Comparison of the Ct-values for genomic and subgenomic SARS-CoV-2 RNA reveals limited predictive value for the presence of replication competent virus.

SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19. In addition to the full length positive-sensed, single-stranded genomic RNA (gRNA), viral subgenomic RNAs (sgRNAs) that are required for expression of the 3' region of the genome are synthesized in virus-infected cells. However, whether these sgRNA-species might be used as a measure of active virus replication and to predict infectivity is still under debate. The commonly used methods to monitor and quantitate SARS-CoV-2 infections are based on RT-qPCR analysis and the detection of gRNA. The infectivity of a sample obtained from nasopharyngeal or throat swabs is associated with the viral load and inversely correlates with Ct-values, however, a cut-off value predicting the infectivity highly depends on the performance of the assay. Furthermore, gRNA derived Ct-values result from nucleic acid detection and do not necessarily correspond to active replicating virus. We established a multiplex RT-qPCR assay on the cobas 6800 omni utility channel concomitantly detecting SARS-CoV-2 gRNAOrf1a/b, sgRNAE,7a,N, and human RNaseP-mRNA used as human input control. We compared the target specific Ct-values with the viral culture frequency and performed ROC curve analysis to determine the assay sensitivity and specificity. We found no advantage in the prediction of viral culture when using sgRNA detection compared to gRNA only, since Ct-values for gRNA and sgRNA were highly correlated and gRNA offered a slightly more reliable predictive value. Single Ct-values alone only provide a very limited prediction for the presence of replication competent virus. Hence, careful consideration of the medical history including symptom onset has to be considered for risk stratification.

Generation of a Sleeping Beauty Transposon-Based Cellular System for Rapid and Sensitive Screening for Compounds and Cellular Factors Limiting SARS-CoV-2 Replication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile cellular infection model that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The model is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression (A549-AT). Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV-2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected A549-AT cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel model cell line allows rapid and sensitive monitoring of SARS-CoV-2 infection and the screening for host factors essential for viral replication.