Sero-surveillance of emerging viral diseases in camels and cattle in Nouakchott, Mauritania: an abattoir study.

This study reports the monitoring of several emerging viral pathogens in Mauritania, which was carried out by the analysis of bovine and camel samples taken at the slaughterhouse of Nouakchott. Blood and serum were collected by random sampling from 159 camels and 118 cattle in March 2013 at the large animals abattoir in Nouakchott. Serological tests for Rift Valley Fever (RVF), Peste des Petits Ruminants (PPR), West Nile disease (WND), epizootic haemorrhagic disease (EHD) and African horse sickness (AHS) were carried out using commercial ELISA kits. The samples, which resulted positives for PPR, WND and AHS, were tested with the confirmatory virus neutralization test (VNT). According to ELISA results, serological prevalence of RVF was 45% (95% CI 52.3-37.7) in camels and 16% (95% CI 22.6-9.4) in cattle. The difference between the observed prevalences in camels and in cattle was significant (p value ≤ 0.01). PPR was absent in camels and had 12% prevalence (95% CI, 17.86-6.14) in cattle. Furthermore, camels showed 92% (95% CI, 96.1-87.9) prevalence of WNV, 73% (95% CI, 82.3-63.64) of EHD and 3% (95% CI, 5.6-0.4) of AHS. This data are of relevance since provided useful feedbacks on the circulation of the pathogens in field. Moreover, this survey provided new information on the susceptibility of camels to several emerging pathogens and on the possible use of this species as sentinel animal.

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.

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.

Sero-epidemioloical survey on African horse sickness virus among horses in Khartoum State, Central Sudan.

BACKGROUND: African horse sickness virus (AHSV) is an infectious non contagious insect-transmitted double-stranded (ds) RNA orbivirus of the family Reoviridae. AHSV causes an often fatal hemorrhagic infection with high mortality among selected breeds of Arabian horses. This study was conducted to avail some information with regard to the prevalence and associated risk factors of AHSV among ecotype breeds of horses in central Sudan. METHODS: Sera were collected from 320 horses, which were selected randomly from four localities and employed in the study. A competitive enzyme-linked immunosorbent assay (cELISA) was used to screen sampled sera for AHSV-specific immunoglobulin G (Ig G) antibodies. RESULTS: Seropositivity to AHSV Ig G was detected in 275 out of the 320 horse sera, thus accounting for a prevalence rate of 85.9%. Potential risk factors to AHSV infection were reported to be associated with horse breed (OR = 5.0, CI = 0.07-2.104, p-value = 0.039) and activity of the horse (OR = 3.21, CI = 0.72-1.48, p- value = 0.008). CONCLUSIONS: The high prevalence of AHSV in Khartoum State of Central Sudan necessitates the need for continuous surveillance for AHSV infection to prevent a possible disease outbreak in this region of the African continent.

African Horse Sickness Virus: History, Transmission, and Current Status.

African horse sickness virus (AHSV) is a lethal arbovirus of equids that is transmitted between hosts primarily by biting midges of the genus Culicoides (Diptera: Ceratopogonidae). AHSV affects draft, thoroughbred, and companion horses and donkeys in Africa, Asia, and Europe. In this review, we examine the impact of AHSV critically and discuss entomological studies that have been conducted to improve understanding of its epidemiology and control. The transmission of AHSV remains a major research focus and we critically review studies that have implicated both Culicoides and other blood-feeding arthropods in this process. We explore AHSV both as an epidemic pathogen and within its endemic range as a barrier to development, an area of interest that has been underrepresented in studies of the virus to date. By discussing AHSV transmission in the African republics of South Africa and Senegal, we provide a more balanced view of the virus as a threat to equids in a diverse range of settings, thus leading to a discussion of key areas in which our knowledge of transmission could be improved. The use of entomological data to detect, predict and control AHSV is also examined, including reference to existing studies carried out during unprecedented outbreaks of bluetongue virus in Europe, an arbovirus of wild and domestic ruminants also transmitted by Culicoides.

Molecular and serological surveillance of African horse sickness virus in eastern and central Saudi Arabia.

