Abstract
Influenza virus has a set of ribonucleoproteins (RNPs) consisting of viral RNAs, influenza virus polymerase subunits, and nucleoprotein. Intracellular reconstitution of the whole set of RNPs via plasmid transfection results in the generation of influenza virus. By the use of reverse genetics and dual promoters, we constructed a 23.6-kb eight-unit plasmid that contains all the required constituents to generate influenza virus in chicken cells. Our "one-plasmid" system generated higher titers of influenza virus in chicken cells than the "eight-plasmid" system, enabling a simpler approach for generating vaccine seeds. Our study identified plasmid size as a potential limiting factor affecting transfection efficiency and hence the influenza viral yield from chicken cells.