A robust method for the rapid generation of recombinant Zika virus expressing the GFP reporter gene

•The reverse genetic method ISA is a convenient method for rapid production of ZIKV molecular clones.•Molecular clones are reliable tools for investigation on ZIKV viral determinants of human disease.•The ISA method allows the rapid production of recombinant ZIKVGFP expressing the GFP reporter gene.•ZIKVGFP is suitable for dynamics study on viral replication in host cells from mosquito to human.•ZIKVGFP is suitable to test antiviral activity of inhibitors as well as of neutralizing antibodies.

Zika virus (ZIKV) infection is a major public health problem with severe human congenital and neurological anomalies. The screening of anti-ZIKV compounds and neutralizing antibodies needs reliable and rapid virus-based assays. Here, we described a convenient method leading to the rapid production of molecular clones of ZIKV. To generate a molecular clone of ZIKV strain MR766NIID, the viral genome was directly assembled into Vero cells after introduction of four overlapping synthetic fragments that cover the full-length genomic RNA sequence. Such strategy has allowed the production of a recombinant ZIKV expressing the GFP reporter gene that is stable over two culturing rounds on Vero cells. Our data demonstrate that the ZIKV reporter virus is a very reliable GFP-based tool for analyzing viral growth and measuring the neutralizing antibody as well as rapid screening of antiviral effect of different classes of inhibitors.