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The ability of each new SARS-CoV-2 variant to evade host humoral immunity is the focus of intense research. Each variant may also harbor unique replication capabilities relevant for disease and transmission. Here, we demonstrate a new approach to assessing viral replication kinetics using real-time cell analysis (RTCA). Virus-induced cell death is measured in real time as changes in electrical impedance through cell monolayers while images are acquired at defined intervals via an onboard microscope and camera. Using this system, we quantified replication kinetics of five clinically important viral variants: WA1/2020 (ancestral), Delta, and Omicron subvariants BA.1, BA.4, and BA.5. Multiple measures proved useful in variant replication comparisons, including the elapsed time to, and the slope at, the maximum rate of cell death. Important findings include significantly weaker replication kinetics of BA.1 by all measures, while BA.5 harbored replication kinetics at or near ancestral levels, suggesting evolution to regain replicative capacity, and both an altered profile of cell killing and enhanced fusogenicity of the Delta variant. Together, these data show that RTCA is a robust method to assess replicative capacity of any given SARS-CoV-2 variant rapidly and quantitatively, which may be useful in assessment of newly emerging variants. [ABSTRACT FROM AUTHOR]

This study investigates the roles of T, B, and Natural Killer (NK) cells in the pathogenesis of severe COVID-19, utilizing mouse-adapted SARS-CoV-2-MA30 (MA30). To evaluate this MA30 mouse model, we characterized MA30-infected C57BL/6 mice (B6) and compared them with SARS-CoV-2-WA1 (an original SARS-CoV-2 strain) infected K18-human ACE2 (K18-hACE2) mice. We found that the infected B6 mice developed severe peribronchial inflammation and rapid severe pulmonary edema, but less lung interstitial inflammation than the infected K18-hACE2 mice. These pathological findings recapitulate some pathological changes seen in severe COVID-19 patients. Using this MA30-infected mouse model, we further demonstrate that T and/or B cells are essential in mounting an effective immune response against SARS-CoV-2. This was evident as Rag2−/− showed heightened vulnerability to infection and inhibited viral clearance. Conversely, the depletion of NK cells did not significantly alter the disease course in Rag2−/− mice, underscoring the minimal role of NK cells in the acute phase of MA30-induced disease. Together, our results indicate that T and/or B cells, but not NK cells, mitigate MA30-induced disease in mice and the infected mouse model can be used for dissecting the pathogenesis and immunology of severe COVID-19. [ABSTRACT FROM AUTHOR]