Reverse Genetics of the Avian Influenza Virus
Reverse genetics is the creation of a virus from a full-length cDNA copy of the viral genome, referred to as an infectious clone, and is the most powerful genetic tool in modern virology. The generation of influenza A viruses by reverse genetics has lagged mainly due to the inherent technical difficulties of providing a sufficient amount of all eight viral RNAs from cloned cDNA. A breakthrough was made in 1999 by utilizing the cellular enzyme RNA polymerase I for the synthesis of influenza viral RNAs. Although slightly different methods are being used in different laboratories for the rescue of the influenza virus, the basic concept of synthesizing viral RNA using RNA polymerase I remains the same. Coupled with in vitro mutagenesis, reverse genetics can be applied widely to accelerate progress in understanding the influenza virus life cycle, the generation of live-attenuated vaccines, and the use of influenza virus as vaccine and gene delivery vectors.
Keywordsinfluenza reverse genetics infectious clone mutagenesis cloning
The authors would like to thank Dr. Gerd Hobom (Institut fur Mikro- und Molekularbiologie, Giessen, Germany) for kindly providing the pHH21 vector and Dr. Yoshihiro Kawaoka (University of Wisconsin) for providing 293T cells and plasmids for A/WSN/33 (H1N1) reverse genetics.
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