ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause acute respiratory disease and multiorgan failure. Finding human host factors that are essential for SARS-CoV-2 infection could facilitate the formulation of treatment strategies. Using a human kidney cell line-HK-2-that is highly susceptible to SARS-CoV-2, we performed a genome-wide RNAi screen and identified virus dependency factors (VDFs), which play regulatory roles in biological pathways linked to clinical manifestations of SARS-CoV-2 infection. We found a role for a secretory form of SARS-CoV-2 receptor, soluble angiotensin converting enzyme 2 (sACE2), in SARS-CoV-2 infection. Further investigation revealed that SARS-CoV-2 exploits receptor-mediated endocytosis through interaction between its spike with sACE2 or sACE2-vasopressin via AT1 or AVPR1B, respectively. Our identification of VDFs and the regulatory effect of sACE2 on SARS-CoV-2 infection shed insight into pathogenesis and cell entry mechanisms of SARS-CoV-2 as well as potential treatment strategies for COVID-19.
Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Host Microbial Interactions/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vasopressins/immunology , Virus Internalization , COVID-19/immunology , COVID-19/virology , Cell Line , Humans , Protein BindingABSTRACT
BACKGROUND: SARS-CoV-2 infection is associated with myocardial injury, but there is a paucity of experimental platforms for the condition.MethodsâandâResults:Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) infected by SARS-CoV-2 for 3 days ceased beating and exhibited cytopathogenic changes with reduced viability. Active viral replication was evidenced by an increase in supernatant SARS-CoV-2 and the presence of SARS-CoV-2 nucleocaspid protein within hiPSC-CMs. Expressions of BNP, CXCL1, CXCL2, IL-6, IL-8 and TNF-α were upregulated, while ACE2 was downregulated. CONCLUSIONS: Our hiPSC-CM-based in-vitro SARS-CoV-2 myocarditis model recapitulated the cytopathogenic effects and cytokine/chemokine response. It could be exploited as a drug screening platform.
Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/complications , Induced Pluripotent Stem Cells/virology , Myocarditis/complications , Myocytes, Cardiac/virology , Pneumonia, Viral/complications , Angiotensin-Converting Enzyme 2 , Betacoronavirus/genetics , COVID-19 , Cell Survival , Cells, Cultured , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Cytokines/metabolism , Cytopathogenic Effect, Viral , Drug Evaluation, Preclinical/methods , Humans , Induced Pluripotent Stem Cells/metabolism , Myocarditis/metabolism , Myocarditis/virology , Myocytes, Cardiac/metabolism , Nucleocapsid Proteins/metabolism , Pandemics , Peptidyl-Dipeptidase A/metabolism , Phosphoproteins , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Virus ReplicationABSTRACT
To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.