SPECIFIC DETECTION and REPLICATION KINETICS of SARS-CoV-2 USING smRNA-FISH

ABSTRACT

Background: SARS-CoV-2 is a positive-sense single-stranded RNA virus and its replication begins after the synthesis of virally encoded polymerase complex that is required for replication and transcription of genomic RNA (gRNA) within the infected cells. Despite the global interest in the study of SARS-CoV-2, the kinetics of SARS-CoV2 RNA replication and transcription during the early phase of viral infection is poorly understood. Here, we used the single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) for sensitive detection of SARS-CoV-2 at single molecule level and to determine the replication of genomic RNA (gRNA) and sub-genomic RNA (sgRNA) in the infected cells, at very early stages of infection. Methods: We designed highly specific smRNA-FISH probes targeted to gRNA and Spike gene sgRNA of SARS-CoV-2 virus, using stellaris method and optimized the method to simultaneously visualize these two RNAs at single cell and single molecule level. Because of the high sensitivity of our probes, we applied smRNA-FISH technology to detect SARS-CoV-2 positive cells from autopsy samples obtained from diseased COVID-19 patients. Furthermore, we used high-resolution and high-speed scanning microscopy to detect extent of infection in cell models of SARS-CoV-2 and in COVID-19 patient samples. Results: A time course analysis SARS-CoV-2 replication indicated that single molecules of gRNA could be detected as little as 30 min to 2 hr. post-infection. Distinct "Replication Centers" (RC) began to appear one to two hours post-infection and the sgRNAs began to migrate out of these RCs. Replication after the initial delay appeared to be rapid and gRNA and sgRNAs dispersed throughout the cell within 4-5 hours post infection forming multiple RCs. We found that our RNA-FISH correctly detected the SARS-CoV-2 positive samples from patient autopsy samples that were characterized by qRT-PCR or immunological detection methods. The signals of spike gRNA and sgRNA along with the spike proteins co-localized within the same cells of the SARS-CoV-2 infected patients within the cells of lung, kidney, and heart autopsy samples. Conclusion: We propose that the specific probes and the methodology that we have developed will be highly applicable to the study of SARS-CoV-2 replication in depth and to characterize SARS-CoV-2 infection in COVID-19 patient samples. This study may open a novel direction towards COVID-19 pathophysiology, drug screening and diagnostics.