Viral Models and Cell Cultures

Antiviral studies of Diph@HsaFtH were carried out using representatives of the following virus families: (i) Flaviviridae (WNV, strains 13–104 and Eg-101, and TBEV, strain Hypr, all provided by the Collection of Arboviruses, Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic), ZIKV, the Brazilian strain Paraiba_01, kindly provided by prof. Paolo M. de A. Zanotto (University of Sao Paolo, Brazil) and ZIKV, strain MR-766 (a representative of the African ZIKV lineage isolated in 1947 in Uganda provided by the European Virus Archive, Marseille, France), and (ii) Coronaviridae (SARS-CoV-2, strain SARS-CoV-2/human/Czech Republic/951/2020 kindly provided by Dr. Jan Weber from Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic). In all experiments, 0.1 multiplicity of infection (MOI) of the viruses was used for experimental infections. UKF-NB-4, Caco-2, and Vero cells were used for the cell studies. UKF-NB-4 cells (kindly provided by Prof. T. Eckschlager, Motol University Hospital, Prague, Czech Republic) were cultured in IMDM supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Caco-2 and Vero cells (ATCC, Manassas, VA, USA) were cultured in DMEM supplemented with 20% (Caco-2) or 10% (Vero) FBS, 1% L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were cultured at 37 °C in a 5% CO₂ atmosphere. For all experiments, ~30,000 cells per well in a multi-well plate were added and cultured for 24 h to create a homogeneous monolayer. The use of viral strains and cell lines was approved by institutional review boards of Veterinary Research Institute and Mendel University in Brno.