Engineering infectious foot-and-mouth disease virus in vivo from a full-length genomic cDNA clone of the A/AKT/58 strain

• Published in: Science China. Life sciences Vol. 52; no. 2; pp. 155 - 162
• Main Authors: Bai, XingWen, Li, PingHua, Cao, YiMei, Li, Dong, Lu, ZengJun, Guo, JianHong, Sun, DeHui, Zheng, HaiXue, Sun, Pu, Liu, XiangTao, Luo, JianXun, Liu, ZaiXin
• Format: Journal Article
• Language: English
• Published: Beijing Science China Press 01-02-2009; Springer Nature B.V
• Subjects: A/AKT/58; AKT protein; Animals; Antigenicity; Base Sequence; Biomedical and Life Sciences; cDNA; Cell Line; clones; Cricetinae; disease; DNA Primers; DNA, Complementary - genetics; DNA-directed RNA polymerase; Fluorescent Antibody Technique, Indirect; Foot & mouth disease; foot-and-mouth; Foot-and-mouth disease virus; Foot-and-Mouth Disease Virus - genetics; Foot-and-Mouth Disease Virus - pathogenicity; Genetic Engineering; Genomics; In Memoriam: Professor Ray Wu; infectious; Life Sciences; Mice; Microscopy, Electron; Pathogenicity; Reverse Transcriptase Polymerase Chain Reaction; RNA polymerase; RNA viruses; strain; Suckling behavior; transcription; Transfection; Virulence; virus; Viruses; vivo; foot-and-mouth disease virus; A/AKT/58 strain; transcription; infectious cDNA clones
• ISSN: 1674-7305; 1006-9305; 1869-1889; 1862-2798
• DOI: 10.1007/s11427-009-0007-6

Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells. The in vivo rescued viruses showed high pathogenicity for 3-day-old unweaned mice (LD50=10?7.5). However, the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicity for suckling-mice (LD50=10?6), and the in vivo transcribed RNA recovered from pCA/FMDV co-transfected with pCT7RNAP showed no significant differences from the wild type virus. These data showed that recovery of the infectious foot-and-mouth disease virus directly from the use of in vivo techniques was better than from in vitro methods. Furthermore, the reverse genetic procedure technique was simplified to a faster one-step procedure based on co-transfection with pCT7RNAP. These results suggest that in vivo RNA tran- scripts may be more valuable for engineering recombinant foot-and-mouth disease virus than in vitro RNA transcripts, and may contribute to further understanding of the biological properties, such as replication, maturation and quasispecies, of the foot-and-mouth disease virus.