Engineering infectious foot-and-mouth disease virus in vivo from a full-length genomic cDNA clone of the A/AKT/58 strain.
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 transcripts 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.
Bai
XingWen
X
Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China. baixingwen@163.com
Li
PingHua
P
Cao
YiMei
Y
Li
Dong
D
Lu
ZengJun
Z
Guo
JianHong
J
Sun
DeHui
D
Zheng
HaiXue
H
Sun
Pu
P
Liu
XiangTao
X
Luo
JianXun
J
Liu
ZaiXin
Z
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics* / Q000472:pathogenicity; D005818:Genetic Engineering; D051379:Mice; D008854:Microscopy, Electron; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014774:Virulence
D017931:DNA Primers; D018076:DNA, Complementary