Assays

ZIKV RNA was extracted from virus working stocks using the QIAmp viral RNA minikit (Qiagen, Hilden, Germany). Isolated RNA was then digested with DNase I and purified using the Qiagen RNeasy kit. RNA quality was analyzed on the 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) using the Agilent RNA 6000 Pico assay and quantified on a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) prior to rRNA depletion and RNA library prep using the KAPA HyperPrep kit (Roche Diagnostics Corporation, Indianapolis, ...

Hybridoma supernatant was harvested as the source of ZV-13 antibody once ~95% cell death was observed. The supernatant was clarified by centrifuging for 10 min at 400 × g at 4°C and then filtered with a 0.22-μm filter prior to antibody purification via protein A affinity chromatography using the Pierce Protein A IgG purification kit and NAb Protein A Plus spin columns (all from Thermo Fisher-Life Technologies). Purified ZV-13 antibody was concentrated and buffer-exchanged into 1× phosphate-buffered ...

A549 cells were seeded in 96-well plates at a density of 2 × 104 cells per well. The following day, virus stocks were serially diluted in DMEM containing 2% FBS (2% DMEM), and cells were inoculated with dilutions in triplicate at 37°C for 2 h. After the incubation, cells were overlaid with DMEM supplemented with 1% carboxymethylcellulose (Fisher Scientific), 2% FBS, 10 mM HEPES, and 1× penicillin/streptomycin. At 48 hpi, the methylcellulose overlay and medium were removed, and cells were fixed ...

A549 cells were seeded in 6-well plates at a density of 6 × 105 cells per well. The following day, virus stocks were serially diluted in 2% DMEM, and cells were inoculated and fixed as described above. Following fixation, cells were washed with 1× PBS, and an anti-flavivirus envelope antibody, 4G2 (Ab00230-2.0; Absolute Antibody, Oxford, UK), diluted 1:500 in permeabilization/wash/block buffer (see recipe above) was added to the cells. Following incubation with primary antibody, donkey anti-mouse ...

A549 cells were seeded at a density of 3 × 105 cells per well in 12-well plates. Cells were uninfected, mock-infected, or infected with ZIKV variants the next day at an MOI of 5 FFU/cell, and virus inocula prepared in DMEM were allowed to adsorb at 37°C for 2 h with rocking. Uninfected samples are seeded cells that remained untouched throughout the infection time course, whereas mock-infected samples were inoculated with DMEM. Following adsorption, the inocula were removed, and cells were washed ...

A549 cells were seeded at a density of 3 × 105 cells per well in 12-well plates. Cells were precooled at 4°C prior to the addition of ice-cold virus inoculum. Inocula were allowed to adsorb to cells at 4°C for 1 h. Inocula were then removed, and cells were washed with ice-cold 1× PBS before fresh prewarmed cDMEM was added to cells. Cells were returned to 37°C until the appropriate time points to evaluate viral entry, genome replication, and virion production. For viral attachment assays, intracellular ...

A549 cells were infected as described above. At the appropriate time points, 1 mL of cell culture supernatants was harvested and centrifuged at 2,000 rpm for 10 min at 4°C. Extracellular RNA was extracted from the clarified supernatants using the QIAmp viral RNA minikit to quantify extracellular viral copy numbers. Cells were washed with ice-cold stringent wash buffer (1 M NaCl, 50 mM sodium bicarbonate, pH 9.5) for 3 min to remove cell surface-associated viruses. After ice-cold 1× PBS washes, ...