African Horse Sickness Virus Serotype Research Articles

First Report of an Outbreak of African Horsesickness Virus Serotype 2 in the Northern Hemisphere

A case of a rapidly fatal disease in polo horses caused by African horsesickness (AHS) virus serotype 2 is described. The pattern of polo tournaments, environmental/management conditions (moist and warm), as well as probable importation strategies with no regard for import control and quarantine, favored introduction and spread of the virus. The outbreak, which involved a large number of horses, was characterized by severe respiratory distress, fever, supraorbital edema, and death. This is the first time a widespread epidemic of AHS has been reported in Nigeria and this is the first report of AHSV serotype 2 in the northern hemisphere. In addition, we amplified the complete genome of the virus using RNA extracted from clotted blood. This report indicates that AHS is expanding its geographical territory northwards and assuming a new microbial ecology.

Virus recovery rates for wild‐type and live‐attenuated vaccine strains of African horse sickness virus serotype 7 in orally infected South African Culicoides species

Previously reported virus recovery rates from Culicoides (Avaritia) imicola Kieffer and Culicoides (Avaritia) bolitinos Meiswinkel (Diptera, Ceratopogonidae) orally infected with vaccine strain of African horse sickness virus serotype 7 (AHSV-7) were compared with results obtained from concurrently conducted oral infections with five recent AHSV-7 isolates from naturally infected horses from various localities in South Africa. Culicoides were fed sheep bloods spiked with 10(7.6) TCID(50)/mL of a live-attenuated vaccine strain AHSV-7, and with five field isolates in which virus titre in the bloodmeals ranged from 10(7.1) to 10(8.2) TCID(50)/mL). After an extrinsic incubation of 10 days at 23.5 degrees C, virus recovery rates were significantly higher in C. imicola (13.3%) and C. bolitinos (4.2%) infected with the live-attenuated virus than in midges infected with any of the field isolates. The virus recovery rates for the latter groups ranged from 0% to 9.5% for C. imicola and from 0% to 1.5% for C. bolitinos. The C. imicola population at Onderstepoort was significantly more susceptible to infection with AHSV-7 isolated at Onderstepoort than to the virus strains isolated from other localities. Results of this study suggest that tissue culture attenuation of AHSV-7 does not reduce its ability to orally infect competent Culicoides species and may even lead to enhanced replication in the vector. Furthermore, oral susceptibility in a midge population appears to vary for geographically distinct isolates of AHSV-7.

Investigaciones sobre brotes de peste equina en la zona de vigilancia de Sudáfrica

Confirmed outbreaks of African horse sickness (AHS) occurred in the surveillance zone of the Western Cape in 1999 and 2004, both of which led to a two-year suspension on the export of horses. Light trap surveys in the outbreak areas showed that known vector competent Culicoides species, notably C. imicola, were abundant and present in numbers equal to those in the traditional AHS endemic areas. Isolations of AHS virus serotypes 1 and 7, equine encephalosis virus, and bluetongue virus from field-collected C. imicola in the surveillance zone demonstrated that this species was highly competent and could transmit viruses belonging to different serogroups of the Orbivirus genus. Molecular identification of recovered virus isolates indicated that at least two incursions of AHS into the surveillance zone had taken place in 2004. The designation of an AHS-free zone in the Western Cape remains controversial since it can be easily compromised, as evidenced by the two recent outbreaks. In light of the results reported in the present study, the policy of maintaining a large population of unvaccinated horses in the surveillance zone should be reconsidered, as it leaves them vulnerable to infection with AHS virus, which is the most pathogenic of all equine viruses.

