Combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-SARS-CoV-2 effects in vitro

Nicola Clementi; Elena Criscuolo; Roberta Antonia Diotti; Roberto Ferrarese; Matteo Castelli; Roberto Burioni; Massimo Clementi; Nicasio Mancini

Virus isolation. An aliquot (0.8 mL) of the transport medium of the nasopharyngeal swab (COPAN's kit UTM® universal viral transport medium -COPAN) of a mildly symptomatic SARS-CoV-2 infected patient was mixed with an equal volume of DMEM without FBS and supplemented with double concentration of P/S and Amphotericin B. The mixture was added to 80% confluent Vero E6 cells monolayer seeded into a 25 cm 2 tissue culture flask. After 1 h adsorption at 37°C, 3 mL of DMEM supplemented with 2% FBS and Amphotericin B were added. Twenty-four hours post-infection (hpi) another 2 mL of DMEM supplemented with 2% FBS and Amphotericin B were added. Live images were acquired (Olympus CKX41 inverted phase-contrast microscopy) daily for evidence of cytopathic effects (CPE), and aliquots were collected for viral RNA extraction and In-house one-step real-time RT-PCR assay (10.1016/ S0140-6736(20)30154-9). Five days post-infection (dpi) cells and supernatant were collected, aliquoted and stored at -80°C (P1). For secondary (P2) virus stock, Vero E6 cells seeded into 25 cm 2 tissue culture flasks were infected with 0.5 mL of P1 stored aliquot, and infected cells and supernatant were collected 48 hpi and stored at -80°C. For tertiary (P3) virus stock, Vero E6 cells seeded into 75 cm 2 tissue culture flasks were infected with 1.5 mL of P2 stored aliquot and prepared as above described.