title: Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof.
abstract: Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, D-17493 Greifswald - Insel Riems, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Nolden
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Diagnostic Virology, D-17493 Greifswald - Insel Riems, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Pfaff
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, D-17493 Greifswald - Insel Riems, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Nemitz
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, D-17493 Greifswald - Insel Riems, Germany.
authors.forename: C M
authors.initials: CM
authors.lastname: Freuling
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Diagnostic Virology, D-17493 Greifswald - Insel Riems, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Höper
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, D-17493 Greifswald - Insel Riems, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Müller
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, D-17493 Greifswald - Insel Riems, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Finke
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004926:Escherichia coli / Q000378:metabolism; D005589:Foxes; D017930:Genes, Reporter; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D012328:RNA Viruses / Q000235:genetics*; D011820:Rabies virus / Q000235:genetics*; D011994:Recombinant Proteins; D011995:Recombination, Genetic; D059386:Reverse Genetics / Q000379:methods*; D014779:Virus Replication; D000097573:Red Fluorescent Protein
chemical_list: D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein