Phd in ADVANCES IN INFECTIOUS DISEASES, MICROBIOLOGY, LEGAL
MEDICINE AND PUBLIC HEALTH SCIENCES

Thesis title: In Vitro Assessment of SARS-CoV-2: Efficacy Evaluation of New Antivirals in Novel and Established Models

ABSTRACT The emergence of a novel Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) has led to one of the most significant pandemics of human history. SARS-CoV-2 continues to evolve under immune selective pressure, the risk of vaccine escape variants increases, particularly with ongoing high transmission levels. Given the clinical importance of emerging viral variants with enhanced transmissibility, a deeper understanding of the early stages of viral replication is urgently needed. This study aims to identify the replication kinetics of SARS-CoV-2 infection during the initial phases in 2D and 3D cellular models, VERO C1008 [Vero 76, clone E6, Vero E6] and AESC-based respiratory epithelia in air-liquid interface (ALI) culture conditions respectively. We used the viral isolate BetaCov/Italy/CDG1/2020|EPIISL412973|2020-02-20, obtained from a COVID-19 patient, to infect VERO E6 cells in Labteck chamber slides. Experiments were conducted in a certificate Biosafety level 3 laboratory (BSL-3) at Istituto Superiore di Sanità. Infected Vero E6 cells were analyzed at different time points, at the very early stages of viral infection (0 - 30 minutes post virus adsorption). We performed Immunofluorescence assay (IFA) to label viral and cellular proteins and to track the progression of viral protein synthesis throughout the viral life cycle. The results showed that after 5 minutes post-infection, Vero-E6 expressed nucleocapsid (N) and membrane (M) viral proteins, which were distributed throughout the cytoplasm, accompanied by significant alterations in cellular compartments. Notably, moderate colocalization of N and M proteins with the endoplasmic reticulum (ER) and Golgi apparatus was observed as early as 5 minutes after infection. Host cellular involvement begins in the initial stages of infection. These results emphasize the importance of studies concerning the interaction of viral proteins and cellular structures involved during the very early stages of the SARS-CoV-2 viral cycle. Similar experiments were conducted on air-liquid interface (ALI) cultures of AESC-based respiratory epithelial cells derived from nasal tissue and the results allowed us to propose a model for the mechanism of viral entry and budding in respiratory epithelium. Additionally, this study aims to investigate the antiviral effect of Paulownia tomentosa Steud extract against SARS-CoV-2. Our results indicated that P. tomentosa extract can inhibit viral replication by directly interacting with both the 3-chymotrypsin-like protease and spike protein in Vero E6 and also in ALI cultured cell conditions.