Construction of an infectious Chikungunya virus cDNA clone and stable insertion of mCherry reporter genes at two different sites.
Chikungunya virus (CHIKV) has caused massive epidemics in the Indian Ocean region since 2005. It belongs to the genus Alphavirus and possesses a positive-stranded RNA genome of nearly 12 kb in size. To produce genetically modified viruses for the study of various aspects of the CHIKV life cycle, a reverse genetic system is needed. We report the generation of a T7 RNA polymerase-driven infectious cDNA clone of CHIKV. Electroporation of in vitro-transcribed RNA resulted in the recovery of a recombinant virus with growth characteristics comparable to the parental strain. Using the established cDNA clone, the red fluorescent marker gene mCherry was introduced into two different sites within the CHIKV nsP3 gene. Both constructs allowed the rescue of stable fluorescent reporter viruses with growth characteristics similar to the wild-type virus. The latter reporter viruses represent valuable tools for easy follow-up of replicating CHIKV useful in several applications of CHIKV research.
Institute of Virology, University of Bonn Medical Center, Bonn, Germany.
Beate Mareike
BM
Kümmerer
Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.|Institute of Virology, University of Bonn Medical Center, Bonn, Germany.
Klaus
K
Grywna
Institute of Virology, University of Bonn Medical Center, Bonn, Germany.
Sabine
S
Gläsker
Institute of Virology, University of Bonn Medical Center, Bonn, Germany.
Janett
J
Wieseler
Institute of Virology, University of Bonn Medical Center, Bonn, Germany.
Christian
C
Drosten
D018354:Alphavirus Infections / Q000821:virology*; D000818:Animals; D002460:Cell Line; D065632:Chikungunya Fever; D002646:Chikungunya virus / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D016679:Genome, Viral; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D000097573:Red Fluorescent Protein
D018076:DNA, Complementary; D008164:Luminescent Proteins; D017361:Viral Nonstructural Proteins; D000097573:Red Fluorescent Protein