African Horse Sickness Virus Serotype Research Articles

Expression of nonstructural protein NS3 of African horsesickness virus (AHSV): evidence for a cytotoxic effect of NS3 in insect cells, and characterization of the gene products in AHSV infected Vero cells.

The smallest genome segment of African horsesickness virus (AHSV), segment 10 (S10), encodes two minor nonstructural proteins, NS3 and NS3A. While the cognate bluetongue virus (BTV) proteins have been suggested to play a role in the release of virus particles from infected cells, no function has yet been ascribed to AHSV NS3/NS3A. When the AHSV-3 S10 gene was expressed in a baculovirus system only a single NS3 protein (24 K) was synthesized, at lower levels than expected. It was shown that this could be due to a membrane association of NS3, leading to an alteration in host cell membrane permeability and eventual cell death. Based on computer predictions a general model for the membrane-associated topology of NS3 of five different orbiviruses was proposed. Studies on AHSV-3 infected Vero cells showed that equimolar amounts of NS3 and NS3A were synthesized. No evidence was found for the glycosylation of NS3. The S10 genes and NS3/3A proteins of AHSV-3 and AHSV-7 were shown to be closely related, and clearly distinct from the cognate proteins of the other 7 AHSV serotypes. This distinguishes the AHSV S10 gene product from that of BTV NS3, which appears to be much more conserved.

Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3.

The gene encoding the outer capsid protein, VP2, of African horsesickness virus serotype 3 (AHSV-3) has been sequenced in its entirety from cDNA clones of the segment 2 RNA, and compared with the previously published VP2 gene sequence of AHSV-4. AHSV-3 genome segment 2 was shown to be 3221 nucleotides in length, encoding a protein of 1057 amino acids with a 50.5% identity to the amino acid sequence of AHSV-4 VP2. Two areas of high variability (approximately 35% identity) were identified. The N-proximal variable region (amino acids 128 to 309) exhibited significant hydrophilicity, suggesting a possible role in the determination of the serotype-specific immune response. VP2 of AHSV-3 has furthermore been expressed in a baculovirus expression system. The expressed protein was shown to react specifically with an anti-AHSV-3 serum in Western blots. Antibodies raised in rabbits and guinea-pigs against the recombinant VP2 neutralized the virus in a plaque reduction assay, confirming the identity of VP2 as the major neutralization-specific antigen of AHSV.

Presence of African horse sickness virus in equine tissues, as determined by in situ hybridization.

In a retrospective study, a negative-sense digoxigenin-labeled RNA probe, corresponding to the gene encoding nonstructural protein-1 of African horse sickness virus (AHSV) serotype 4, was applied to formalin-fixed, paraffin-embedded tissue taken from horses in the terminal stages of infection with AHSV. Fifteen infected ponies and one noninfected control were studied. Ponies exhibited a range of clinical signs and lesions. Thirteen ponies were infected with serotype 4, one with serotype 1, and one with serotype 2. Ponies were monitored clinically and euthanatized when severely clinically ill. The following tissues were available for study by in situ hybridization and histopathology: lung, heart, spleen, neck muscle, and supraorbital fat. Histologically, the most striking changes were pulmonary edema and, in some, acute myocardial necrosis. In situ hybridization revealed virus distributed widely in sections of lung and heart examined, with relatively less in spleen, neck muscle, or supraorbital fat. Virus was localized to target cells with morphologic features compatible with endothelium in all organs except spleen, where it was found in both endotheliumlike cells and large mononuclear cells.

Vaccinia virus expression of the VP7 protein of South African bluetongue virus serotype 4 and its use as an antigen in a capture ELISA.

Recombinant vaccinia viruses expressing the VP7 core protein of South African bluetongue virus serotype 4 (SA-BTV4) were identified by polymerase chain reaction amplification. Expression of VP7 was verified by radio-immunoprecipitation and a F(ab')2-based ELISA. Antibodies to VP7 were detected in sera from sheep that had been infected with 20 different virulent BTV serotypes by using the vaccinia virus (VV) expressed VP7 as antigen in a capture ELISA. F(ab')2-immobilised VV-expressed SA-BTV4 VP7 cross-reacted with sera directed against all 9 African horsesickness virus serotypes and epizootic haemorrhagic disease virus serotype 2.

