The use of African horse sickness virus NS3 protein, expressed in bacteria, as a marker to differentiate infected from vaccinated horses.

Segment 10 of the double-stranded RNA (dsRNA) genome from African horse sickness virus serotype 4 (AHSV-4) was cloned and sequenced. The sequence of the coding region showed a total length of 667 bp. Nucleotide comparisons showed a 95% sequence similarity between serotypes 4 and 9, and 76% between serotypes 4 and 3. cDNA clones containing the coding region were cloned in the vector pET3xb and expressed in Escherichia coli. The NS3 gene product was synthesised at very high level as an insoluble fusion protein. The recombinant protein was used in a differential ELISA to distinguish horses that were infected with AHSV-4 or vaccinated with live-modified virus from those vaccinated with a purified inactivated vaccine. The results obtained indicate that recombinant NS3 can indeed differentiate between infected and vaccinated animals implying that this recombinant could be developed as a diagnostic reagent, and it would allow the mobility of vaccinated horses. Thus, economical losses associated with this disease could be avoided.

Expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed.

The peplomer protein (S) and the transmembrane protein (M) of transmissible gastroenteritis virus (TGEV) of swine were identified by iodination and serologically on the surface of infected cells. Of a total of 4 monoclonal antibodies (mAb) directed against four antigenic sites of S protein (Correa et al., 1988), 3 specific for sites A, B and D attached to the plasma membrane of infected cells, as disclosed by indirect immunofluorescence and by complement-mediated cytolysis. Four of the mAbs assayed were specific for the viral protein M and two of them gave plasma membrane immunofluorescence and mediated cytolysis in the presence of complement. The viral nucleoprotein N could not be demonstrated on the surface of infected cells either by iodination or employing 3 mAbs against this protein. Finally, a time course infection experiment demonstrated that S and M proteins were expressed on the surface of infected cells at 4 h after infection, before infective virus was released from infected cells.

Serological and virological responses in mules and donkeys following inoculation with African horse sickness virus serotype 4.

Two groups, comprising 4 donkeys and 4 mules (group 1) and 4 donkeys and 3 mules (group 2), were used to determine the duration of viraemia and to monitor the development of antibodies following inoculation with African horse sickness virus (AHSV). One group of animals was given a single dose of attenuated AHSV serotype 4 (AHSV 4) vaccine. The second group was inoculated with a virulent field strain of AHSV 4. Both groups were subsequently challenged with the virulent field strain of AHSV 4, 51 and 58 days, respectively, after their primary inoculation. Blood and serum samples, collected on alternate days after the primary inoculations and also after subsequent challenge, were assayed for virus and antibodies. Seven of the 8 AHSV vaccinated (group 1) and 7 of the 7 AHSV inoculated (group 2) animals showed humoral antibody responses after primary inoculation. Although no infectious virus could be isolated from either group for the duration of the study, reverse transcription-PCR data obtained for the second group did show the presence of AHSV viral RNA from as early as day 5 in mules and day 9 in donkeys after the primary inoculation. Viral RNA was detected consistently up to day 47 in some animals and intermittently thereafter. There was no evidence of a second viraemia in any of the animals after challenge. The detection of specific antibodies, against AHSV 4 NS3 protein, in all animals confirmed that both donkeys and mules were infected and that the virus had replicated.

Validation of ELISA for the detection of African horse sickness virus antigens and antibodies.

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.

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

A sandwich enzyme-linked immunsorbent 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.

Pathogenesis of African horse sickness: ultrastructural study of the capillaries in experimental infection.

African horse sickness (AHS) was induced in five horses by inoculation, to determine the ultrastructural changes in endothelial cells of capillaries in the myocardium, lung, spleen and liver. The animals developed cardiac and mixed forms of the disease. Alterations detected in the endothelial cells of the vessels of infected animals included: the presence of structures associated with viral infection, hypertrophy, degenerative changes, appearance of cytoplasmic projections, changes in permeability, alteration of intercellular junctions, loss of endothelium, subendothelial deposition of cell debris and fibrin, and vascular repair. In association with these changes, oedema, haemorrhages and microthromboses were detected, particularly in the myocardium and lung. This study showed that infection of, and changes to, the capillary endothelial cells of the organs under study was independent of the form in which the disease manifested itself but was dependent on the organ and blood vessel type. Thus, different levels of viral tropism were observed for the endothelial cells of the vessels in different organs. Viral infection was commonest in the endothelial cells of myocardial vessels, followed by those in the lung, whereas in the spleen and liver, endothelial cell infection was rare and, in the case of the liver, limited to the interstitial capillaries.

The role of pulmonary intravascular macrophages in the pathogenesis of African horse sickness.

African horse sickness (AHS) is a disease of equids, characterized by severe pulmonary oedema and caused by an orbivirus. To determine the role of pulmonary intravascular macrophages (PIMs) in the development of pulmonary microvascular changes in this disease, five horses were given an intravenous inoculation of 10(6)TCID50of serotype 4 of AHS virus. Viral replication was detected in endothelial cells, PIMs, interstitial macrophages and fibroblasts. Alveolar and interstitial oedema, and changes in pulmonary microvasculature, consisting mainly of the sequestration of neutrophils and the formation of platelet aggregates and fibrinous microthrombi, were related to endothelial changes and to a high degree of PIM activation. This suggested that the PIMs, once activated, contributed to these vascular changes by releasing chemical inflammatory mediators.

Detection of African swine fever virus antibodies by immunoblotting assay.

An immunoblotting assay has been adapted to detect antibodies against African swine fever virus. The electrophoretic transfer of proteins and the immunoreaction conditions were optimized, using 4 mA/cm2 of current intensity and 10 micrograms of soluble cytoplasmic antigen of infected cells per strip. Filters of polyvinylidene difluoride showed the highest capacity for protein absorption, but nitrocellulose filters showed lower backgrounds. The specificity and the pattern of the proteins induced by African swine fever virus that react with the antisera were determined in immunoblotting assay, IP30 being the most reactive protein.

Proteins specified by swine transmissible gastroenteritis virus: identification of non-structural proteins by two-dimensional electrophoresis.

Four virus-induced non-structural proteins with apparent molecular weights of 11-14 kilodaltons (kDa) were identified by two-dimensional electrophoresis in cells infected by TGEV. Differences in the number of non-structural proteins were observed among virulent and attenuated TGEV strains as well as with two antigenically related feline and canine coronaviruses.

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.