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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.

The mortality rate in susceptible populations of horses during an epizootic of African horse sickness (AHS) may be in excess of 90%. Rapid and reliable assays are therefore essential for the confirmation of clinical diagnoses and to enable control strategies to be implemented without undue delay. One of the major objectives of a recent European Union funded project was the validation of newly developed diagnostic assays which are rapid, sensitive, highly reproducible and inexpensive, for the detection of African horse sickness virus (AHSV) antigens and antibodies. The Laboratorio de Sanidad y Produccion Animal (LSPA) in Algete, Spain was charged with the responsibility of co-ordinating and supplying samples of viruses and antisera to the participating laboratories in Spain, France and the United Kingdom. The panels comprised 76 antigen samples for assay by indirect sandwich ELISAs and 53 serum samples for antibody detection by either indirect or competitive ELISAs. Results generated by ELISA for each laboratory were analysed in LSPA in terms of their relative sensitivities and specificities. There was a good agreement between the ELISAs used for either antigen or antibody detection. The participating groups agreed that any field sample giving a doubtful result would always be retested by ELISA and an alternative assay.

An established mouse model system was used to evaluate the effectiveness of the major outer core protein VP7 of African horse sickness virus (AHSV) serotype 9 as a subunit vaccine. Balb C mice were immunised with VP7 crystals purified from AHSV infected BHK cells. In groups of mice, each of which was immunised with > or = 1.5 micrograms of the protein in Freund's adjuvant, > or = 80% of mice survived challenge with a virulent strain of a heterologous AHSV serotype (AHSV 7), that killed > or = 80% of the mice in the uninoculated control groups. This level of protection was significantly greater than that observed in mice inoculated with equivalent amounts of either denatured VP7 (50% survival), or GST/VP7 fusion protein (50-70% survival), or which were vaccinated with AHSV 9 (40-50% survival). The VP7 protein folding, or its assembly into crystals, are thought to play some role in the effectiveness of the protective response observed. Titres of circulating antibodies against AHSV VP7 were determined by competitive ELISA but did not appear to correlate with the levels of protection observed. Passive transfer of these antibodies to syngeneic recipients also failed to protect Balb C mice from the AHSV 7 challenge. The observed protection is therefore unlikely to be due to an antibody mediated immune response.

African horse sickness virus (AHSV) antigen was demonstrated immunohistochemically in formalin-fixed, paraffin-embedded sections of tissues collected from three ponies suffering from the peracute form of the disease and from one pony affected by the fever form. The pattern of the antigen distribution indicated a particular organ tropism characterised by an accumulation of AHSV antigen in cardio-pulmonary tissues of the animals with the peracute disease and in the spleen of the pony with the fever form. AHSV antigen was identified in endothelial cells of small blood vessels, particularly capillaries and in large mono-nuclear cells resembling macrophages or reticular cells of lymphatic tissues. Occasional circulating mononuclear cells with the morphology of monocytes were also positively stained within the larger vessels. The immunohistochemical results confirm earlier work suggesting that AHSV may have different tropisms to particular organs during various forms of the disease and that different target cell populations exist in vivo. Immunohistochemistry may be an additional useful method for diagnostic and research purposes in AHS.

The distribution of viral antigen was studied in various tissues of three ponies, aged 3-4 years, infected experimentally with a virulent strain of African horse sickness virus (AHSV) serotype 4. Tissues were collected from the animals in the terminal stage of the peracute form of the disease and from one noninfected horse, included as a control. A polyclonal antibody with specificity for AHSV, plus the nonstructural protein NS2, was used in a sensitive avidin-biotin-peroxidase-complex (ABC) method performed on formalin-fixed, paraffin-embedded tissue sections. AHSV antigen was located primarily in endothelial cells of capillaries and small venous and arteriolar vessels, particularly of cardiopulmonary tissues. Viral antigen was also identified in cells resembling macrophages and in reticular cells of spleen and lymph nodes. The pattern of viral antigen labeling in some lymph nodes along the mantle zone of lymphoid follicles was compatible with the morphology of cellular processes of follicular dendritic cells. In some tissues, viral antigen was detected occasionally in circulating cells, probably monocytes, within the larger vessels. These findings suggest that endothelial cells, and to a lesser extent mononuclear cells, are the main target cells of AHSV infection during the late stage of the peracute form of the disease.

