title: Simplified recombinational approach for influenza A virus reverse genetics.
abstract: Influenza A virus (FLUAV) reverse genetics requires the cloning of all eight viral genome segments into genomic expression plasmids using restriction enzyme cleavage and ligation. Herein is described the construction of a pair of plasmid vectors and their use in RecA Escherichia coli for direct recombination with influenza cDNA for reverse genetics. This approach is simpler; avoiding restriction digestion and ligation while maintaining the required orientation of genome segments. For this recombinational approach two plasmid constructs were generated, pHH21A and pHH21G, that both possess a 25 nucleotide recombination cassette comprised of the consensus 5' and 3' ends of the negative strand divided by a StuI cleavage site, but that differ at position 4 from the 3' end due to the presence of an A or G nucleotide (plus sense) to correspond to differences among genome segments. Using the described procedure it was possible to clone viral cDNA genomes of several avian and human FLUAVs into genomic expression plasmids in a single recombination step. This novel approach to generating sets of genomic plasmid constructs for reverse genetics reduces the time and complexity of procedures thus avoiding complications that would delay rescue of viral genomes for vaccine production or biological characterization and analysis.
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5.
authors.forename: Shuai
authors.initials: S
authors.lastname: Wang
authors.forename: Qinfang
authors.initials: Q
authors.lastname: Liu
authors.forename: Juan
authors.initials: J
authors.lastname: Pu
authors.forename: Yishan
authors.initials: Y
authors.lastname: Li
authors.forename: Liya
authors.initials: L
authors.lastname: Keleta
authors.forename: Yu-Wen
authors.initials: YW
authors.lastname: Hu
authors.forename: Jinhua
authors.initials: J
authors.lastname: Liu
authors.forename: Earl G
authors.initials: EG
authors.lastname: Brown
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens / Q000821:virology; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D004372:Ducks / Q000821:virology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics; D053123:Influenza A Virus, H3N8 Subtype / Q000235:genetics; D053127:Influenza A Virus, H9N2 Subtype / Q000235:genetics; D009980:Influenza A virus / Q000145:classification / Q000235:genetics*; D005585:Influenza in Birds / Q000821:virology; D007251:Influenza, Human / Q000821:virology; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary