Vero 76 cells

CHIKV (strain 181/25 was obtained from BEI resources, Genbank no: EF452493.1) was propagated in Vero 76 cells (ATCC CRL-1587) grown in MEM-E with 10% FBS. After CPE was monitored, the cell supernatant was harvested and clarified with a low-speed centrifugation at 4,000 ×g for 20 min. The cell supernatant was stored at -80°C until use and viral RNA was isolated from the supernatants. RNA was isolated using RNAzol RT (MRC) with the Direct-zol RNA miniprep kit (Zymo Research) according to manufacturer’s instructions, with one additional wash step.

For PEDV propagation, Vero 76 cells (ATCC CRL-1587) were maintained
in MEM plus 10 % fetal bovine serum and antibiotics. Three-day old confluent
monolayers of Vero 76 cells in 150 cm² flasks were
washed 3 times with serum free minimum essential media (MEM) prior to
inoculation. Monolayers were infected at ~0.1 moi of PEDV in MEM containing 2
ug/ml TPCK treated trypsin, incubated at
37 °C for approximately 48 h until obvious CPE was apparent. Flasks were frozen
at −80 °C until needed.

HSV-1 strain 17+ was originally transferred from John Hay (SUNY Buffalo, NY, USA) to FDA (Bethesda, MD, USA) and propagated in Vero 76 cells (ATCC CRL-1587), and first-passage stocks were transferred to UCSF (San Francisco, CA, USA); first-passage stocks were then transferred to the Bertke lab (Virginia Tech, Blacksburg, VA, USA). Stocks were propagated on Vero 76 cells and titrated by standard plaque assay on Vero 76 cells in quadruplicate to determine concentration. SARS-CoV-2 (Isolate USA-WA1/2020, NR-52281) was procured from BEI, and then propagated and titrated on Vero-E6 cells (ATCC CRL-1586). Both viruses were thawed on ice and diluted in Dulbecco’s modified Eagle medium (DMEM, Fisher Scientific, Waltham, MA, USA) to prepare inocula.

HSV-1 strain 17+ was originally isolated and transferred from John Hay (SUNY, Buffalo, NY, USA) to the Krause lab (FDA, Bethesda, MD, USA). HSV-2 strain 333 was originally isolated and transferred from Gary Hayward (Johns Hopkins, Baltimore, MD, USA) to the Krause lab (FDA, Bethesda, MD, USA). Virus was propagated in Vero 76 cells (CRL-1586, ATCC) in the Krause lab and Passage 1 aliquots were stored at −80 °C; most new virus stocks propagated in the Krause lab were propagated from the first passage aliquots, resulting in Passage 2. A Passage 2 aliquot was transferred to the Margolis lab (UCSF, San Francisco, CA, USA); new virus stocks were propagated and stored at −80 °C (Passage 3). A Passage 3 aliquot was transferred to the Bertke lab (Virginia Tech, Blacksburg, VA, USA); new virus stocks were propagated and stored at −80 °C (Passage 4). All virus stocks used in the Bertke lab were propagated from the Passage 4 stocks in Vero 76 cells (Passage 5). Stocks were titrated on Vero 76 cells in quadruplicate to determine concentration. Stock viruses were diluted in Neurobasal A medium (Thermo Fisher, Waltham, MA, USA) for inoculation of primary adult murine neuronal cultures.

Spike experiments were used to detect ZIKV in human plasma specimens. The supernatant of Vero-76 cells (ATCC, cat: CRL-1587) infected by ZIKV strain PRVA BC59 was titrated by focus formation assays [14 (link)], then added in different amounts to the plasma of 40 healthy pediatric volunteers taken before the arrival of ZIKV to the Americas. The range of FFU tested was 50–100,000 FFU/mL, all with a final constant volume of 200 µL that was used for purification with the automated protocol. To corroborate the specificity of the automated isolation, 209 plasma samples from healthy volunteers taken before 2015, to whom ZIKV was not added in vitro, were also included for automated RNA purification.

A placental-derived, Asian lineage ZIKV strain R103451 (Zika Virus, R103451, NR-50355, BEI Resources, NIAID, NIH, Manassas, VA, USA) was passaged once and expanded upon acquisition in Vero-76 cells (CRL-1587, ATCC, Manassas, VA, USA) and stored in sheep serum at −80 °C. Virus was quantitated by PFU assay, in which ZIKV was mixed as eight 10-fold serial dilutions in commercial cell culture media (Modified Eagle’s Media, MEM, Gibco), inoculated into duplicate wells of a 12-well plate containing 95% confluent Vero-76 cells, and incubated at 37 °C with 5% CO₂ for 1 h. The inoculum was removed and 1 mL of 0.05% methylcellulose overlay was added to each well. After incubation for 72 h, wells were rinsed and stained with 0.1% Coomassie blue (Thermo Fisher Scientific, Waltham, MA, USA) in 50% methanol, 43% ethanol, and 7% acetic acid for visualization. Plaques were counted and the PFU was calculated as previously described [52 (link)].

Challenge dose and infectious virus in swab samples were determined by agarose plaque assay, run on fresh (i.e. not frozen then thawed) samples the day of collection. Required dilutions of each specimen were prepared in virus diluent [1X minimum essential media (Corning Life Sciences, Pittston, PA) containing 10% fetal bovine serum (Cytiva Life Sciences, Marlborough, MA), 1% GlutaMAX (Gibco, Waltham, MA), and 1% NEAA (Sigma, St. Louis, MO)] in duplicate (swab specimens) or triplicate (all-glass impinger specimens from Study Day 1), were added to plates containing Vero 76 cells (ATCC, Manassas, VA) at greater than or equal to 85% confluency. Two days later, the cells were stained with neutral red (Thermo Fisher Scientific, Waltham, MA), and plaque counts were obtained the day after staining.