title: Partial and full PCR-based reverse genetics strategy for influenza viruses.
abstract: Since 1999, plasmid-based reverse genetics (RG) systems have revolutionized the way influenza viruses are studied. However, it is not unusual to encounter cloning difficulties for one or more influenza genes while attempting to recover virus de novo. To overcome some of these shortcomings we sought to develop partial or full plasmid-free RG systems. The influenza gene of choice is assembled into a RG competent unit by virtue of overlapping PCR reactions containing a cDNA copy of the viral gene segment under the control of RNA polymerase I promoter (pol1) and termination (t1) signals - herein referred to as Flu PCR amplicons. Transfection of tissue culture cells with either HA or NA Flu PCR amplicons and 7 plasmids encoding the remaining influenza RG units, resulted in efficient virus rescue. Likewise, transfections including both HA and NA Flu PCR amplicons and 6 RG plasmids also resulted in efficient virus rescue. In addition, influenza viruses were recovered from a full set of Flu PCR amplicons without the use of plasmids.
authors.affiliation: Virginia-Maryland Regional College of Veterinary Medicine, Department of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America.
authors.forename: Hongjun
authors.initials: H
authors.lastname: Chen
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Ye
authors.forename: Kemin
authors.initials: K
authors.lastname: Xu
authors.forename: Matthew
authors.initials: M
authors.lastname: Angel
authors.forename: Hongxia
authors.initials: H
authors.lastname: Shao
authors.forename: Andrea
authors.initials: A
authors.lastname: Ferrero
authors.forename: Troy
authors.initials: T
authors.lastname: Sutton
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Perez
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000235:genetics*; D004285:Dogs; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D009439:Neuraminidase / Q000235:genetics*; D009975:Orthomyxoviridae / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000235:genetics; D059386:Reverse Genetics; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D019267:Hemagglutinin Glycoproteins, Influenza Virus; C485184:enhanced green fluorescent protein; C483468:hemagglutinin, human influenza A virus; D049452:Green Fluorescent Proteins; D012318:RNA Polymerase I; D009439:Neuraminidase