Modification of PCV-2 virulence by substitution of the genogroup motif of the capsid protein

Allemandou, Aude; Grasland, Béatrice; Hernandez-Nignol, Anne-Cécile; Kéranflec'h, André; Cariolet, Roland; Jestin, André

Eighty-percent confluent PK15 cells, seeded in six-well plates, were transfected with 1 μg of each clone DNA using Lipofectamine LTX reagent (Invitrogen) according to the manufacturer's instructions. Twenty-four hours post-transfection, supernatants were harvested and stored at -80°C until virus titration. Cells were fixed with 80% ice-cold acetone for 10 min at -20°C and stored for the immunoperoxydase monolayer assay (IPMA) at -20°C. Cells expressing the capsid of each parental clone and of each mutant clone were revealed by IPMA with polyclonal anti-PCV-2b antibody using a previously described protocol [21] . Infectivity of both parental and mutant clones was assessed by infecting fresh PK15 cells with the transfection supernatants. Production of infectious virus was assessed by IPMA, which was specifically used to detect the viral capsid.