Preparation of recombinant African horse sickness virus VP7 antigen via a simple method and validation of a VP7-based indirect ELISA for the detection of group-specific IgG antibodies in horse sera

This paper describes the production and purification of a group-specific recombinant protein VP7 of African horse sickness virus serotype 3 (AHSV-3) and validation of an I-ELISA for the detection of IgG-antibodies to VP7 in horse sera. Baculovirus-expressed VP7 crystals were purified from infected insect cells. Analytical accuracy of the I-ELISA was examined using sera ( n = 38) from an experimentally infected horse, from foals born to vaccinated mares, from guinea-pigs immunized with nine serotypes of AHSV, and from sera of animals infected with other orbiviruses. Compared to traditional serological assays, the I-ELISA was more sensitive in detection of the earliest immunological response in an infected horse and declining levels of maternal immunity in foals. Antibodies to all nine serotypes of AHSV could be detected. Cross-reactivity to related orbiviruses was not observed. Diagnostic accuracy of the I-ELISA was assessed by testing sera from vaccinated horses ( n = 358) residing in AHS-enzootic areas and from unvaccinated horses ( n = 481) residing in an AHS-free area. Sera were categorised as positive or negative for antibodies to AHSV using virus neutralisation tests. The TG-ROC analysis was used for the selection of the cut-off value. At a cut-off of 11.9 of the high positive control serum (percentage positivity), the I-ELISA specificity was 100%, sensitivity 99.4%, and the Jouden index was 0.99.

A group-specific, indirect sandwich ELISA for the detection of equine encephalosis virus antigen

A polyclonal antibody-based, group-specific, indirect, sandwich ELISA (S-ELISA) for the detection of equine encephalosis virus (EEV) antigen was developed. Purified EEV particles were titrated in the S-ELISA and the limit of detection was determined to be approximately 9.0 ng of antigen/ml (0.45 ng/well). Positive S-ELISA reactions were recorded with seven serologically distinct EEV serotypes. No cross-reactions were recorded with other arboviruses including African horse sickness virus (AHSV) serotypes 1–9, bluetongue serotypes 1–24, epizootic haemorrhagic disease serotypes 1–8 and isolate 318, and selected isolates of Palyam, Eubenangee, Corriparta, Warrego, Akabane and bovine ephemeral fever viruses. The assay proved to be sensitive and specific for the rapid detection of EEV in cell cultures and in homogenated suckling mouse brain (MB). The data generated in this study suggest that the ELISA will be valuable for epidemiological studies of EE and will assist in making a reliable differential diagnosis between EEV and AHSV infections.

A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes

The outer capsid protein VP2 of African horsesickness virus (AHSV) is a major protective antigen. We have cloned full-length VP2 genes from the reference strains of each of the nine AHSV serotypes. Baculovirus recombinants expressing the cloned VP2 genes of serotypes 1, 2, 4, 6, 7 and 8 were constructed, confirming that they all have full open reading frames. This work completes the cloning and expression of the first full set of AHSV VP2 genes. The clones of VP2 genes of serotypes 1, 2, 5, 7 and 8 were sequenced and their amino acid sequences were deduced. Our sequencing data, together with that of the published VP2 genes of serotypes 3, 4, 6 and 9, were used to generate the first complete sequence analysis of all the (sero)types for a species of the Orbivirus genus. Multiple alignment of the VP2 protein sequences showed that homology between all nine AHSV serotypes varied between 47.6 % and 71.4 %, indicating that VP2 is the most variable AHSV protein. Phylogenetic analysis grouped together the AHSV VP2s of serotypes that cross-react serologically. Low identity between serotypes was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralization. The data presented here impact on the development of new vaccines, the identification and characterization of antigenic regions, the development of more rapid molecular methods for serotype identification and the generation of comprehensive databases to support the diagnosis, epidemiology and surveillance of AHS.

VP2 gene phylogenetic characterization of field isolates of African horsesickness virus serotype 7 circulating in South Africa during the time of the 1999 African horsesickness outbreak in the Western Cape

We present the first VP2-gene phylogenetic analysis of African horsesickness (AHS) viruses within a serotype. Thirteen AHSV 7 isolates were obtained from cases that occurred in South Africa during 1998–1999, and three were historical AHSV 7 isolates. The goals were to start a database of isolates of known location and time of isolation and to determine if we could identify the origin of an AHS outbreak in the surveillance area in the Western Cape. We prepared full-length cDNA copies of the VP2-genes of the isolates. Nucleic acid sequence data of a 786 bp region was used to characterize the genetic relationships between the isolates. The nucleic acid identities between the isolates ranged from 95.5 to 100%. Isolates from common geographical regions grouped together. Characterization of field isolates revealed the presence of two AHSV 7 lineages in South Africa during this period. The grouping of the viruses into two clades accurately reflected the geographical groupings of the isolates. The average nucleic acid divergence between the clades was 4.3%. Within the clades the divergence was 0.5 and 0.1%, respectively. The data suggests that the AHS outbreak in the Western Cape could have been an incursion from the Kwazulu Natal Province.

