NDLI: Efficient and stable rescue of classical swine fever virus from cloned cDNA using an RNA polymerase II system

Content Provider	Springer Nature Link
Author	Li, Chao Huang, Junhua Li, Yongfeng He, Fan Li, Dan Sun, Yuan Han, Wen Li, Su Qiu, Hua Ji
Copyright Year	2012
Abstract	Conventional reverse genetics for classical swine fever virus (CSFV) is based on the transfection of permissive cells with either in vitro or intracellularly synthesized RNA transcripts from a viral genomic cDNA clone. These strategies are complicated, inefficient and time-consuming. This study is aimed to develop an improved reverse genetics method for the direct, rapid and efficient recovery of CSFV from cloned cDNA. The cDNA clone pBRCISM was constructed, which harbors the full-length genomic sequence from the CSFV Shimen strain flanked by the cytomegalovirus promoter (an RNA polymerase II promoter), a chimeric intron, and hammerhead ribozyme sequences at the 5′-end and the hepatitis delta virus ribozyme and SV40 polyadenylation signal sequences at the 3′-end. Infectious progeny virus was rescued from PK-15 cells directly transfected with pBRCISM, and its morphology, one-step growth characteristics and pathogenicity were indistinguishable from the parent virus and virus rescued from classical reverse genetics. The reverse genetics based on RNA polymerase II yielded a 120-fold increase in the titer of nascent virus in 12-h less time than a reverse genetics method based on in vitro transcription. The full-length cDNA clone remained stable and infectious after 20 passages in bacterial cells, in contrast to the instability of the full-length clone without the intron after 9 passages. The improved reverse genetics method developed in the present study is efficient, stable, convenient and cost-effective and will be valuable for the rapid recovery of CSFV mutants.
Starting Page	901
Ending Page	907
Page Count	7
File Format	PDF
ISSN	03048608
Journal	Archives of Virology
Volume Number	158
Issue Number	4
e-ISSN	14328798
Language	English
Publisher	Springer Vienna
Publisher Date	2012-11-29
Publisher Place	Vienna
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Virology Medical Microbiology Infectious Diseases
Content Type	Text
Resource Type	Article
Subject	Virology Medicine

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