Besides pandemic SARS-CoV-2, also endemic seasonal human common cold coronaviruses (hCoVs) have a significant impact on human health and economy. Studies on hCoVs and the identification of antivirals are therefore crucial to improve human well-being. However, hCoVs have long been neglected and the methodology to study virus infection, replication and inhibition warrants being updated. We here evaluated the established plaque-based assays to determine viral titers and cell-to-cell spread and developed protocols for the immunodetection of the viral nucleocapsid protein by flow cytometry and in-cell ELISA to study infection rates at early time points. The developed protocols allow detection of hCoV-229E infection after 2, and hCoV-NL63 and -OC43 infection after 3 days at a single cell level or in a 96 well microtiter format, in large sample numbers without being laborious or expensive. Both assays can be applied to assess the susceptibility of cells to hCoV infection and replication, and to determine the efficacy of antiviral compounds as well as neutralizing antibodies in a sensitive and quantitative manner. Application revealed that clinically applied SARS-CoV-2 targeting monoclonal antibodies are inactive against hCoVs, but that the viral polymerase targeting antivirals remdesivir and molnupiravir are broadly active also against all three hCoVs. Further, the in-cell ELISA provided evidence that nirmatrelvir, previously shown to broadly inhibit coronavirus proteases, also prevents replication of authentic hCoVs. Importantly, the protocols described here can be easily adapted to other coronavirus strains and species as well as viruses of other families within a short time. This will facilitate future research on known and emerging (corona)viruses, support the identification of antivirals and increase the preparedness for future virus outbreaks.
Keywords: Antiviral testing; Drug screening; In-cell ELISA; Seasonal coronaviruses.
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