Phylogenetic analysis of segment 10 from African horsesickness virus and cognate genes from other orbiviruses

Utilizing the reverse transcriptase-polymerase chain reaction (RT-PCR) procedure, we have synthesized full-length copies of segment 10 from African horsesickness virus (AHSV) serotypes 1, 4 and 8. The genes were cloned, sequenced and compared with the sequence of the cognate gene from AHSV serotypes 3 and 9. Sequences were analyzed to assess evolutionary relationships among serotypes using cladistics. Based on this analysis the data support a close relationship between serotypes 4 and 9 and between serotypes 1 and 8 and a closer relationship of serotype 3 to the 4 and 9 group.

African horse sickness virus structure

African horse sickness virus (AHSV), of which there are nine serotypes (AHSV-1, -2, etc.), is a member of Orbivirus genus within the Reoviridae family. Both in morphology and molecular constituents AHSV particles are comparable to those of bluetongue virus (BTV), the prototype virus of the genus. The two viruses have seven structural proteins (VP1–7) organized in two layered capsid. The outer capsid is composed of VP2 and VP5. The inner capsid, or core, is composed of two major proteins, VP3 and VP7, and three minor proteins, VP1, VP4 and VP6. Within the core is the virus genome. This genome consists of 10 double-stranded (ds)RNA segments of different sizes, three large, designated L1–L3, three medium, M4–M6, and four small, S7–S10. In addition to the seven stuctural proteins that are coded by seven of the RNA species, four non-structural proteins, NS1, NS2, NS3 and NS3A, are coded by three RNA segments, M5, S8 and S10. The two smallest proteins (NS3 and NS3A) are synthesized by the S10 RNA segment, probably from different in-frame translation initiation codons. Nucleotide sequences of eight RNA segments (L2, L3, M4, M5, M6, S7, S8 and S10) and the predicted amino acid sequences of the encoded gene products are also available, mainly representing one serotype, AHSV-4. In this review the properties of the AHSV genes and gene products are discussed. The sequence and hybridization analyses of the different AHSV dsRNA segments indicate that the segments that code for the core proteins, as well as those that code for NS1 and NS2 proteins, are highly conserved between the different virus serotypes. However, the RNA encoding NS3 and NS3A, and the two segments encoding the outer capsid proteins, are more variable between the AHSV serotypes. A close phylogenetic relationship between AHSV, BTV and epizootic haemorrhagic disease virus (EHDV), three Culicoides-transmitted orbiviruses, has been revealed when the equivalent sequences of genes and gene products are compared. Recently, the four major AHSV capsid proteins have been expressed using recombinant baculoviruses. Biochemically and antigenically these proteins are similar to the authentic proteins. Since the AHSV VP7 protein is highly conserved among the different serotypes, it has been utilized as a diagnostic reagent. The expressed VP7 protein has also been purified to homogeneity and crystallized for three-dimensional X-ray analysis. The expressed outer capsid proteins, VP2 and VP5, have been purified and used to raise antisera in rabbits. The VP2 antisera neutralize virus infections in vitro indicating the importance of this protein for vaccine development.

Purification and properties of virus particles, infectious subviral particles, cores and VP7 crystals of African horsesickness virus serotype 9

Methods were developed for the purification, at high yield, of four different particle types of African horsesickness virus serotype 9 (AHSV-9). These products included virus particles purified on CsCl gradients which contain proteins apparently directly comparable to those of bluetongue virus (VP1 to VP7); virus particles purified on sucrose gradients which also contain, as a variable component, protein NS2; infectious subviral particles (ISVPs), containing chymotrypsin cleavage products of VP2; and cores, obtained by treating purified ISVPs with 1 M-MgCl2 to remove the components of the outer capsid layer (VP5 and VP2 cleavage products). Additional protein bands migrating with apparent M(r)s lower than that of VP5 were detected during SDS-PAGE analysis of virus particles. These appear to be conformational variants of VP5 and are identified as VP5' and VP5". BHK-21 cells infected with this strain of AHSV-9 produce large quantities of flat, usually hexagonal crystals of VP7, a major group antigen and core protein; these were also purified. Either 20 mg of virus particles, 20 mg of ISVPs or 10 mg of cores as well as 20 mg of VP7 crystals could be purified from approximately 8 x 10(9) infected cells. None of the preparations of particles or crystals showed any detectable contamination with BHK-21 cell proteins or antigens, as determined by SDS-PAGE or indirect ELISA. Virus particle and ISVP preparations had similar specific infectivities for BHK-21 cells (approximately 1 x 10(9) TCID50/A260 unit) but the infectivity of cores was approximately 10(5)-fold lower.

