Based on the reverse genetic manipulation,7chimeric full-length cDNA plasmids of FMDVcontaining site-directed mutations in the major capsid protein VP1were constructed by usingoverlapping-PCR methods and exchange-cassette strategy. Five rescued FMDV mutants were generatedand designated as rNX83(K83E in VP1protein of O/NX/CHA/99strain), rNX139(R139S in VP1protein of O/NX/CHA/99strain), rNX8&9(K83E and R139S in VP1protein of O/NX/CHA/99strain),rZheJ139(S139R in VP1protein of O/ZheJ/CHA/8/2008strain) and rHK83(E83K in VP1protein ofO/HK/HB/CHA/99strain), respectively. To identify the molecular determinant(s) in VP1thatcontributes to the HS-binding ability of these viruses, plaque assays of these chimeric FMDV mutantswere proformed in BHK-21cells and CHO-K1cells. The experiments that rNX83and rNX8&9passaged8times in BHK-21cells and in suckling mice were performed in order to detect the geneticstability of chimeric and mutant FMDVs. The results showed:(1) rNX83, rZheJ139, rNX8&9andrHK83viruses formed large plaques in BHK-21cells and no plaques produced in CHO-K1cells. TherNX139virus formed large and small blend plaques in BHK-21cells and produced plaques in CHO-K1cells. Plaques formation of rNX139virus in BHK-21cells could be partly inhibited by addition ofheparin sodium and peptides in certain concentration. However, the plaques formation of rNX83,rZheJ139, rNX8&9and rHK83viruses in BHK-21cells could not be inhibited by heparin sodium andcould be completely inhibited by RGD-containing peptides.(2) Reverse mutations appeared betweenthe third generation and the fourth generation when rNX83and rNX8&9viruses passaged in BHK-21cells. But this phenomenon did not appear in suckling mice. Revertant events reappear when the8thgeneration viruses in suckling mice inoculated in BHK-21cells.In conclusion, our experiment confirmed the facts as follows:(1) O/NX/CHA/99strain can infectcells via HS-mediated adsorption by utilizing the key amino acid residue K83in VP1protein. Aminoacid residue K83in VP1protein of O/NX/CHA/99strain was correlated with plaque phenotypes inBHK-21cells.(2) Chimeric FMDVs of O/NX/CHA/99strain with mutations (K83E, K83E&R139S) inVP1protein can revert to the wide type in BHK-21cells gradually and acquire the ability of formingplaques in CHO-K1cells. Whereas, the genetic stability of K83E mutation is shown in suckling mice.Chimeric and mutant FMDVs (K83E, K83E&R139S) can not perform plaques in CHO-K1cells. |