African horse sickness virus (AHSV) is one of the most devastating viral diseases of the family Equidae. Infection with AHSV threatens not only the Saudi equine industry but also the equine industry worldwide. This is due to the high morbidity and mortality rates among the infected population of up to 100%. The World Organisation for Animal Health (OIE) lists AHSV among its notifiable diseases; this requires Member Countries to monitor the situation with regard to AHSV very carefully in order to avoid the spread of the virus. The OIE also suggests the systematic monitoring of AHSV in the equine population at regular intervals. The main aim of the current study is to perform molecular and serological surveillance on different horse populations in eastern and central regions of Saudi Arabia. To achieve this aim, the authors collected 361 serum samples, 103 whole blood samples and 323 swabs from Al-Hasa, Dammam, Al-Jubail, Al-Qateef, Riyadh and Al-Qassim. Commercial enzyme-linked immunosorbent assay (ELISA) kits were used to detect AHSV antibodies and commercial real-time reverse transcriptase-polymerase chain reaction (RT-PCR) kits were used to detect AHSV nucleic acids in blood and swabs. The results of this study demonstrate the absence of anti-AHSV antibodies in the sera of tested animals. Furthermore, no viral nucleic acids were detected in the collected blood and swab samples, as evaluated by real-time AHSV-RT-PCR. Moreover, all tested samples collected during 2014-2016 were negative for AHSV. This confirms that the horse populations studied in the eastern and central regions of Saudi Arabia during 2014-2016 were AHSV free.

Le virus de la peste équine est responsable d'une des maladies virales les plus dévastatrices affectant les membres de la famille des Equidae. Les infections par le virus de la peste équine sont une menace pour le secteur équin saoudien et plus largement pour celui du monde entier. La gravité de cette menace est due aux taux de morbidité et de mortalité extrêmement élevés dans les populations atteintes, pouvant atteindre 100 %. L'infection par le virus de la peste équine fait partie des maladies à déclaration obligatoire de l'Organisation mondiale de la santé animale (OIE) ; de ce fait, les Pays membres doivent suivre la situation sanitaire de leur cheptel au regard du virus de la peste équine afin d'éviter sa propagation. L'OIE recommande également de réaliser un dépistage systématique et régulier du virus de la peste équine dans la population équine. Les auteurs présentent les résultats d'une étude basée sur la surveillance moléculaire et sérologique de plusieurs populations de chevaux dans les régions orientale et centrale de l'Arabie saoudite. Pour les besoins de cette étude, les auteurs ont prélevé 323 échantillons de sérum, 103 échantillons de sang entier et 323 écouvillons de chevaux provenant des localités d'Al-Hasa, Dammam, Al-Jubail, Al-Qatif, Riyad et Al-Qasim. Une épreuve immuno-enzymatique (ELISA) sous forme de kits du commerce a été utilisée pour détecter la présence d'anticorps dirigés contre le virus de la peste équine ; la présence dans le sang et les écouvillons d'acides nucléiques spécifiques du virus de la peste équine a été détectée au moyen d'une amplification en chaîne par polymérase couplée à une transcription inverse (RT­PCR) du commerce. Les résultats de cette étude ont montré l'absence d'anticorps dirigés contre le virus de la peste équine dans le sérum des animaux testés. De même, la RT­PCR en temps réel n'a pas détecté d'acides nucléiques spécifiques du virus de la peste équine dans les prélèvements de sang ni les écouvillons analysés. En outre, tous les échantillons collectés entre 2014 et 2016 et soumis à un test ont donné des résultats négatifs pour le virus de la peste équine. Ces résultats confirment que les populations de chevaux étudiées entre 2014 et 2016 dans les régions orientale et centrale de l'Arabie saoudite étaient indemnes de peste équine.