Effect of temperature on the transmission of orbiviruses by the biting midge, Culicoides sonorensis.

The influence of temperature on the likelihood of Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae) transmitting African horse sickness virus (AHSV) serotypes 4 and 6, bluetongue virus (BTV) serotypes 10 and 16 and epizootic haemorrhagic disease of deer virus (EHDV) serotype 1 was investigated. Extrinsic incubation periods (EIP), vector competence and vector survival were determined at 15, 20, 25 and 30 degrees C. The effect of humidity on vector survival was also investigated by maintaining adult C. sonorensis at 40, 75 and 85% r.h. at each temperature. Higher temperatures were associated with a shorter EIP for all virus serotypes except AHSV6, to which C. sonorensis was orally refractory, increased vector competence for AHSV4 and EHDV1, but not for BTV10 or BTV16, and a reduction in vector survival. Humidity interacted with temperature in influencing vector survival, such that at low temperatures, lower humidity (40 and 75% r.h.) was detrimental for survival (up to 18% reduction in longevity), whereas at high temperatures, high humidity (85% r.h.) was detrimental (up to 36% reduction in longevity). In general, the transmission potential of C. sonorensis for AHSV4, EHDV1, BTV10 and BTV16 was greater at higher temperatures, because although vector survival was reduced, this was more than compensated for by the accompanying decrease in duration of the EIP.

The protective efficacy of a recombinant VP2-based African horsesickness subunit vaccine candidate is determined by adjuvant

We previously demonstrated that soluble baculovirus-expressed African horsesickness virus (AHSV) serotype 5 VP2 protein (AHSV5 rVP2) elicits neutralising antibodies in guinea pigs. We have now determined the immunogenicity of soluble AHSV5 rVP2 in horses when administered in three different adjuvant types, ISA-50, aluminium phosphate and different saponin preparations. Doses of 10 and 50 μg of rVP2 administered with saponin induced full protection to a lethal challenge, albeit with dose-related side effects. The results establish that soluble rVP2 is the biologically active form and that it can induce complete protection when it is delivered with saponin adjuvants. We conclude that the use of the soluble biologically active form of AHSV rVP2 and the choice of adjuvant will be crucial factors in determining efficacy, safety and the production cost of recombinant AHSV subunit vaccines.

Definition of neutralizing sites on African horse sickness virus serotype 4 VP2 at the level of peptides.

The antigenic structure of African horse sickness virus (AHSV) serotype 4 capsid protein VP2 has been determined at the peptide level by PEPSCAN analysis in combination with a large collection of polyclonal antisera and monoclonal antibodies. VP2, the determinant for the virus serotype and an important target in virus neutralization, was found to contain 15 antigenic sites. A major antigenic region containing 12 of the 15 sites was identified in the region between residues 223 and 400. A second domain between residues 568 and 681 contained the three remaining sites. These sites were used for the synthesis of peptides, which were later tested in rabbits. Of the 15 synthetic peptides, three were able to induce neutralizing antibodies for AHSV-4, defining two neutralizing epitopes, 'a' and 'b', between residues 321 and 339, and 377 and 400, respectively. A combination of peptides representing both sites induced a more effective neutralizing response. Still, the relatively low neutralization titres make the possibility of producing a synthetic vaccine for AHSV unlikely. The complex protein-protein interaction of the outer shell of the viral capsid would probably require the presence of either synthetic peptides in the correct conformation or peptide segments from the different proteins VP2, VP5 and VP7.

African horse sickness epidemiology: vector competence of south african Culicoides species for virus serotypes 3, 5 and 8.

