Horse serum

Cells
Permanent HCT-8 [HRT-18] (ATCC-CCL-244™, LGC Standards, Middlesex, UK) was kept at 5% CO₂ and 37 °C in RPMI 1640 (Roswell Park Memorial Institute Medium, ATCC-30-2001) to which L-glutamine 0.3 g·L⁻¹ (Sigma-Aldrich, Darmstadt, Germany), Penicillin 100 UI, horse serum 10% (ATCC-30-2021), and Streptomycin 0.1 mg·mL⁻¹ (Sigma-Aldrich, Darmstadt, Germany) were added. Madine–Darby canine kidney MDCK (ATCC-CCL-34™) cell line was a gift from Dr. Cyril Bharbesange, National Reference Center for Influenza, Sciensano, Belgium. Cells were grown in DMEM (Gibco, Washington, DC, USA) containing fetal bovine serum (FBS) 5%, (Gibco), sodium bicarbonate 3.7 mg·mL⁻¹, HEPES buffer 10 µM (AppliChem GmbH, Darmstadt, Germany), Penicillin 100 U·mL⁻¹, and Streptomycin 100 µg·mL⁻¹. Both cell lines were seeded at a density of 2.5 × 105/mL at 37 °C in a 5% CO₂ incubator Thermo Forma 310 (Thermo Fisher Scientific, Waltham, MA, USA), in 96-well plates (Corning^® Costar^®, New York, NY, USA), Amphotericin B 25 µg·mL⁻¹ for 24 h until monolayer confluency is reached.
Viruses
Human coronavirus OC-43 (HCoV-OC43) (ATCC: VR-1558) strain was propagated in HCT-8 cells in a RPMI 1640 maintenance solution; the medium was supplemented with horse serum 2%, Penicillin 100 U/mL, and Streptomycin 100 µg·mL⁻¹. The cells were lysed by 2 freeze and thaw cycles 5 days after infection; the virus was titrated according to the Reed and Muench formula [52 ]. The infectious titer of the stock virus was 10^6.0 cell culture infectious doses 50% in 1 mL (CCID50.mL⁻¹).
Allantoic fluid and MDCK-derived seasonal influenza A virus (IAV) strain A/Panama/07/99 (H3N2) (National Center for Infectious and Parasitic Diseases—NCIPD, Sofia, Bulgaria) were used. The infectious titer of the stock virus was T = 10^−5.0 lg CCID50.mL⁻¹ and used as a stock suspension or at a working dose of 100 CCID50 mL⁻¹.
Cytotoxicity assay
Cytotoxicity at the 72nd hour on HCT-8 and MDCK cells was evaluated first by visual microscopic observation and then by cell viability assessment after treatment with varying concentrations of test samples following neutral red (NR) dye uptake assay as described previously [53 (link)]. As a reference antiviral inhibitor of coronavirus replication, the stock solution of Veklury^® (Gilead Science Inc., Cork, Ireland UC) with a concentration of 150 mg·mL⁻¹ was used; it was prepared in double distilled water at a final remdesivir (REM) concentration of 8.3 × 10⁻³ M. As a reference antiviral inhibitor of influenza virus replication, oseltamivir phosphate (OS) (Hoffman-LaRoche, Basel, Switzerland) was used.
Antiviral activity assay
The cells were cultivated in 96-well plates. After a confluent monolayer was formed, cell infection with 0.1 mL viral suspension in tenfold falling dilutions was carried out. The non-adsorbed virus was eliminated after an hour, and 0.1 mL/well maintenance medium was added to the cells. The plates were incubated at 33 °C for 5 days for HCoV-OC43 and at 37 °C for 3 days in a 5% CO₂ atmosphere for IAV. Unintended cells were used as control, cultivated under the same conditions (cells infected with the maximum concentration of the virus demonstrating the maximum cytopathic effect). Microscopic monitoring of the cellular monolayer was used to determine the infectious viral titer. The visually defined cytopathic effect (CPE) was confirmed by NR Uptake Assay [53 (link)]. The optical density (OD) of each well was reported at 540 nm in a microplate reader (Biotek Organon, West Chester, PA, USA).
The antiviral activity of the test formulation was evaluated with the aid of a CPE inhibition test. A 100-cell culture infectious dose of 50% (CCID50) in 0.1 mL (containing a different virus strain) was applied to infect a confluent cell monolayer in 96-well plates. The non-adsorbed virus was removed after an hour of virus adsorption for IAV or 2 h for HCov-OC-43, and the test formulation was added in various concentrations; incubation was carried out for 48 h at 37 °C and 5% CO₂ for IAV, or 120 h at 33 °C and 5% CO₂ for HCov-OC-43. The CPE was determined using a neutral red uptake assay, and the percentage of CPE inhibition for each concentration of the test sample was calculated according to a protocol described previously [19 (link)]. IC₅₀ (the 50% inhibitory concentration) was defined as the concentration causing 50% viral replication inhibition as compared to the virus control.
Virucidal Assay
The test formulation was used in its maximum tolerated concentration (MTC) and combined in a 1:1 ratio with 1 mL containing virus (10⁵ CCID50). The samples were stored at room temperature for different time intervals (15, 30, 45, and 60 min). The residual infectious virus content in each sample was determined by the end-point dilution method, and ∆lgs compared to the untreated controls were evaluated.
Statistical analysis
Data were recorded using Gen5^® and further processed by Excel^® Microsoft. The values of CC₅₀ were calculated using non-linear regression analysis (GraphPad Software, San Diego, CA, USA). The values were presented as means ± SD from three independent experiments.