Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof
| Autor: | Stefan Finke, Conrad M. Freuling, Tobias Nolden, Thomas Müller, Florian Pfaff, Sabine Nemitz, Dirk Höper |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
DNA Complementary Viral pathogenesis viruses Green Fluorescent Proteins Oligonucleotides Foxes Genome Viral Biology Recombinant virus Virus Replication Polymerase Chain Reaction Orthobunyavirus Virus Article Cell Line 03 medical and health sciences Mice 0302 clinical medicine Dogs Genes Reporter Complementary DNA Escherichia coli Animals RNA Viruses Cloning Molecular Genetics Recombination Genetic Multidisciplinary RNA virus biology.organism_classification Virology Reverse genetics Recombinant Proteins Reverse Genetics Kinetics Luminescent Proteins 030104 developmental biology Viral replication Rabies virus Mutation 030217 neurology & neurosurgery |
| Zdroj: | Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|