title: Megaprimer-mediated capsid swapping for the construction of custom-engineered chimeric foot-and-mouth disease virus.
abstract: Foot-and-mouth disease (FMD) is a highly contagious, economically important disease of transboundary importance. Regular vaccination with chemically inactivated FMD vaccine is the major means of controlling the disease in endemic countries like India. However, the selection of appropriate candidate vaccine strain and its adaptation in cell culture to yield high titer of virus is a cumbersome process. An attractive approach to circumvent this tedious process is to replace the capsid coding sequence of an infectious full-genome length cDNA clone of a good vaccine strain with those of appropriate field strain, to produce custom-made chimeric FMD virus (FMDV). Nevertheless, the construction of chimeric virus can be difficult if the necessary endonuclease restriction sites are unavailable or unsuitable for swapping of the capsid sequence. Here we described an efficient method based on megaprimer-mediated capsid swapping for the construction of chimeric FMDV cDNA clones. Using FMDV vaccine strain A IND 40/2000 infectious clone (pA(40/2000)) as a donor plasmid, we exchanged the capsid sequence of pA(40/2000) with that of the viruses belonging to serotypes O (n = 5), A (n = 2), and Asia 1 (n = 2), and subsequently generated infectious FMDV from their respective chimeric cDNA clones. The chimeric viruses exhibited comparable infection kinetics, plaque phenotypes, antigenic profiles, and virion stability to the parental viruses. The results from this study suggest that megaprimer-based reverse genetics technology is useful for engineering chimeric vaccine strains for use in the control and prevention of FMD in endemic countries.
authors: ICAR-Project Directorate on Foot-and-mouth Disease, Mukteswar, Nainital, 263138, Uttarakhand, India. jkubiswal@gmail.com.
authors: Jitendra K
authors: JK
authors: Biswal
authors: ICAR-Project Directorate on Foot-and-mouth Disease, Mukteswar, Nainital, 263138, Uttarakhand, India.
authors: Saravanan
authors: S
authors: Subramaniam
authors: ICAR-Project Directorate on Foot-and-mouth Disease, Mukteswar, Nainital, 263138, Uttarakhand, India.
authors: Gaurav K
authors: GK
authors: Sharma
authors: ICAR-Project Directorate on Foot-and-mouth Disease, Mukteswar, Nainital, 263138, Uttarakhand, India.
authors: Sonalika
authors: S
authors: Mahajan
authors: ICAR-Project Directorate on Foot-and-mouth Disease, Mukteswar, Nainital, 263138, Uttarakhand, India.
authors: Rajeev
authors: R
authors: Ranjan
authors: Indian Council of Agricultural Research, Krishi Bhavan, New Delhi, 110 001, India.
authors: Jyoti
authors: J
authors: Misri
authors: ICAR-Project Directorate on Foot-and-mouth Disease, Mukteswar, Nainital, 263138, Uttarakhand, India. pattnaikb@gmail.com.
authors: Bramhadev
authors: B
authors: Pattnaik
mesh_terms: D036022:Capsid Proteins / Q000235:genetics*; D017931:DNA Primers; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics*; D050296:Microbial Viability; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids; D011995:Recombination, Genetic; D014773:Virology / Q000379:methods*
chemical_list: D036022:Capsid Proteins; D017931:DNA Primers
keywords: Capsid Swapping; Chimeric FMDV; FMD virus; Megaprimer-PCR