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IntroductionThe severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike (S) protein is essential in mediating membrane fusion of the virus with the target cells. Several reports demonstrated that SARS-CoV-2 S protein fusogenicity is reportedly closely associated with the intrinsic pathogenicity of the virus determined using hamster models. However, the association between S protein fusogenicity and other virological parameters remains elusive.MethodsIn this study, we investigated the virological parameters (e.g., S1/S2 cleavage efficiency, plaque size, pseudoviral infectivity, pseudovirus entry efficiency, and viral replication kinetics) of eleven previous variants of concern (VOCs) and variants of interest (VOIs) correlating with S protein fusogenicity.Results and discussionS protein fusogenicity was found to be strongly correlated with S1/S2 cleavage efficiency and plaque size formed by clinical isolates. However, S protein fusogenicity was less associated with pseudoviral infectivity, pseudovirus entry efficiency, and viral replication kinetics. Taken together, our results suggest that S1/S2 cleavage efficiency and plaque size could be potential indicators to predict the intrinsic pathogenicity and S protein fusogenicity of newly emerged SARS-CoV-2 variants.

ABSTRACT Increasing the reliable testing capacity for SARS-CoV-2 is important for the diagnosis and control of COVID-19. We developed an automated, customizable, easy-to-implement molecular testing system, named PCRpack, for the high-throughput testing of SARS-CoV-2. PCRpack includes a liquid handling instrument enclosed in a negative-pressure clean booth (Biomek i5), a laboratory information management system (SimpPCR), other equipment, and documents that are needed for testing operation. An in vitro diagnostic assay was employed to detect SARS-CoV-2. System performance was evaluated based on liquid handling accuracy and precision, analytical sensitivity, clinical diagnostic performance, and testing capacity per day. Clinical diagnostic performance was determined against the reference extraction-based reverse transcription-PCR assay using 3,965 upper respiratory samples. Analytical sensitivity analysis showed a lower limit of detection of 1,000 genome copies/mL of sample. The accuracy and precision of sample or reagent dispensing in PCRpack ranged from −2.24% to 0.73% and 0.83% to 4.52%, respectively. In the evaluation of clinical samples, PCRpack showed a positive percent agreement of 96.6% and a negative percent agreement of 100% compared with the reference assay. The average turnaround times per 94 samples and the maximum numbers of tests within an 8-hour shift of one operator were 2 hours and 28 minutes vs 2 hours and 1 minute and 564 vs 376 samples for PCRpack and the manual method, respectively. The developed PCRpack system shows high liquid handling and clinical diagnostic performance and is a promising testing system for increasing the SARS-CoV-2 testing capacity and testing future emerging pathogens. IMPORTANCE Accurate and fast molecular testing is important for the diagnosis and control of COVID-19. During patient surges in the COVID-19 pandemic, laboratories were challenged by a higher demand for molecular testing under skilled staff shortages. We developed an automated multipurpose molecular testing system, named PCRpack, for the rapid, high-throughput testing of infectious pathogens, including SARS-CoV-2. The system is provided in an all-in-one package, including a liquid handling instrument, a laboratory information management system, and other materials needed for testing operation; is highly customizable; and is easily implemented. PCRpack showed robust liquid handling performance, high clinical diagnostic performance, a shorter turn-around time with minimal hands-on time, and a high testing capacity. These features contribute to the rapid implementation of the high-performance and high-throughput molecular testing environment at any phase of the pandemic caused by SARS-CoV-2 or future emerging pathogens.

Summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein mediates membrane fusion between the virus and the target cells, triggering viral entry into the latter. Here, we describe a SARS-CoV-2 spike-protein-mediated membrane fusion assay using a dual functional split reporter protein to quantitatively monitor the fusion kinetics of the viral and target cell membranes in living cells. This approach can be applied in various cell types, potentially predicting the pathogenicity of newly emerging variants.For complete details on the use and execution of this protocol, please refer to Kimura et al. (2022b), Kimura et al. (2022c), Motozono et al. (2021), Saito et al. (2022a), Saito et al. (2022b), Suzuki et al. (2022), and Yamasoba et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.