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Development of a cell line stably expressing T7 RNA polymerase using retroviral genetransfer
technology for efficient rescue of infectious foot-and-mouth disease virus from cloned cDNA
Haixue Zheng1, Hong Tian1, Ye Jin1, Jinyan Wu1, Youjun Shang1, Xiangtao Liu1, Zhidong Zhang2
1Lanzhou
Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
for Animal Health, Pirbright Laboratory, Ash Road, W oking, Surrey, GU24 0NF, UK.
2Institute
Introduction
•Reverse-genetics systems for the rescue of recombinant FMDV have proven to be of great value for foot-and-mouth disease virus (FMDV) research and
vaccine development. The reverse-genetics techniques based on in vitro transcription system or similar techniques were successfully developed for recovery
of FMDV O, A, SAT1and SAT2. However, these reverse genetics systems has low efficiency of virus recovery. To overcome this, using retroviral gene
transfer technology, we developed a stable BHK-21 cell line (designated as BHKT7) constitutively expressing cytoplasmic bacteriophage T7 RNA polymerase
(T7 RNAP) for efficient rescue of infectious FMDV from cloned cDNA.
Materials and methods
•T7 RNAP gene was inserted in the retroviral vector at first. Then, the retrovirus packaging cell line was co-infected with the recombinant retroviral plasmids
and pVSV-G (the pseudotype virus enveloped with G glycoprotein of the vesicular stomatitis virus) was obtained. BHK-21 cell was infected by the pseudotype
virus, and T7 RNAP gene was integrated into the chromosome of the BHK-21, resulting in a BHKT7.
Results
•By using retroviral genetransfer technology, the T7 RNAP gene was integrated into the chromosome of BHK-21. T7 RNAP was constitutively expressed in
cytoplasm of BHKT7 (Fig1). The transcriptional activity in the different passage-time BHKT7 was confirmed by detection of expression level of EGFP reporter
gene under T7 promoter (Fig2). The different passage-time BHKT7 cell line was then directly transfected with linearized full-length cDNA of FMDV and
infectious FMDV was efficiently recovered from the cDNA. The recued FMDV recovered from transfected BHKT7 cells was designated as rFMDV (Fig 3, 4,
5).
A
B
rFMDV
Fig.1. Detection of T7 RNAP by immunofluonrescence. (A) Normal BHK-21
cells; (B) BHKT7 cells.
A
The second passage
B
The fourth passage
Parental virus
Fig. 3. CPE was observed on BHK-21 cells infected with rFMDV, which
was the same as parental virus did.
A
B
Fig. 4. Detection of viral protein in BHK cells infected with rFMDV by
immunofluonrescence. (A) BHK-21 cells infected with rFMDV; (B)
BHKT7 cells control.
The seventh passage
D
The twentieth passage
Fig.2. Analysis of the transcriptional activity in the different passage-time
BHKT7 by detection of expression level of EGFP reporter gene under T7
promoter using flow cytometry.
Discussion
•A cell line expressing T7 RNAP was developed using transient expressional
vector or vaccinia virus. To our best knowledge, we firstly utilized retroviral
gene transfer technology to integrate T7 RNAP into the cellular chromosome.
As a result, T7 RNAP was constitutively expressed even cells was passaged
a few times.
Conclusion
•Our data show that the BHKT7 cell line can be used as T7 polymerase donor
for recovery of infectious RNA virus directly from full-length cDNAs.
Viral titre (TCID50)
C
Parental virus
rFMDV
Hours after inoculation
Fig.5. Growth kinetics of the rFMDV was compared to parental stain on
BHK-21 cells. BHK-21 cells were infected at 0.1 MOI with either rFMDV or
paternal virus. Cell culture supernatants were harvested at 2, 4, 6, 8, 10, 12
and 24 h after infection. The titre of both viruses peaked at 10 h after
infection (105.4 and 105.6, respectively).
Acknowledgements
•This work was supported by the Chinese National Basic Research 973 Program (No.393 2005CB523202) and National Key Technology R&D Program of
China (No. 2006BAD06A03). Z. Zhang was supported by UK BBSRC china partnership award.