title: Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone.
abstract: The recent Zika virus (ZIKV) outbreak has been linked to severe pathogenesis. Here, we report the construction of a plasmid carrying a cytomegalovirus (CMV) promoter-expressed prototype 1947 Uganda MR766 ZIKV cDNA that can initiate infection following direct plasmid DNA transfection of mammalian cells. Incorporation of a synthetic intron in the nonstructural protein 1 (NS1) region of the ZIKV polyprotein reduced viral cDNA-associated toxicity in bacteria. High levels of infectious virus were produced following transfection of the plasmid bearing the wild-type MR766 ZIKV genome, but not one with a disruption to the viral nonstructural protein 5 (NS5) polymerase active site. Multicycle growth curve and plaque assay experiments indicated that the MR766 virus resulting from plasmid transfection exhibited growth characteristics that were more similar to its parental isolate than previously published 2010 Cambodia and 2015 Brazil cDNA-rescued ZIKV. This ZIKV infectious clone will be useful for investigating the genetic determinants of ZIKV infection and pathogenesis and should be amenable to construction of diverse infectious clones expressing reporter proteins and representing a range of ZIKV isolates.IMPORTANCEThe study of ZIKV, which has become increasingly important with the recent association of this virus with microcephaly and Guillain-Barré syndrome, would benefit from an efficient strategy to genetically manipulate the virus. This work describes a model system to produce infectious virus in cell culture. We created a plasmid carrying the prototype 1947 Uganda MR766 ZIKV genome that both was stable in bacteria and could produce high levels of infectious virus in mammalian cells through direct delivery of this DNA. Furthermore, growth properties of this rescued virus closely resembled those of the viral isolate from which it was derived. This model system will provide a simple and effective means to study how ZIKV genetics impact viral replication and pathogenesis.
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Megan C
authors.initials: MC
authors.lastname: Schwarz
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Marion
authors.initials: M
authors.lastname: Sourisseau
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Espino
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Essanna S
authors.initials: ES
authors.lastname: Gray
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Chambers
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Domenico
authors.initials: D
authors.lastname: Tortorella
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Evans
keywords: Zika virus; cell culture; flavivirus; infectious clones