Construction of a Full-length Infectious Clone of Zika Virus Stably Expressing EGFP Marker in a Eukaryotic Expression System

Abstract Background: Zika virus is among the most widely transmitted arboviruses in the world and closely associated with diseases, such as encephalitis, fetal microcephaly, and Guillain–Barré syndrome. The pathogenic mechanism of the virus has not been fully elucidated, and there are no vaccines or specific drugs targeting the virus. To address these issues, the application of reverse genetics is needed for viral reconstruction and reproduction.Methods: Polymerase chain reaction (PCR) was used to merge the full-length Zika virus genome, CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into a pBAC11 vector through recombination to produce recombinant pBAC-ZIKA-EGFP. The ZIKA–EGFP was rescued by transfection of 293T cells with pBAC-ZIKA-EGFP, and at 7-days post-transfection, the supernatant (P0 generation) was passed through a 0.45-μm membrane and used to infect Vero cells (to produce the P1 generation). Fluorescence-based quantitative PCR, 50% tissue culture infectious dose, and plaque assays were used to measure differences in replication ability and pathogenicity relative to the rescue virus (ZIKA–WT), the sequence of which is consistent with that of the wild-type Zika virus. Additionally, caffeic acid phenethyl ester (CAPE), a nuclear factor kappaB (NF-kB) inhibitor, was used to examine its effect on viral replication.Results: The results showed that ZIKA–EGFP could effectively infect Vero cells, SH-SY5Y cells and C6/36 cells, and cause cytopathic effects on them. ZIKA–EGFP exhibited stable replication and EGFP expression during cell passage for at least six generations, with no significant difference in replication ability relative to the ZIKA–WT. Fluorescent cell foci were observed in the plaque assay while the ZIKA–EGFP was in the absence of phage plaque formation. The inhibition of NF-kB inhibitor on ZIKA-EGFP was observed by fluorescence microscopy, which was consistent with the results of fluorescence quantitative PCR.Conclusions: We constructed an infectious clone of the full-length genome of Zika virus which could replicate with stable EGFP expression in eukaryotic cells during passage. The infectious clone, remaining main characteristics of wild type ZIKA virus could be appied on the studies of reverse genetics, drug screening and gene function of ZIKA virus.