*2.1. Cells and Reagents*

Vero cells (CCL-81, ATCC, Manassas, VA, USA), A549-Dual ™ cells (a549d-nfis, InvivoGen, San Diego, CA, USA) and human embryonic kidney HEK-293 cells (CRL-1573, ATCC, Manassas, VA, USA) were cultured at 37 ◦C under a 5% CO2 atmosphere in MEM medium, supplemented with 5% to

10% heat-inactivated foetal bovine serum (FBS). A549-Dual ™ (A549DUAL) cells were maintained in growth medium supplemented with nonessential amino acids, 10 <sup>μ</sup>g·mL−<sup>1</sup> blasticidin and 100 <sup>μ</sup>g·mL−<sup>1</sup> zeocin (InvivoGen, San Diego, CA, USA). Chloroquine phosphate was purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Rat antibody specifically raised against ZIKV E protein Domain III was developed *in-house* and used in immunoblot with reducing conditions [28]. Mouse anti-pan flavivirus envelope E protein monoclonal antibody (mAb) 4G2 was purchased from RD Biotech (Besancon, France) and used in immunoblot with nonreducing conditions. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were purchased from Vector Laboratories (Burlingame, CA, USA).

## *2.2. Design of ZIKV Molecular Clones*

ZIKV molecular clones (MR766, GenBank accession number LC002520, and BR15, GenBank accession number KU365778) were designed and produced according to the Infectious Subgenomic Amplicon method as previously described [23,29,30]. To introduce BR15 E-152/156/158 residues into MR766 (MR766E-152I/156T/158H), we used mutagenesis primers (forward primer: <sup>5</sup>-ggctcccagcacagtgggatgatcgttaatgacacaggacatgaaactg-3 and reverse primer: <sup>5</sup>-cagtttcatgtcctgtgtcattaacgatcatcccactgtgctgggagcc-3) to generate two overlapping fragments Z1MR766-E-MUT1 and Z1MR766-E-MUT2 from the Z1MR766 fragment encoding the MR766 structural proteins in which encoding region of the E protein received the IVNDTGH motif (amino acids 152 to 158) from BR15. To generate BR15E-152T/156I/158Y, a new Z1BR15-E-I152T/T156I/H158Y fragment was synthesised in which the sequence was modified so that encoding region of the E protein received the TVNDIGY motif (amino acids 152 to 158) from MR766. Synthetic genes were cloned into plasmid pUC57 by GeneCust (Boynes, France). Fragments were amplified by PCR from their respective plasmids using a set of primer pairs that was designed so that fragments overlapped with each other of about 30 to 50 nucleotides.

## *2.3. Recovering of Molecular Clones BR15E-152T*/*156I*/*158Y and MR766E-152I*/*156T*/*158H*

Molecular clones were produced as previously described [23,29]. Briefly, purified PCR fragments were electroporated into Vero cells. After 5 days, cell supernatants were recovered usually in absence of cytopathic e ffect and used to infect fresh Vero cells in a first round of amplification (P1). Viral clones were recovered at the onset of cytopathic e ffect and amplified for another round on Vero cells to produce a second round of amplification (P2), which was used for described studies. To produce MR766E-152I/156T/158H and BR15E-152T/156I/158Y mutant viral clones, Vero cells were respectively electroporated with PCR fragments Z1MR766-E-MUT1, Z1MR766-E-MUT2, Z2MR766, Z3MR766, and Z4MR766 and with Z1BR15-E-MUT, Z2BR15, Z3BR15, and Z4BR15. Recovered mutant viruses MR766E-152I/156T/158H and BR15E-152T/156I/158Y respectively consist of viral sequence of MR766 in which E-152/156/158 residues of BR15 ZIKV strain were introduced and viral sequence of BR15 in which E-152/156/158 residues were replaced with its counterpart from MR766 ZIKV strain.
