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

Abstract

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 βD-βE loop and 110–112 in the βF-βG 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. Foot-and-mouth disease (FMD) is a major threat for world economies. Local farmers in endemic areas relying on livestock for their livelihoods are affected by FMD outbreaks. Improved vaccines are essential for effective control and should be produced based on currently circulating field viruses. Current vaccines are produced by chemical inactivation of virus grown in large-scale production cultures of BHK-21 cells. However, not all field viruses can be adapted to cell cultures. The significance of our research is in identifying amino acid residues responsible for enhancing cell culture adaptation of FMD field viruses. This study engineered chimeric viruses containing the antigenic region of a field strain and novel cell culture receptor binding sites. This facilitated rapid virus amplification within a few passages in BHK-21 cells to create master virus seed stocks, circumventing the need for isolation on primary cell lines prior to further adaptation.