ENGINEERING FOOT-AND-MOUTH DISEASE VIRUS RNA TO ALLOW BINDING TO HEPARAN SULFATE

The picornavirus foot-and-mouth disease virus (FMDV) is the cause of an economically important and highly contagious disease that primarily affects livestock. This makes research contributing to vaccine development of FMDV important. Current vaccines are not capable of producing long term immunity and require many vaccination doses throughout the life of the animal. Dendritic cells are a vital component of the immune system for the development of sustained immunity against a virus because of their role in internalization and presentation of viral antigens. FMDV enters epithelial cells through integrin receptors at the cell surface. Cell culture adapted strains of FMDV can acquire the ability to enter cells via binding to heparan sulfate glycosaminoglycan (HS), and internalization by dendritic cells of HS- binding FMDVs has been shown to be more efficient than that of non-heparan sulfate binding virus. In serotype O, HS binding associates with the addition of positively charged amino acids on the surface of the viral capsid. My host lab is interested in constructing a serotype C FMDV with the ability to bind HS. To this end, I worked in the introduction of mutations VP3 E173K and VP3 Q218K into the infectious FMDV clone plasmid pMT28 by site-directed mutagenesis. Plasmid pGem E173K, which contains a fragment (nucleotides 2826-3760) of pMT28 with mutation VP3 E173K, was used as a template for site-directed mutagenesis using the PCR mediated kit NZYMutagenesis kit (nyztech, Portugal) to add the mutation Q218K (single nucleotide substitution C3202A). This generated the plasmid pGem E173K Q218K. Both pGem E173K Q218K and pMT28 E173K (WT) were digested with the restriction enzymes AvrII and SfiI to produce the FMDV DNA fragment containing the mutations VP3 E173K, VP3 Q218K, and also the linearized plasmid pMT28. Following cloning of the fragment containing the mutations into the infectious clone pMT28, mutant infectious FMDV clone pMT28 E173K Q218K was produced. Mutant FMDV viral RNA was in-vitro transcribed from pMT28, which is permitting to recover virus in the BLS3 facilities at CISA-INIA to assess whether these two positively charged amino acid substitutions are sufficient to allow HS binding, using a soluble-HS binding assay. The development of type C C-S8c1 FMDV with the ability to bind HS can contribute to a better understanding of the virus-DCs interactions, and to the development of improved vaccines for livestock.