%0 Journal Article
%T A yellow fever¨CZika chimeric virus vaccine candidate protects against Zika infection and congenital malformations in mice
%A Christian Alfano
%A Dieudonn¨¦ B. Kum
%A Dominique Schols
%A Ivan Gladwyn-Ng
%A Ji Ma
%A Johan Neyts
%A Kai Dallmeier
%A Laurent Nguyen
%A Michael A. Schmid
%A Niraj Mishra
%A Rafael E. Marques
%A Robbert Boudewijns
%A Suzanne Kaptein
%J Archive of "NPJ Vaccines".
%D 2018
%R 10.1038/s41541-018-0092-2
%X Construction and propagation of YF-ZIKprM/E. a Schematic representation of the YF-ZIKprM/E construction: ZIK-5¡äA contains the capsid anchor (C anch) sequence of YFV-17D whereas ZIK-5¡äB contains the C-signal sequence of ZIKV. ZIKV Yap (2007) sequence nt 313 to 2382 was cloned into YFV-17D between nucleotides 422 and 2451. b Immunofluorescence assay at selected time points after transfection of Vero E6 cells with YF-ZIKprM/E plasmid. At 2, 8, and 14 days post transfection (dpt), cultures were split and transferred to larger flasks and further cultured (Supplementary Table 1). After each split, a part of the cells was seeded in 8-well chamber slides and stained for viral antigen using pan-flavivirus monoclonal antibody (mAb) 4G2 (at 1, 3, and 5, days post split [dps]). Upper panels represent intracellular passages 1 and 2, respectively. Supernatant of passage 4 was used to infect na£¿ve Vero E6 cells and virus infection and spread was monitored at indicated time points (bottom panel). c Antigenicity of YF-ZIKprM/E: Vero E6 cells were infected with either YFV-17D, ZIKV BeH819015,45 or YF-ZIKprM/E at multiplicity of infection (MOI) of 0.1 and stained 2dpi with pan-flavivirus antibody 4G2, anti-YFV NS1 mAb 1A5 or JEV E-specific mAb MA1-71256 that shows strong cross-reactivity only with ZIKV and YF-ZIKprM/E, but not with the YFV E protein. d Extracellular passaging of YF-ZIKprM/E in Vero E6 cells and quantification of viral RNA. Arrows indicate the first detection of ZIKV E protein mutations S455L and A40T, respectively. e YF-ZIKprM/E chimeric virus (Passage 5) forms smaller plaques (developed and stained 7dpi; complete well of a 6-well plate shown: diameter = 34.8 mm) on BHK-21J cells than the parental YFV-17D, similar to those formed by ChimeriVax-JE. f Replication kinetics of YFV-17D (red circles) and YF-ZIKprM/E (blue squares) on mammalian (Vero E6) and mosquito (C6/36) cell lines. Cells were infected with YFV-17D and YF-ZIKprM/E (Passage 5) at MOI of 0.01 and extracellular progeny viral RNA was quantified over time by qRT-PCR. Data are presented as mean values with error bars indicating SEM of n£¿=£¿2 replicates. Dotted line denotes the limit of detection (L.O.D) of the qRT-PCR assa
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292895/