Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

doi:10.1016/S2095-3119(13)60258-0

Journal of Integrative Agriculture ›› 2013, Vol. 12 ›› Issue (5): 877-883.DOI: 10.1016/S2095-3119(13)60258-0

Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

HUANG Jun-hua, LI Yong-feng, HE Fan, LI Dan, SUN Yuan, HAN Wen , QIU Hua-ji

Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, P.R.China

收稿日期:2012-05-11 出版日期:2013-05-01 发布日期:2013-05-01
通讯作者: Correspondence QIU Hua-ji, E-mail: huajiqiu@hvri.ac.cn
作者简介:HUANG Jun-hua, E-mail: jhhuang@ymail.com
基金资助:
This study was supported by the National Basic Research Program of China (2005CB523202).

Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

HUANG Jun-hua, LI Yong-feng, HE Fan, LI Dan, SUN Yuan, HAN Wen , QIU Hua-ji

Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, P.R.China

Received:2012-05-11 Online:2013-05-01 Published:2013-05-01
Contact: Correspondence QIU Hua-ji, E-mail: huajiqiu@hvri.ac.cn
About author:HUANG Jun-hua, E-mail: jhhuang@ymail.com
Supported by:
This study was supported by the National Basic Research Program of China (2005CB523202).

摘要/Abstract

摘要： The reverse genetics for classical swine fever virus (CSFV) is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the lowcopy vector pOK12, producing pOKShimen-RzT . Direct transfection of pOKShimen-RzT into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.

关键词: classical swine fever virus , reverse genetics , T7 RNA polymerase , stable cell line

Abstract: The reverse genetics for classical swine fever virus (CSFV) is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the lowcopy vector pOK12, producing pOKShimen-RzT . Direct transfection of pOKShimen-RzT into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.

Key words: classical swine fever virus , reverse genetics , T7 RNA polymerase , stable cell line

HUANG Jun-hua, LI Yong-feng, HE Fan, LI Dan, SUN Yuan, HAN Wen , QIU Hua-ji. Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA[J]. Journal of Integrative Agriculture, 2013, 12(5): 877-883.

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Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

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