Development of a hamster kidney cell line expressing stably T7 RNA polymerase using retroviral gene transfer technology for efficient rescue of infectious foot-and-mouth disease virus.
Reverse genetics systems, with the ability to manipulate viral genomes at the DNA molecular level, are an important platform for study of the assembly and function of viruses. Genome manipulation, such as gene recombination, mosaicism, and mutation may interfere with replication, assembly and release of viruses. An efficient, convenient and economical method of virus rescue is undoubtedly required for increasing the efficiency of rescuing recombinant viruses. To develop an efficient, helper virus-free viral recovery system (reverse genetics), a retroviral gene transfer technology was used to establish a stable BHK-21 cell line (designated as BHKT7) which expressed constitutively bacteriophage T7 RNA polymerase (T7 RNAP). An improved method for rescue of infectious foot-and-mouth disease virus (FMDV) was then developed. FMDV full-length cDNA under control of a T7 promotor, was transfected into BHKT7 of differing passages. FMDV virus was rescued efficiently from the BHKT7 cells, the passage number not having an effect on the efficiency of recovery. As a result, the cell line was stable even after multiple passages, expressing sufficient T7 RNAP to support ex vivo transcription and efficient rescue. The reverse genetics system described below is efficient, stable, and convenient. The system could provide not only the basis of gene function research into FMDV, but could also be used for reverse genetics research into other positive-strand RNA viruses, without the need for helper viruses.
Zheng
Haixue
H
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Tian
Hong
H
Jin
Ye
Y
Wu
Jinyan
J
Shang
Youjun
Y
Yin
Shuanghui
S
Liu
Xiangtao
X
Xie
Qingge
Q
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics* / Q000254:growth & development; D017930:Genes, Reporter; D005814:Genes, Viral; D005822:Genetic Vectors; D051379:Mice; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics*
D018076:DNA, Complementary; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases