Efficient generation of influenza virus with a mouse RNA polymerase I-driven all-in-one plasmid

• Published in: Virology journal Vol. 12; no. 1; p. 95
• Main Authors: Zhang, Xiangmin, Curtiss, Roy
• Format: Journal Article
• Language: English
• Published: London BioMed Central 22.06.2015; BioMed Central Ltd
• Subjects: Animals; Antigens, Viral - biosynthesis; Antigens, Viral - genetics; Biomedical and Life Sciences; Biomedicine; CHO Cells; cost effectiveness; Cricetulus; Cricetulus griseus; DNA-directed RNA polymerase; Dogs; Epidemics; Genetic aspects; hamsters; Influenza; Influenza A virus; Influenza vaccines; Influenza Vaccines - genetics; Influenza Vaccines - immunology; Influenza viruses; kidneys; Madin Darby Canine Kidney Cells; Mice; Orthomyxoviridae - genetics; pandemic; Physiological aspects; Plasmids; Proteins; reverse genetics; Reverse Genetics - methods; RNA; RNA Polymerase I - metabolism; RNA, Viral - biosynthesis; RNA, Viral - genetics; tissue culture; Vaccines, Attenuated - genetics; Vaccines, Attenuated - immunology; Virology; viruses; Mouse RNA polymerase I promoter; Influenza virus; Reverse genetics
• ISSN: 1743-422X; 1743-422X
• DOI: 10.1186/s12985-015-0321-5

Background The current influenza vaccines are effective against seasonal influenza, but cannot be manufactured in a timely manner for a sudden pandemic or to be cost-effective to immunize huge flocks of birds. We propose a novel influenza vaccine composing a bacterial carrier and a plasmid cargo. In the immunized subjects, the bacterial carrier invades and releases its cargo into host cells where the plasmid expresses viral RNAs and proteins for reconstitution of attenuated influenza virus. Here we aimed to construct a mouse PolI-driven plasmid for efficient production of influenza virus. Results A plasmid was constructed to express all influenza viral RNAs and proteins. This all-in-one plasmid resulted in 10 5 –10 6 50 % tissue culture infective dose (TCID 50 )/mL of influenza A virus in baby hamster kidney (BHK-21) cells on the third day post-transfection, and also reconstituted influenza virus in Madin–Darby canine kidney (MDCK) and Chinese hamster ovary (CHO) cells. A 6-unit plasmid was constructed by deleting the HA and NA cassettes from the all-in-one plasmid. Cotransfection of BHK-21 cells with the 6-unit plasmid and the two other plasmids encoding the HA or NA genes resulted in influenza virus titers similar to those produced by the 1-plasmid method. Conclusions An all-in-one plasmid and a 3-plasmid murine PolI-driven reverse genetics systems were developed, and efficiently reconstituted influenza virus in BHK-21 cells. The all-in-one plasmid may serve as a tool to determine the factors inhibiting virus generation from a large size plasmid. In addition, we recommend a simple and robust “1 + 2” approach to generate influenza vaccine seed virus.