PMC:7075884 / 30265-33179 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7075884","sourcedb":"PMC","sourceid":"7075884","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7075884","text":"Cells, viruses and GA\nHEp2 cells, obtained from the American Type Culture Collection (ATCC Rockville, MD), were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS). HEK293T/17 (293T) cells obtained from the ATCC were grown in DMEM supplemented with 10% FBS. HSV-1 strain F, a limited-passage isolate, is the prototype strain used in this laboratory28. GFP-HSV-1 (strain 17), was a generous gift from Dr. Peter O’Hare29. Vero cells obtained from the ATCC were cultured in complete DMEM (cDMEM) containing 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin/streptomycin, and 10 mM Hepes buffer at 37 °C with 5% CO2. Fetal-derived Normal Human Astrocytes (NHA, Lonza) were maintained in Astrocyte Growth Medium (AGM, Lonza) supplemented with 0.3% FBS, 30 µl/mL ascorbic acid, 1 µl/mL rhEGF, 30 µg/mL gentamicin, 15 μg/mL amphotericin B, 2.5 µl/mL insulin, and 10 µl/mL L-glutamine. Early passages (1–4) were used in these experiments.\nHCMV studies were performed using cell-associated low-passage clinical isolates CH1914 and BI-615. CH19 is resistant to the first-line HCMV antiviral drug, ganciclovir (GCV), while BI-6 is susceptible to GCV. Additional experiments were performed using CMVPT30-GFP16, which expresses green fluorescent protein and produces extracellular virus.\nZIKV strain PRVABC59 obtained from the American Type Culture Collection (ATCC; Manassas, VA) was propagated in Vero cells grown in T-150 flasks by infection at 1:50 dilution of viral stock (MOI 0.25) in the absence of FBS. The medium was removed and replaced 6 h post-infection (hpi) with fresh cDMEM. Supernatant was collected at 72 hpi, clarified by centrifugation at 350 × g for 5 min, and filtered through a 0.45-μm surfactant-free cellulose acetate membrane. For mock infections, supernatant was collected from uninfected Vero cells and prepared by the same protocol used to make virus stocks. Virus was titered by focus assay. Briefly, infected Vero cells, 24 hpi were fixed and permeabilized using Cytofix/Cytoperm Solution Kit (BD Biosciences) according to the manufacturer’s instructions and stained with a mouse monoclonal antibody (mAb) specific for flavivirus group envelope proteins (1:250; EMD Millipore; clone D1–4G2-4-15) followed by incubation with an anti-mouse IgG - PE (1:1000 dilution). Samples were run through an LSR II flow cytometer and data were collected with FACSDiva software (BD Biosciences) and analyzed using FlowJo Software (TreeStar). Human Adenovirus Type5 (dE1/E3), Cat. No.: 1060, was purchased from Vector Biolabs.\nGA C13:0, C15:1, and C17:1, were purchased from Sigma Aldrich, St. Louis, MO. GA was diluted in methanol or DMSO to a concentration of 50 mM. Effects of GA in all assays and models used in this study have been compared to vehicle-treated (i.e. methanol or DMSO) controls. 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