An established mouse model was used to evaluate the effectiveness of the major outer core protein of African horsesickness virus (AHSV), VP7, as a subunit vaccine. Adult female BALB/c mice were immunized with VP7 crystals purified from BHK cells infected with AHSV serotype 9 (AHSV-9), using three inoculations in Freund's adjuvant. Eighty to one hundred per cent of the immunized mice were protected against a heterologous challenge with a known lethal dose of AHSV-7. The protected immunized mice did not develop any clinical signs characteristic of virulent AHSV infection in this model during the study. In contrast, 80-100% mortality was observed in the non-immunized mice that received the same challenge virus. Subsequent studies indicated that a single inoculation of 1.5 micrograms purified AHSV VP7 in Freund's complete adjuvant was sufficient to protect at least 90% of mice from AHSV-7 challenge. If the antigen was presented in the absence of Freund's complete adjuvant, 70% of the mice were still protected by one inoculation of VP7 crystals. Titres of circulating antibody against AHSV VP7, determined by competitive ELISA, did not appear to correlate with protection and passive antibody transfer from immunized BALB/c mice failed to protect syngeneic recipients from AHSV-7 challenge. Therefore, the observed protection is unlikely to be due to an antibody-mediated immune response. The number of viraemic mice and the duration of viraemia post-challenge was significantly reduced in vaccinated mice compared to non-vaccinated controls. However, the levels of viraemia were similar.

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.

A full-length cDNA clone of genome segment 7 of African Horse Sickness Virus, serotype 9 (AHSV9) was obtained using the PCR technique. The clone was sequenced and found to be 98.27% homologous to the previously published sequence of the equivalent cDNA clone from AHSV4 at the nucleotide level and to exhibit 99.7% identity at the amino acid level. The cDNA clone was transferred to pGEX-2T (Pharmacia), a bacterial expression vector, such that the reading frame of AHSV9 VP7 was continuous with that of the bacterial glutathione-S-transferase (GST) protein, under the control of the bacterial tac promoter. On induction with IPTG a fusion protein consisting of GST and VP7 was synthesised, which was readily purified on a GST-sepharose column (Pharmacia). The fusion protein reacted equally well in an indirect ELISA using monoclonal antibodies specific for AHSV9 VP7 or polyclonal guinea pig antisera raised against AHSV9 infectious sub-viral particles. This protein was also shown to be a suitable substitute for virus antigen, prepared from infected BHK cell extracts, in a competitive ELISA. Antibodies titres recorded for AHSV9 positive and negative horse sera were similar in the competitive ELISA using either bacterial AHSV VP7 or BHK extracted virus as the source of antigen, in combination with monoclonal or polyclonal antibodies, respectively, as the detectors.

Four ponies were each inoculated with a different serotype of African horse sickness virus (AHSV) which had been passaged through cell culture in order to achieve attenuation. Three of the ponies died suddenly after showing mild clinical signs, the fourth pony remained clinically normal and was killed at day 38. Infectious AHSV was isolated from blood samples collected at intervals from all four ponies. Positive antigen ELISA reactions were only observed with blood samples from two of the ponies on the two days preceding death. Specific AHSV antibodies were detected by ELISA in serum samples from the other two ponies although one eventually died. African horse sickness viral antigens were detected by ELISA in post-mortem tissue samples collected from all four ponies. No infectious virus could be detected in tissue samples taken post-mortem from the pony which survived African horse sickness (AHS) infection. In the event of a suspected outbreak of AHS it is recommended that sera and heparinized blood should be tested for specific antibodies and AHSV antigen respectively. When available, post-mortem tissues, including spleen, heart, lung and liver, should also be tested for AHSV antigen. Although the ELISA used for the detection of AHSV antigen is highly sensitive and specific, negative ELISA results should be confirmed by virus isolation attempts.

Serially collected epithelial samples from lesions in the mouth and on the feet of calves experimentally infected with foot-and-mouth disease (FMD) type O1 BFS 1860 were assayed for the presence of FMD viral antigen using a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and a complement fixation (CF) test. The amount of infectious virus in each sample was also determined. FMD viral antigen was detected by ELISA in 70 per cent of the mouth samples and 92 per cent of samples from the feet. The CF test was less sensitive; it detected antigen in 44 per cent of mouth and 85 per cent of foot samples. In mouth samples the amount of antigen decreased rapidly becoming undetectable by the fourth day of sampling whereas in foot samples the quantity of antigen declined more slowly, and could be detected until the seventh day of sampling. Therefore it was concluded that the age of lesion and the site from which epithelial samples are collected are both important determinants in the laboratory diagnosis of FMD. In cattle, foot lesions are more likely than mouth lesions to yield antigen and to remain positive for a longer period.

A rapid double sandwich enzyme-linked immunosorbent assay (ELISA) has been used for the identification and type differentiation of foot-and-mouth disease (FMD) viruses in epithelial tissue samples submitted for diagnosis from the field. No difficulty was experienced in the direct typing of freshly harvested epithelium from recently ruptured vesicles by the complement fixation (CF) test or ELISA. The ELISA was more sensitive and specific, but proved no more efficient than the traditional CF test in the direct typing of samples of poorer quality from many countries overseas where communications are often difficult. However, when both tests were used concurrently, FMD virus typings were confirmed in 27 more samples. Some possible reasons for the failure of ELISA to detect virus in certain cases are discussed.