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J Vis Exp. 2017 Jun 7;(124). doi: 10.3791/55857.

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone.

Author information

1
Department of Microbiology, Immunology, and Pathology, Colorado State University.
2
Division of Vector-Borne Diseases, Centers for Disease Control and Prevention.
3
Department of Microbiology, Immunology, and Pathology, Colorado State University; gregory.ebel@colostate.edu.

Abstract

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

PMID:
28654045
PMCID:
PMC5608285
DOI:
10.3791/55857
[Indexed for MEDLINE]
Free PMC Article

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