Expression and Characterization of the Two Outer Capsid Proteins of African Horsesickness Virus: The Role of VP2 in Virus Neutralization

African horsesickness virus (AHSV) is a gnat-transmitted member of the Orbivirus genus of the Reoviridae family. The virus has a genome of 10 double-stranded RNA species (L1-L3, M4-M6, S7-S10). The L2 and M6 genes of AHSV serotype 4 (AHSV-4) which encode the outer capsid proteins VP2 and VP5, respectively, were inserted into recombinant baculoviruses downstream of the baculovirus polyhedrin, or p10 promoters. Recombinant baculoviruses expressing VP2, VP5, or VP2 and VP5 proteins of AHSV-4 were isolated. The expressed AHSV proteins were similar in size and antigenic properties to those of viral AHSV-4. Expressed VP2 and VP5 proteins were purified to homogeneity and utilized to differentiate sera from vaccinated and infected horses. Antigens were also used to determine whether any other AHSV serotypes are related to AHSV-4. The results indicated that AHSV-4 is distantly related to some serotypes (e.g., AHSV-2, -6, and -9) but not to others (e.g., AHSV-5 end -7). Hyperimmune monospecific antisera raised in rabbits with purified VP2 neutralized the infectivity of a virulent strain of AHSV-4 isolated from an infected horse during a recent outbreak of the disease in Spain.

Baculovirus expression of non-structural protein NS2 and core protein VP7 of African horsesickness virus serotype 3 and their use as antigens in an indirect ELISA

Non-structural protein NS2 and core protein VP7 of African horsesickness virus serotype 3 (AHSV3) were expressed in Spodoptera frugiperda cells by recombinant baculoviruses containing the relevant genes. These proteins were purified and analysed by polyacrylamide gel electrophoresis and Western blot. NS2 and VP7 were used separately as antigens in an indirect ELISA for the detection of AHSV antibodies. Both antigens cross-reacted with hyperimmune guinea-pig antisera to infected cell lysates of all nine known AHSV serotypes and to antisera obtained from horses immunized with attenuated virus of seven AHSV serotypes.

Identification of African Horse Sickness Virus in Cell Culture using a Digoxigenin-Labeled RNA Probe

A digoxigenin-labeled RNA probe was synthesized from a plasmid containing a portion of the African horse sickness virus (AHSV) serotype 4 genome segment coding for nonstructural protein 1. In an in situ hybridization procedure, this probe hybridized successfully to Vero cells infected with each of the 9 serotypes of AHSV. There was no hybridization with noninfected cell cultures or cell cultures infected with bluetongue virus.

Rapid detection of African horsesickness virus by the reverse transcriptase polymerase chain reaction (RT-PCR) using the amplimer for segment 3 (VP3 gene).

The complete sequence of the major core protein (VP3) gene of African horsesickness virus serotype 4 (AHSV-4; vaccine strain) was determined by analysis of a complete cDNA clone representing segment 3. The RNA was 2,789 bp long and a comparison of its sequence with that of bluetongue virus serotype 10 (BTV-10) revealed 58% nucleotide similarity. Based on these data, the reverse transcriptase-polymerase chain reaction (RT-PCR) technique was applied to the specific detection of AHSV using a pair of primers designed for AHSV-4 VP3 gene. Approximately 230 bp of PCR products were amplified by RT-PCR from the total RNA extracts (mRNA and dsRNA) of Vero cells infected with eight serotypes of AHSV. No product was observed analogous to other orbiviruses. The supernatant of the infected cell culture fluid without any RNA purification was also suitable as a template for RT-PCR after being denatured at 94 degrees C for 5 min. The sensitivity of this method was between 10(0) and 10(1) TCID50 when viral RNA from the supernatant of infected cell culture was subjected to RT-PCR. The whole procedure for detecting the virus RNA by RT-PCR could be carried out within 5 h. The RT-PCR with AHSV VP3 gene as a target was found to be a simple, highly specific and sensitive assay for AHSV.

Diagnosis of the African horse sickness virus serotype 4 by a one-tube, one manipulation RT-PCR reaction from infected organs

A single tube reverse transcription — polymerase chain reaction (RT-PCR) method for detection of African horse sickness virus (AHSV) in splenic tissues from infected horses is described. Double stranded RNA was extracted from infected organs of horses and used to produce complementary DNA (cDNA) with the two primers selected for the PCR. The 1179 bp amplified product (the segment 7 which encodes for VP 7), detected by electrophoresis on agarose gel and ethidium bromide staining, was hydrolysed with eight restriction endonucleases for characterization of the AHSV. The sensitivity of this method is discussed. Application of the RT-PCR method should improve detection and shorten the time required to confirm a clinical diagnosis of AHSV infection.

