ABSTRACT
A simple and robust cell culture system is essential for generating authentic SARS-CoV-2 stocks for evaluation of viral pathogenicity, screening of antiviral compounds, and preparation of inactivated vaccines. Evidence suggests that Vero E6, a cell line commonly used in the field to grow SARS-CoV-2, does not support efficient propagation of new viral variants and triggers rapid cell culture adaptation of the virus. We generated a panel of 17 human cell lines overexpressing SARS-CoV-2 entry factors and tested their ability to support viral infection. Two cell lines, Caco-2/AT and HuH-6/AT, demonstrated exceptional susceptibility, yielding highly concentrated virus stocks. Notably, these cell lines were more sensitive than Vero E6 cells in recovering SARS-CoV-2 from clinical specimens. Further, Caco-2/AT cells provided a robust platform for producing genetically reliable recombinant SARS-CoV-2 through a reverse genetics system. These cellular models are a valuable tool for the study of SARS-CoV-2 and its continuously emerging variants.
ABSTRACT
Since the beginning of the COVID-19 pandemics, variants have emerged. Some of them display increased transmissibility and/or resistance to immune response. Most of the mutations involved in the functional adaptation are found in the receptor-binding motif (RBM), close to the interface with the receptor ACE2. We thus developed a fast molecular assay to detect mutations in the RBM coding sequence. After amplification, the amplicon is heat-denatured and hybridized with an amplicon of reference. The presence of a mutation can be detected using a mismatch-specific endonuclease and the cleavage pattern is analyzed by capillary electrophoresis. The method was validated on RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants produced in vitro before being implemented for clinical samples. The assay showed 97.8% sensitivity and 97.8% specificity. The procedure can be set up for high-throughput identification of the presence of mutations and serve as a first-line screening to select the samples for full genome sequencing.
ABSTRACT
The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a significant threat to global health. While respiratory aerosols or droplets are considered as the main route of human-to-human transmission, secretions expelled by infected individuals can also contaminate surfaces and objects, potentially creating the risk of fomite-based transmission. Consequently, frequently touched objects such as paper currency and coins have been suspected as potential transmission vehicle. To assess the risk of SARS-CoV-2 transmission by banknotes and coins, we examined the stability of SARS-CoV-2 and bovine coronavirus, as surrogate with lower biosafety restrictions, on these different means of payment and developed a touch transfer method to examine transfer efficiency from contaminated surfaces to fingertips. Although we observed prolonged virus stability, our results indicate that transmission of SARS-CoV-2 via contaminated coins and banknotes is unlikely and requires high viral loads and a timely order of specific events.