The oral susceptibilities of 17 Culicoides species to infection with African horse sickness virus (AHSV) serotypes 3, 5 and 8 were determined by feeding field-collected midges on AHSV infected horse blood. The mean titres of virus in the bloodmeals for the three serotypes of AHSV were between 5.7 and 6.5 log10 TCID50/ml. Virus was detected, after 10 days incubation at 23.5 degrees C, in the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) that had fed on blood containing AHSV 5 (8.5%) and 8 (26.8%), and in the Culicoides bolitinos Meiswinkel that had fed on AHSV 3 (3.8%), 5 (20.6%) and 8 (1.7%). Although 44.4% of the C. imicola were shown to have ingested AHSV 3 immediately after feeding, no virus was detected in 96 C. imicola after incubation. The relatively high titres of virus recorded in individual midges of both species after 10 days incubation suggested a fully disseminated infection. Previously, C. imicola was considered to be the only field vector of AHSV in Africa. Identifying C. bolitinos as a potential vector for AHSV is an important finding, which if proven will have a significant impact on our understanding of the epidemiology of AHS. No AHSVs could be detected in the other 15 species of Culicoides assayed, which suggests that some of the southern African Culicoides species are refractory to AHSV infection. However, further work with larger numbers of each species will be necessary to confirm this observation.

Development of probes for typing African horsesickness virus isolates using a complete set of cloned VP2-genes

A set of cloned full-length VP2-genes from the reference strain of each of the nine serotypes of African horsesickness virus (AHSV) was used to develop probes for typing AHSV isolates. The VP2-gene probes hybridised serotype-specific to purified viral dsRNA from its corresponding serotype. No cross-hybridisation was observed between the different AHSV serotypes or with RNA from equine encephalosis virus or bluetongue virus (BTV) which are related viruses within the genus Orbivirus that co-circulate with AHSV in South Africa. The probes were able to detect AHSV isolates from recent field cases of AHS in South Africa, despite being derived from historical reference strains. With regard to sensitivity and time considerations: radioactive 32P-labelling resulted in a marginal increase in sensitivity over digoxigenin-labelled probes. By infecting cell cultures at different multiplicities of infection (m.o.i.) and harvesting at various times post infection, it was established that AHSV RNA could be detected 16 h post infection (p.i.) at a m.o.i. of 1.00 pfu per cell and 48 h p.i. at a m.o.i. of 0.01 pfu per cell. Typing of AHSV isolates by means of VP2-gene probe hybridisation can be completed in 4 days, which is less than half the time required for conventional isolation and serotyping. This report on the use of a complete set of cloned AHSV VP2-gene probes is the first demonstration of typing for a whole specie (serogroup) in a genus of the family Reoviridae.

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

Cloning, sequencing and expression of the gene that encodes the major neutralisation-specific antigen of African horsesickness virus serotype 9

A marked improvement in the efficiency of cloning the large double stranded RNA (dsRNA) genome segments of African horsesickness virus (AHSV) was achieved when the dsRNA polyadenylation step was carried out with undenatured rather than strand-separated dsRNA. It is a prerequisite to use dsRNA of very high purity because in the presence of even trace amounts of single stranded RNA, the dsRNA appears to be poorly polyadenylated as judged by its effectiveness as a template for oligo-dT-primed cDNA synthesis. The full-length VP2 gene of AHSV-9, cloned by this approach, was sequenced and it was found to show the highest percentage identity (60%) to VP2 of AHSV-6, providing an explanation of why these two serotypes show some cross protection. The VP2 protein was also expressed in Spodoptera frugiperda (Sf9) cells by means of a baculovirus recombinant. The yield of the expressed VP2 was high, but the protein was found to be largely insoluble. Nine smaller, truncated VP2 peptides were subsequently expressed in insect cells, but no significant improvement in solubility of the peptides, as compared to that of the full-sized protein, was observed. A western immunoblot analysis of the overlapping peptides indicated the presence of a strong linear epitope located within a large hydrophilic domain between amino acids 369 and 403.

