2.4. SARS-COV-2 Inactivation Test

Vero E6 cells were used to prepare virus stock and to test the viability of the virus by microscopic observation of the cytopathic effect caused by the virus. The cells were cultured at 37 °C and 5% CO₂ in 2% fetal bovine serum and 1% v/v penicillin–streptomycin in Dulbecco’s modified Eagle’s medium. The Hong Kong index SARS-COV-2 virus was used in the tests and 0.5% (w/v) bovine serum albumin and 0.1% (w/v) glucose in Earle’s balanced salt solution with a pH of 7.4 was used as a viral transport medium.

Inactivation of the virus by the CuO coating was examined as follows. The CuO or control coating was initially disinfected with 70% ethanol in water, followed by drying in an air atmosphere at 37 °C overnight. A 5 μL droplet containing 6.2 × 10⁷ (7.8 log unit) TCID₅₀/mL of the virus was spotted on the test solid at 22–23 °C and 60–70% humidity, and after a predefined time, the coating was immersed in 300 μL of viral transport medium to elute the virus. The active virus within the eluted droplet was assessed using a 50% tissue culture infective dose (TCID₅₀) assay^21,22 using Vero E6 cells. The TCID₅₀ assay consists of making a series of 3.16× (i.e., half-log) dilutions of the eluted virus. Cells on 96-well plates were exposed to one of the dilutions, with quadruplicate²³ of each dilution. The cells were then incubated at 37 °C and 5% CO₂. After 5 days, the cells were assessed for any cytopathic effect. The dilution at which 50% (2 of 4) of Vero E6 cell cultures showed a cytopathic sign is called TCID₅₀/mL. Three independent samples (i.e., a new solid sample and a new inoculation of virus) were tested for each time point, and the virus inactivation at each time point was calculated based on the reduction of log(TCID₅₀/mL) as follows