El virus de la peste equina provoca una de las enfermedades víricas más devastadoras que afectan a la familia de los équidos. La infección por este virus amenaza al sector equino no solo de Arabia Saudí, sino del mundo entero, dado que en las poblaciones infectadas las tasas de morbilidad y mortalidad pueden llegar al 100%. La Organización Mundial de Sanidad Animal (OIE) tiene incluida esta infección en su lista de enfermedades de declaración obligatoria, lo que obliga a sus Países Miembros a seguir muy de cerca la situación sanitaria al respecto para evitar que el virus se disemine. La OIE también sugiere hacer periódicamente controles sistemáticos de la presencia del virus en la población equina. Los autores describen un estudio encaminado básicamente a realizar operaciones de vigilancia molecular y serológica de diferentes poblaciones de caballos de las regiones oriental y central de Arabia Saudí. Para ello, los autores obtuvieron 361 muestras de suero, 103 muestras de sangre entera y 323 hisopados en las áreas de Al Hasa, Dammam, Jubail, Qatif, Riad y Casim. Para detectar anticuerpos contra el virus de la peste equina utilizaron un estuche comercial de ensayo inmunoenzimático (ELISA) y para detectar la presencia de ácidos nucleicos del virus en muestras sanguíneas e hisopados un estuche comercial de reacción en cadena de la polimerasa con retrotranscriptasa (RT­PCR) en tiempo real. Los resultados del estudio demuestran la ausencia de anticuerpos contra el virus en el suero de los animales analizados. La técnica de RT­PCR en tiempo real tampoco deparó indicio alguno de la presencia de ácido nucleico vírico en las muestras de sangre e hisopados. Además, todas las muestras analizadas obtenidas entre 2014 y 2016 resultaron negativas para el virus, lo que confirma que las poblaciones equinas estudiadas durante ese periodo en las regiones central y oriental de Arabia Saudí estaban libres del virus de la peste equina.

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.

Requirements and comparative analysis of reverse genetics for bluetongue virus (BTV) and African horse sickness virus (AHSV).

BACKGROUND: Bluetongue virus (BTV) and African horse sickness virus (AHSV) are distinct arthropod borne virus species in the genus Orbivirus (Reoviridae family), causing the notifiable diseases Bluetongue and African horse sickness of ruminants and equids, respectively. Reverse genetics systems for these orbiviruses with their ten-segmented genome of double stranded RNA have been developed. Initially, two subsequent transfections of in vitro synthesized capped run-off RNA transcripts resulted in the recovery of BTV. Reverse genetics has been improved by transfection of expression plasmids followed by transfection of ten RNA transcripts. Recovery of AHSV was further improved by use of expression plasmids containing optimized open reading frames. RESULTS: Plasmids containing full length cDNA of the 10 genome segments for T7 promoter-driven production of full length run-off RNA transcripts and expression plasmids with optimized open reading frames (ORFs) were used. BTV and AHSV were rescued using reverse genetics. The requirement of each expression plasmid and capping of RNA transcripts for reverse genetics were studied and compared for BTV and AHSV. BTV was recovered by transfection of VP1 and NS2 expression plasmids followed by transfection of a set of ten capped RNAs. VP3 expression plasmid was also required if uncapped RNAs were transfected. Recovery of AHSV required transfection of VP1, VP3 and NS2 expression plasmids followed by transfection of capped RNA transcripts. Plasmid-driven expression of VP4, 6 and 7 was also needed when uncapped RNA transcripts were used. Irrespective of capping of RNA transcripts, NS1 expression plasmid was not needed for recovery, although NS1 protein is essential for virus propagation. Improvement of reverse genetics for AHSV was clearly demonstrated by rescue of several mutants and reassortants that were not rescued with previous methods. CONCLUSIONS: A limited number of expression plasmids is required for rescue of BTV or AHSV using reverse genetics, making the system much more versatile and generally applicable. Optimization of reverse genetics enlarge the possibilities to rescue virus mutants and reassortants, and will greatly benefit the control of these important diseases of livestock and companion animals.

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.

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.

A new duplex real-time RT-PCR assay for sensitive and specific detection of African horse sickness virus.

