Antigenic properties and virulence of foot-and-mouth disease virus rescued from full-length cDNA clone of serotype O, typical vaccine strain

Abstract

We cloned the full-length cDNA of O Manisa, the virus for vaccinating against foot-and-mouth disease. The antigenic properties of the virus recovered from the cDNA were similar to those of the parental virus. Pathogenesis did not appear in the pigs, dairy goats or suckling mice, but neutralizing antibodies were raised 5-6 days after the virus challenge. The utilization of O Manisa as a safe vaccine strain will increase if recombinant viruses can be manipulated by inserting or removing a marker gene for differential serology or replacing the protective gene from another serotype.

Keywords: Foot-and-mouth disease; Molecular cloning; Vaccine.

Fig. 1

Recovery of virus from FMDV full-length cDNA clone, and antibody response, pathogenesis in animals after infection of the virus. (A) Recovery of virus from full-length cDNA clone (pO-Manisa-FG) from foot-and-mouth disease virus. The small fragment (1-369 nt), including poly(C) of O Manisa, were cloned into pBluescript II KS (+) vector. The large (382-8,206 nt) were subsequently inserted into plasmid. The redundant restriction enzyme sites were removed (B). The LF-BK cells were overlaid with 2 mL of 1.5% SeaPlaque agarose containing 2% fetal bovine serum in Dulbecco's modified Eagle's medium and cultured at 37℃ in 5% CO₂ for three days. The plaques were visualized by staining with a 0.1% crystal violet solution. The recovered virus had a plaque morphology similar to that of the parental virus. For electron microscopy of O Manisa virus, virion purification was performed by sucrose gradient centrifugation. The viruses were concentrated using Amicon ultracentrifuge filters (100 kDa). Samples were observed using electron microscope. The viruses showed size of 28 nm. (C) Growth properties of FMDV, O Manisa-parental virus and O Manisa-FG recovered from pO-Manisa-FG clone. One-step growth kinetics in BHK21 (up-left) or LF-BK cells (up-right). Virus titration by tissue culture (down-left) and quantitative reverse transcription polymerase chain reaction (down-right). There was no significant difference in growth characterization between transfectant virus and the parental virus. (D) VN antibody level after challenge of O Manisa-FG in the pigs. Pigs were challenged by different administration routes (ID injection on the foot-pad with 0.1 mL, and IM injection 1 mL, with 10^5.0 TCID₅₀/0.1 mL). The animals did not show clinical signs. The viruses for VN test were O Manisa-parental virus (left) and O/Andong/SKR/2010 strain of SEA topotype (right). (E) VN antibody level after challenge of O Manisa-FG in the dairy goats. Dairy goats were challenged by ID route of the same method as pig injection and the viruses for VN test were O Manisa-parental virus (left) and O/Andong/SKR/2010 (right). (F) Survival in seven-day-old mice after challenge of recovered, parental virus and O/SKR/2002 of 10^5.0 TCID₅₀/0.1 mL by intraperitoneal route. FMDV, foot-and-mouth disease virus; VN, virus neutralizing; ID, intradermal; IM, intramuscular; SEA, South East Asian.