Detection of African horsesickness virus by reverse transcriptase polymerase chain reaction (RT-PCR) using primers for segment 5 (NS1 gene).

The reverse transcription followed by the polymerase chain reaction (RT-PCR) technique was applied to the detection of African horsesickness virus (AHSV) using primers specific for attenuated AHSV serotype 4 segment 5 (NS1 gene). Total RNA which contains both messenger RNA and genomic dsRNA was extracted by the acid guanidinium-phenol-chloroform method from the AHSV infected Vero cells and was used as templates to optimize the RT-PCR. A pair of primer (NP2-NP32) amplified the product of the expected size from all serotypes of attenuated AHSV when four pairs of primers were tested. Using this primer pair, no RT-PCR product was detected from the RNA samples extracted from ten other orbiviruses infected cells and their virions. In addition, RT-PCR using a serial dilution of RNA samples suggested that AHSV was efficiently detected from 1 to 2 cells of the cell monolayer infected with 10(6) TCID50 of AHSV. The RT-PCR concerning with total RNAs of AHSV NS1 gene was found to be a specific and sensitive method for the detection of AHSV.

Neutralizing Epitopes of African Horsesickness Virus Serotype 4 Are Located on VP2

A panel of monoclonal antibodies (MAbs) was generated against African horsesickness virus serotype 4 (AHSV/4). Three of the MAbs (SA6, OH3, and ME11) strongly neutralized the homologous virus and a heterologous type 4 isolate. The MAbs did not cross-neutralize AHS serotypes 1-3 or 5-9. The MAbs immunoprecipitated a viral protein of 108 kDa which co-migrated with VP2. Pretreatment with SA6 prevented mortality of 71% of day-old mice after intracranial injection of 100 LD50 of AHSV/4, while OH3 and ME11 significantly increased the average survival time of challenged animals. This study demonstrates that neutralizing epitope(s) for AHS are located on VP2 and that antibodies to these epitope(s) are protective in a neonatal mouse model.

The complete nucleotide sequence of African horsesickness virus serotype 4 (vaccine strain) segment 4, which encodes the minor core protein VP4

The complete sequence of RNA segment 4 of African horsesickness virus serotype 4 (AHSV-4) vaccine strain was determined from the full-length cDNA clone inserted into pBR322. The RNA is 1978 bp long ( M r 1.27 × 10 6) and contains an open reading frame encoding a protein of 642 amino acids ( M r 75826) with a net charge of +10 at neutral pH. The 5' and 3' termini of AHSV-4 segment 4, 5'GTTTAT… and …CCTTAC3', were different from orbivirus characteristic terminal sequences, being 5'GTTAAA… and …ACTTAC3'. A comparison of the sequence of AHSV-4 segment 4 with that of bluetongue virus (BTV) serotype 10 revealed 55.4% nucleotide similarity and 48.5% amino acid similarity. In addition, Northern blot hybridization showed that the full-length AHSV-4 segment 4 cDNA cross-hybridized well with the corresponding genes of serotype 1, 2, 3, 4 and 7 but slightly with serotype 5, 6 and 8 of attenuated AHSV.

Rapid generation of monoclonal antibody-secreting hybridomas against African horse sickness virus by in vitro immunization and the fusion/cloning technique

Splenocytes from non-immune mice were stimulated in vitro using an equimolar mixture of factors from mixed lymphocyte reaction (MLR) and from phorbol-12-myristate 13-acetate (PMA) stimulated EL-4 cells, and concomitantly immunized with inactivated African horse sickness virus (AHSV) antigen serotype 4 or viral proteins 2 and 5 from AHSV serotype 9. Fusion with NSO myeloma cells was performed five days after primary or secondary stimulation/immunization. The record of hybridoma growth after a standard method of fusion, expansion of cells and subsequent cloning was compared with a fusion/cloning method in which cells were cloned within 2 to 3 days of the fusion event. Detection of antigen specific antibodies in the hybridoma culture supernatants was successful only with cells derived from primary stimulation/immunizations. Antibodies were detected using an indirect ELISA with the immunizing antigen coated on to the surface of the plates. Monoclonal hybridomas were isolated within 2 to 3 weeks using the fusion/cloning method, compared with the standard method, where it took 4 to 5 weeks. Although the total number of clones isolated from the fusion/cloning method was less than that obtained through the standard method, the yield of specific antibody-producing hybridomas as a percentage of the total picked was often more efficient with the fusion/cloning method. With respect to the immunoglobulin isotype produced, not all of the antibodies could be classified by the ELISA system used; 14% of anti-AHSV positive clones were identified as IgG-secreting cells, 25% as IgM-secreting, 18% were cross-reacting with IgG and IgM, and 43% could not be classified. Similar results in all aspects of the work were obtained whether a crude infected cell extract or purified outer capsid polypeptides VP2/5, from serotype 4 and serotype 9 respectively, were used.

Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting.

The structural and non-structural proteins induced by African horsesickness virus serotype 4 (AHSV-4) in infected Vero cells were analysed by SDS-PAGE. Twenty-two virus-induced polypeptides were detected in infected cells by comparison with the polypeptides of mock-infected cells, of which four major (VP2, VP3, VP5 and VP7) and three minor (VP1, VP4 and VP6) structural proteins and four non-structural proteins (P58, P48, P21 and P20) were shown to be virus-coded, as deduced from electrophoretic and antigenic studies of purified virions and infected cells. The proteins that elicit the major antibody responses both in vaccinated and naturally or experimentally infected horses were shown to be three structural proteins, VP2, VP5 and VP7, and the four major non-structural proteins, P58, P48, P21 and P20, as deduced by radioimmunoprecipitation and immunoblotting assays. The cross-reactivity between AHSV-4 and sera obtained from horses experimentally infected with seven other serotypes was also determined. The results showed that VP5, VP7, P48, P21 and P20 are conserved and can be used to diagnose the infection of any of these eight serotypes.

Group-Reactive ELISAs for Detecting Antibodies to African Horsesickness and Equine Encephalosis Viruses in Horse, Donkey, and Zebra Sera

Group-reactive enzyme-linked immunosorbent assays (ELISAs) were developed to selectively detect antibodies to African horsesickness virus (AHSV) and equine encephalosis virus (EEV), 2 orbiviruses that infect equids. In indirect ELISA, guinea pig antisera to all known AHSV or EEV serotypes recognized immobilized AHSV serotype 3 or EEV Cascara, respectively. Antisera from naturally infected animals did not cross-react with their respective heterologous viruses. The ELISA was used in parallel with the complement fixation (CF) and agar gel immunodiffusion tests to detect antibodies in sera from animals in the field. The ELISA distinguished among those that contained antibodies to AHSV, EEV, or both viruses and was useful with sera that did not yield results in CF tests because of anticomplementary activity. Zebra and donkeys, both potential subclinical carrier animals in Africa, contained AHSV or EEV antibodies. Some sera reacted with 1 of the 2 orbiviruses, whereas others reacted with both. The ELISA can be used in projected epidemiological studies in which many serum samples must be assayed.

Diagnostic methods for African horsesickness virus using monoclonal antibodies to structural and non-structural proteins

A panel of 32 hybridoma cell lines secreting monoclonal antibodies (MAbs) reactive with African horsesickness virus serotype 4 (AHSV-4) has been developed. Four of the MAbs recognized the major core antigen VP7, twenty recognized the outer capsid protein VP2 and eight reacted with the non-structural protein NS1. With the VP7-specific MAbs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected Vero cells and in spleen tissue from AHSV-infected horses. The sensitivity of the assay is 10 ng viral antigen per 100 μl. The NS1-specific MAbs allowed visualization by immunofluorescence of tubulc-like structures in the cytoplasm of infected Vero cells. This can be very useful as a confirmatory diagnostic procedure. The antigenic map of the outer capsid VP2 protein with MAbs is also reported.

Detection of African horsesickness virus in infected spleens by a sandwich ELISA using two monoclonal antibodies specific for VP7

A sandwich enzyme-linked immunosorbent assay (ELISA) for rapid detection of African horsesickness virus (AHSV) in infected spleens or cell culture supernatant was developed. This method uses two monoclonal antibodies (MAbs) which recognize two non-overlapping epitopes of the major core protein (VP7) to coat the solid phase, and one labeled with biotin as second antibody. This ELISA was evaluated for its ability to detect AHSV in infected spleens resulting in a sensitivity of 97.4% and a specificity of 100% compared with virus isolation in cell culture, and can be used for the detection of the nine different AHSV serotypes.