Fig. 1. Construction of ZIKV-reporter viral particles (ZIKV-RVPs).

(A) The genetic organization of the Flavivirus genome is shown. The Flaviviruses genome is translated into a single polyprotein that is proteolytically processed into the illustrated structural and non-structural proteins. The West Nile Virus replicon GFP and Zeocin (GZ) is shown, which is the genome of West Nile virus where the GFP protein has been inserted replacing part of the capsid (C), promembrane (prM) and part of the envelope (E). The Zika Virus structural gens are shown. To create ZIKV-RVPs, human HEK293T cells were transfected the 5 μg of the West Nile virus replicon GZ together with 1 μg of the Zika Virus structural genes. Supernatants containing ZIKV-RVPs were collected 48 h post transfection, and ZIKV-RVPs were tittered in Vero cells using serial dilutions. (B) ZIKV-RVPs infect human microglial CHME3, human fibroblasts HT1080, African green monkey fibroblast-like Vero, and dog Cf2Th cells. CHME3, HT1080, Vero and Cf2Th cells were challenges by ZIKV-RVPs at an MOI of 1. Forty-eight hours post-challenge infection was determined by measuring the percentage of GFP-positive cells using a flow cytometer. Experiments were repeated 5 times and a representative experiment is shown. (C) Neutralizing antibodies against the envelope of ZIKV inhibit the infection by ZIKV-RVPs. To determine whether neutralizing antibodies against the envelope of ZIKV inhibit the infection by ZIKV-RVPs, we challenged Cf2Th cells using ZIKV-RVPs that were pre-incubated with the indicated antibody for an hour at 37 °C. Forty-eight hours post-challenge infection was determined by measuring the percentage of GFP-positive cells using a flow cytometer. Experiments were repeated three times and a representative neutralization curve for each antibody is shown.