Identification and differentiation of the nine African horse sickness virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

This paper describes the first RT-PCR for discrimination of the nine African horse sickness virus (AHSV) serotypes. Nine pairs of primers were designed, each being specific for one AHSV serotype. The RT-PCR was sensitive and specific, providing serotyping within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralization for a coded panel of 56 AHSV reference strains and field isolates. Serotyping was achieved successfully with live and formalin-inactivated AHSVs, with isolates of virus after low and high passage through either tissue culture or suckling mouse brain, with viruses isolated from widely separated geographical areas and with viruses isolated up to 37 years apart. Overall, this RT-PCR provides a rapid and reliable method for the identification and differentiation of the nine AHSV serotypes, which is vital at the start of an outbreak to enable the early selection of a vaccine to control the spread of disease.

African Horsesickness Virus Infection in Adult Mice.

Adult BALB/c mice were inoculated intranasally with African horsesickness virus serotype 4 (AHSV-4), the attenuated-vaccine strain. The 4-week-old mice died after developing nervous signs at 7 to 8 days post infection (p.i). Lesions, such as perivascular accumulation of lymphocytes and necrosis of neurons, were found only in the brains of virus-infected mice, but not in those of controls. Brains, red blood cells (RBC) and white blood cells (WBC) were collected from two mice each day p.i for virus isolation and RT-PCR. Virus could be isolated from brains collected at 2 to 7 days p.i of infection mice and only one of WBC sample collected on 7 day p.i, but no virus was isolated from any RBC samples. In contrast, RT-PCR products were visible in agarose gel electrophoresis stained with ethidium bromide from RBC and WBC samples collected at days 1 to 7 p.i, and from brain samples collected at days 2 to 7 p.i. The amplified PCR products were increased with days p.i when using extracted RNAs from brain samples, but the quantity of the PCR products was relatively constant with the RBC and WBC samples. It was shown that mice were susceptible to AHSV.

Cloning and expression of the M5 RNA segment encoding outer capsid VP5 of epizootic hemorrhagic disease virus Japan serotype 2, Ibaraki virus.

The complete nucleotide sequence of a cDNA clone representing the M5 RNA segment of epizootic hemorrhagic disease virus Japan serotype 2 (EHDV-2), Ibaraki virus, was determined. The M5 segment is 1641 base pairs long with the single open reading frame which predicts a polypeptide of 527 amino acids. The comparison of the amino acid sequence of the VP5 with those of EHDV-1, bluetongue virus serotype 10, and African horse sickness virus serotype 4 revealed that the protein shared 67%, 57% and 42% homologies, respectively. In addition, the VP5 protein was expressed in insect cells by recombinant baculovirus, which could be recognized by the mouse anti-EHDV-2 sera at a position of the expected 59 kDa on immunoblot analysis.

Antigenic Profile of African Horse Sickness Virus Serotype 4 VP5 and Identification of a Neutralizing Epitope Shared with Bluetongue Virus and Epizootic Hemorrhagic Disease Virus

African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned inEscherichia coliusing the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151–200) and II (residues 83–120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85–92 (PDPLSPGE) for MAb 10AE12 and at 179–185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes.

Sequence analysis of the RNA polymerase gene of African horse sickness virus.

The gene encoding the inner core protein VP1 of African horse sickness virus (AHSV) serotype 9 has been cloned, expressed in vitro and entirely sequenced, completing molecular characterization of the AHSV genome. An analysis of the sequence supporting the identity of AHSV VP1 as the putative viral RNA polymerase is presented.

Transmission and distribution of African horse sickness virus serotypes in South African zebra.

The prevalences of African horse sickness (AHS) virus serotypes in zebra foals from the Kruger National Park, South Africa were examined for possible associations between serotypes. Serotypes known to cross-react were combined for analysis. The distributions of serotypes between zebra were not always independent; in 7-8 month old zebra positive pairwise associations were observed between 3 serotypes. This could be generated by biological interactions between serotypes or heterogeneity in host-vector transmission. The data were also used to estimate the basic reproduction number, R0. For AHS virus overall, estimates of R0 ranged from 31-68. This underlines the need for a better understanding of serotype transmission and interactions in AHS.