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Rapid Reverse Genetics Systems for Rhabdoviruses: From Forward to Reverse and Back Again

  • Tobias Nolden
  • Stefan FinkeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1602)

Abstract

Methods to recover recombinant negative strand RNA viruses (rNSVs) from cloned cDNAs have been significantly improved in more than two decades of NSV reverse genetics. In particular, for non-segmented negative strand RNA viruses (NNSVs) like rhabdoviruses, time-consuming generation of reverse genetics systems by stitching PCR subfragments of genomic rhabdovirus cDNAs using ligase-based conventional cloning approaches limited the number of available recombinant virus cDNA clones. As genetic variability is considered an intrinsic feature of RNA viruses, it is thus reasonable to conclude that reverse genetics approaches to investigate natural virus functions and pathogenesis require improved systems that reflect the complexity of naturally occurring wild-type viruses, and that largely exclude adaption to cell culture conditions.

In order to allow rapid cloning of wild-type NSV genome populations into reverse genetics vector plasmids, we developed a system in which cDNA copies of complete rhabdovirus populations are inserted into a plasmid bank by linear-to-linear homologous RecE/T recombination (LLHR). Limited requirements for sequence information a priori, high cloning efficiencies, and the possibility to directly generate recombinant viruses from individual cDNA clones now offer novel opportunities to combine forward genetic dissection of natural rhabdovirus populations and downstream reverse genetics approaches.

Key words

Rhabdovirus Rabies virus (RABV) Reverse genetics Recombination RecE/T recombinase 

Notes

Acknowledgment

This work was supported by an intramural collaborative research grant at the Friedrich-Loeffler-Institut.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Institute of Molecular Virology and Cell BiologyFriedrich-Loeffler-InstitutGreifswaldGermany
  2. 2.ViraTherapeuticsInnsbruckAustria

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