulicaris, in Switzerland, are potential vectors of both bluetongue virus (BTV) and African horse sickness virus (AHSV). The epidemic of BTV in 2006 and 2007 in Europe has highlighted the risk of introduction and spread of vector-borne diseases in previously non-endemic areas. As a measure of prevention, as part of an integrated control programme in the event of an outbreak of African horse sickness (AHS), it is of utmost importance to prevent, or substantially reduce, contact between horses and Culicoides. The aim of the present study was to compare the effect of three protection systems, net, fan, repellent, or combinations thereof, with regard to their potential to reduce contact between horses and Culicoides. Three different equine housing systems, including individual boxes (BX), group housing systems (GR), and individual boxes with permanently accessible paddock (BP) were used. The efficacy of the protection systems were evaluated by comparing the total number counts of collected female Culicoides, of non-blood-fed and blood-fed Culicoides, respectively, with UV black light traps. The study was conducted over 3 summer months during 2012 and 2013 each and focused on the efficacy and practicality of the protection systems. The repellent was tested in 2012 only and not further investigated in 2013, as it showed no significant effect in reducing Culicoides collected in the light traps. Net protection system provided the best overall protection for the total number of female Culicoides, non-blood-fed and blood-fed Culicoides in all tested housing systems. The net, with a pore size of 0.1825 mm(2), reduced the total number of Culicoides collected in the housing systems BP, GR and BX by 98%, 85% and 67%, respectively. However, in the GR housing system, no significant difference between the effectiveness of the fan and the net were determined for any of the three Culicoides categories. The results of the present study demonstrated that horse owners can substantially reduce their horses' exposure to Culicoides, by using net protection in the housing systems BX, BP and GR. In GR housing systems, protection against Culicoides using a fan is also recommended.

Effect of temperature on African horse sickness virus infection in Culicoides.

This paper shows that both the infection rate and the rate of virogenesis of African horse sickness virus (AHSV) within vector Culicoides are temperature dependent. As temperature is reduced from permissive levels the lifespan of the vector itself is extended but the rate of virogenesis decreases and infection rate falls dramatically so that at 10 degrees C virtually all midges are free from virus by 13 days post infection (dpi). When vectors that had been kept at this temperature for 35 days were moved to a permissive temperature for 3 days; however, the apparent zero infection rate increased to 15.5%. It therefore appears that at low temperature (< or = 15 degrees C) AHSV does not replicate but virus may persist in some vectors at a level below that detectable by traditional assay systems and when the temperature later rises to permissive levels virus replication is able to commence. On the basis of this information an overwintering mechanism for AHSV is suggested. The temperature at which the immature stages of Culicoides are reared may also influence infection with AHSV. A 5-10 degrees C rise in larval developmental temperature resulted in an increase in oral infection rate of a normally non-vector species of Culicoides, from < 1% to > 10%. A mechanism is suggested.

Donkeys as reservoirs of African horse sickness virus.

Investigations have been carried out to elucidate the possible role of the donkey in the epidemiology of African horse sickness (AHS). These studies have shown that despite the absence of pyrexia or other observable clinical signs, donkeys become infected with virulent AHS virus serotype 4 (AHSV 4) and that they develop a viraemia which can persist for at least 12 days, albeit at a comparatively lower titre than that recorded for similarly infected ponies. AHSV 4 showed a similar tissue tropism in the pony and donkey but the virus appeared to replicate less efficiently in donkey tissues. The only gross pathological changes observed in the donkeys post mortem were increased fluid accumulation in the serosal lined compartments, particularly the peritoneal cavity, and petechial and ecchymotic haemorrhages on the left hepatic ligament. The absence of infectious virus or viral antigens in any of the tissues collected at 14 and 19 days post inoculation (dpi) from 6 experimental donkeys suggest that, though susceptible to infection, the donkey is unlikely to be a long term reservoir for AHSV. Although AHSV 4 was detected in all 6 donkeys following the primary inoculation, no virus could be isolated from blood collected from two donkeys subsequently challenged with a second virulent virus, AHSV 5. Data generated from virus neutralisation tests showed a second primary antibody response, against AHSV 5, in these donkeys at 12 dpi. In contrast, the boost in antibody levels detected from 5 dpi, as measured by ELISA, was probably due to an anamnestic response against the AHSV group-specific viral proteins. Homogenised spleen tissue, collected post mortem from a donkey 7 dpi with AHSV 4, caused a lethal, cardiac form of AHS when inoculated into a susceptible pony.

Clinical, virological and immune responses of normal and immunosuppressed donkeys (Equus asinus africanus) after inoculation with African horse sickness virus.

To elucidate the role that donkeys may play in African horse sickness virus (AHSV) persistence during inter-epizootic periods we looked for clinical signs of infection and studied the viraemia and neutralising antibody kinetics in 3 immunocompetent and 3 immunosuppressed donkeys inoculated with AHSV-4. None of the donkeys developed signs of AHS. However infectious AHSV was isolated from the blood of the immunocompetent donkeys for up to 17 days post infection (dpi) and viral antigens were detected for up to 28 dpi. Immune cells also increased significantly from 35 to 60 dpi. There was no evidence of a recrudescence of viraemia following immunosuppression of these donkeys at 90 dpi despite a decrease in the numbers of immune cells. Infectious virus was not isolated from the blood of donkeys that had been immunosuppressed, prior to AHSV inoculation. However viral antigens were detected for up to 35 dpi. The titres of AHSV-specific neutralising antibodies and the number of immune cells were also significantly lower than in immunocompetent animals. Our findings suggest that donkeys may be able to play a role in the epidemiology of AHS but the ability of vectors to become infected by feeding upon viraemic donkeys needs to be assessed before the significance of that role can be fully understood.