Symmetrical arrangement of positively charged residues around the 5-fold axes of SAT type foot-and-mouth disease virus enhances cell culture of field viruses.

Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83-85 in the ßD-ßE loop and 110-112 in the ßF-ßG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110-112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions.

Synthesis of empty african horse sickness virus particles.

As a means to develop African horse sickness (AHS) vaccines that are safe and DIVA compliant, we investigated the synthesis of empty African horse sickness virus (AHSV) particles. The emphasis of this study was on the assembly of the major viral core (VP3 and VP7) and outer capsid proteins (VP2 and VP5) into architecturally complex, heteromultimeric nanosized particles. The production of fully assembled core-like particles (CLPs) was accomplished in vivo by baculovirus-mediated co-synthesis of VP3 and VP7. The two different outer capsid proteins were capable of associating independently of each other with preformed cores to yield partial virus-like particles (VLPs). Complete VLPs were synthesized, albeit with a low yield. Crystalline formation of AHSV VP7 trimers is thought to impede high-level CLP production. Consequently, we engineered and co-synthesized VP3 with a more hydrophilic mutant VP7, resulting in an increase in the turnover of CLPs.

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.