Creation of leaderless FMDV replicon for development of replication defective virus (leaderless FMDV): A strategy towards the development of attenuated vaccine with marker facility

Leader protease (L-pro) of foot-and-mouth disease virus (FMDV) which is essential for viral replication and pathogenicity in host is found in two forms, Lab and Lb with similar activity but, both differing only at amino-termini with separate initiation codons, 84 nucleotides apart. After translation of the genomic RNA into single polyprotein, the L protease is released autocatalytic from the N terminus and cleaves the p220 subunit of the eukaryotic initiation factor 4F complex resulting in the shut off host protein synthesis, an essential step for viral pathogenicity. We exploited this function for development of attenuated virus by deleting the gene encoding Lb protease from FMDV Asia 1(63/72) cDNA replicon (Joshi et al, 2013) by PCR mutagenesis approach. The deletions and intactness of the frames were confirmed by sequence analysis. P1-2A polyprotein gene of FMDV 'O' was inserted into the replicon and the full length construct was studied for virulence to baby hamster kidney (BHK) 21 cells. Vaccinia expressing T7 polymerase was used for in vitro RNA generation and infection. The lower cytopathic effect (cpe) as observed by reduced replication efficiency confirmed the effect of Lb deletion when compared with the construct with no deletion.