A new real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for a simple and rapid diagnosis of African Horse Sickness (AHS) was developed. Primers and FAM-labeled TaqMan-MGB probes specific for African horse sickness virus (AHSV) were selected from the consensus sequence of the segment 8 of all 9 serotypes of AHSV reference strains. For the determination of the analytical sensitivity, an in vitro transcript (AHS_ns2T7) of the target region was constructed and tested. Furthermore, the AHS_ns2T7 transcript was used either as positive control or as a standard for quantifying target copies. A commercial heterologous Armored RNA was used as an internal positive control (IPC) for both RNA isolation and RT-PCR steps. The qRT-PCR AHS_ns2 was able to amplify the target sequence up to 0.71 copies/reaction. Its flexibility allowed to amplify a wide dynamic range of RNA copies from 1.5 to 0.001fg. Within this range, the Ct values varied from 18 to 38 cycles with SD values always lower than 0.5 confirming their strong and constant linear correlation with the RNA target. Furthermore the newly designed duplex real-time RT-PCR proved to be strictly AHSV-specific as it did not amplify close related viruses.

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.

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.

Tissue and cell tropism of African horse sickness virus demonstrated by immunoperoxidase labeling in natural and experimental infection in horses in South Africa.

Tissues from 196 experimental and confirmed natural cases of African horse sickness (all 9 serotypes) were examined with a standardized and validated immunohistochemical assay for detection of the causative virus. The study confirmed that heart and lung are the main target tissues for African horse sickness virus (across all serotypes), followed closely by spleen. It also indicated that microvascular endothelial cells and monocyte-macrophages are the main target cells for virus replication. The importance of monocytes as target cells was emphasized, with relatively few tissue macrophages containing antigen in the lung and spleen, respectively. The results were largely in agreement with those of previous studies, but the large number of cases examined permitted more precise description of the location and distribution of antigen in different tissues. Comparison with descriptions of tissue and cell tropism of other orbiviruses indicated similarity with African horse sickness. Immunohistochemistry was shown to be a useful and consistent technique for demonstrating target cells, but the difficulty of identifying cell types-in particular, different types of monocyte-macrophages-is a limitation.

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.

The oral susceptibility of South African field populations of Culicoides to African horse sickness virus.

Twenty-two isolates of African horse sickness virus (AHSV), representing its distinct serotypes, geographical and historical origins, were fed to three populations of South African livestock-associated Culicoides spp. (Diptera, Ceratopogonidae). Infective blood meals included 12 recent isolates, nine historical reference strains and one live attenuated vaccine strain serotype 7 (AHSV-7) of the virus. Field-collected midges were fed through a chicken-skin membrane on sheep blood spiked with one of the viruses, which concentrations ranged from 5.4 to 8.8 log(10)TCID(50)/mL of blood. After 10 days incubation at 23.5 degrees C, AHSV was isolated from 11 Culicoides species. Standard in vitro passaging of AHSV-7, used for the preparation of live attenuated vaccine, did not reduce its ability to infect Culicoides species. Virus recovery rates in orally infected Culicoides midges differed significantly between species and populations, serotypes, isolates and seasons. Significant variations in oral susceptibility recorded in this study emphasize a complex inter-relationship between virus and vector, which is further influenced by multiple intrinsic and extrinsic factors. As it is not possible to standardize all these factors under laboratory conditions, conclusive assessment of the role of field-collected Culicoides midges in the transmission of orbiviruses remains problematic. Nevertheless, results of this study suggest the potential for multi-vector transmission of AHSV virus in South Africa.

Rapid and sensitive detection of African horse sickness virus by real-time PCR.

A highly sensitive and specific TaqMan-MGB real-time RT-PCR assay has been developed and standardised for the detection of African horse sickness virus (AHSV). Primers and MGB probe specific for AHSV were selected within a highly conserved region of genome segment 7. The robustness and general application of the diagnostic method were verified by the detection of 12 AHSV isolates from all of the nine serotypes. The analytical sensitivity ranged from 0.001 to 0.15 TCID(50) per reaction, depending on the viral serotype. Real-time PCR performance was preliminarily assessed by analysing a panel of field equine samples. The same primer pair was used to standardise a conventional RT-PCR as an affordable, useful and simple alternative method in laboratories without access to real-time PCR instruments. The two techniques present novel tools to improve the molecular diagnosis of African horse sickness (AHS).