Single Cell Transcriptome Characteristics Of Different SARS-CoV-2 Strains Infected Cardioids And Intestinal Organoids

The novel coronavirus pneumonia caused by Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection became a global epidemic in 2020,posing a serious threat to human health.SARS-CoV-2 has evolved more than 2900 lineages,and SARSCoV-2 omicron strain is currently the main circulating strain in the world.After SARSCoV-2 infection,the patients have the symptoms of myocarditis,intestinal inflammation and other damage to the extrapulmonary organs.After recovery,they often have sustained damage to the cardiovascular and intestine,the mechanism of which is still unclear.The results showed that omicron BA.1 and BA.5 had lower mortality than the prototype strain,and BA.5 also had stronger transmission advantages.The genomes of BA.1 and BA.5 strains is different in the mutation of spike proteins.Studies based on cell and animal models have found that compared with BA.1,BA.5 has higher replication efficiency in cultured cell lines,stronger pathogenicity to cardiomyocytes and hamsters,and stronger immune escape ability in hamster infection models.However,the replication characteristics of omicron BA.1 and BA.5,and its influence on the heart and intestinal tract are still limited.Based on the cardiac and intestinal organoid culture system,this study reveals the characteristics of virus replication by constructing infection models of different strains,and explores the characteristics of antiviral immunity of cells infected with organoids by different strains from the single-cell level,in order to provide data reference for analyzing the differences in clinical symptoms and pathogenic mechanisms after SARS-CoV-2 infection.First,we established a model of SARS-CoV-2 infection in cardiac and intestinal organoids.The prototype strain,omicron BA.1 and BA.5 were used to infect the heart and intestinal organoids with 0.5 virus infection complex value,and the infected organoids were collected at 8h,24h and 48h after infection(control group was set at each time point).RT-PCR,histopathological sections and immunohistochemistry were used to detect the replication dynamics of three strains in the cardiac and intestinal organoids.The results showed that the viral nucleic acid copies on organoid culture and intracellular viral nucleic acid copies increased rapidly with the infection time,indicating that the three strains were successfully replicated and expressed in these organoids.Pathological section results showed that the infection of the three strains could cause pathological damage such as cell death,nuclear fragmentation and fibrosis in the heart and intestinal organs,and the replication ability of BA.5 was stronger than that of the prototype strain and BA.1 strain,which proved that the infection model of SARS-CoV-2 in the cardiac and intestinal organoids was successfully constructed.Based on single-cell transcriptome analysis,it was found that there are six cell subsets of cardiac organoids,which are cardiomyocytes,cardiac stem cells,fibroblasts,hepatocyte-like cells,epithelial cells and endothelial cells.Analysis of the virus replication level at different time points after infection showed that the proportion of infected cells was the highest(28.92%)with no significant statistical difference.The number of viral reads in cardiomyocytes and cardiac stem cells after infection with the three strains was relatively high,but fibroblasts(30.08%),epithelial cells(15.78%)and hepatocyte-like cells(28.64%)were the highest in the prototype strain,BA.1 and BA.5 infection groups,respectively.Infection with all three strains resulted in the conversion of cardiac stem cells and fibroblasts into myofibroblasts.Pathway activity analysis and transcription factor activity analysis showed that the hypoxia pathway was inhibited first and then activated after infection by the three strains.The prototype strain induced strong changes in the JAKSTAT pathway,activation of TGF pathway and increased expression of fibrosis-related transcription factors 48h after infection.The prototype strain was more capable of mediating apoptosis through the activation of p53,while the activity of Trial pathway was significantly increased after BA.5 infection.In the analysis of the genes related to antiviral innate immune factors and ISG,it was found that the transcriptional activities of IRF1 and STAT2 and the transcriptional levels of ISG in the infected organ cells of BA.1 and BA.5 were lower than those of the original strains,and the bystander effect of lower expression of ISG in uninfected cells was observed.It is suggested that BA.1 and BA.5 infection of cardiac organoids may cause a lower level of innate immune response.Cell communication analysis revealed that infection with all three strains could promote cardiac organoid fibrosis by enhancing the action of TGF-related ligands.BA.5 promotes fibrosis by inducing more permanent TGF pathway activation through ligand action,inhibits endothelial cell proliferation repair,causes microvascular maturation disorders,and causes vascular injury by inhibiting FGF and CXCL12-CXCR4 axis and causing VEGFA and VEGFB-related ligand imbalance.Intestinal organoid cell types include intestinal epithelial cells,embryonic stem cells,mesenchymal fibroblasts,myoepithelial cells,basal layer cells,KRT19+ smooth muscle cells,mesenchymal stem cells,and endothelial cells.The cell infection rate of BA.5(3.84%)was significantly higher than that of the prototype strain(1.785%,P<0.05).The number of viral reads was the highest in intestinal epithelial cells after infection with the three strains,but the infection rates of embryonic stem cells(2.99%),suprabasal cells(4.98%)and myoepithelial cells(5.32%)were the highest in the prototype strain,BA.1 and BA-5 infection groups,respectively.Myoepithelial cells showed a tendency to transform into KRT19+ smooth muscle cells after infection of the three strains,suggesting a tendency of fibrosis in intestinal organoids.Pathway activity analysis and transcription factor activity analysis showed that the anoxic pathway in intestinal organs was activated first and then inhibited after infection with the prototype strain and BA.5 strain,and the TGF-β pathway activity increased after infection with the prototype strain and BA.1 strain,and the JAKSTAT pathway activity increased in 48 h.p.i induced by the infection of the three strains.These results indicated that all three viral infections may cause inflammation,and BA.5 infection can reduce the TGF pathway activity of intestinal organoid cells and inhibit cell repair and proliferation.The correlation analysis between antiviral innate immune factors and ISG found that compared with prototype strain,BA.1 and BA.5 infection could induce stronger IRF3,STAT2 and ISG transcriptional activities,indicating that BA.1 and BA.5 infection could induce stronger inflammation and antiviral immune response.At the same time,the bystander effect of ISG expression decreased significantly in uninfected cells.Cell communication analysis showed that BA.5 infection could promote intestinal organoid fibrosis by enhancing the action of TGF ligand and activating the proliferation of KRT19+SMC.In the early stage of infection,the three strains could enhance the TNFSF10 ligand action through intestinal epithelial cells and induce the activation of Trail pathway in various cell types,leading to cell apoptosis.On the basis of establishing the model of SARS CoV-2 infection in cardioids and intestinal organoids,this study revealed the transcriptional differences between omicron BA.1 and BA.5 and the prototype strains after infecting organoids at the single cell level,and found that BA.5 infection can lead to stronger cardiovascular damage and tissue fibrosis in cardioids and intestinal organoids,providing data reference and theoretical basis for assisting in explaining its clinical pathogenic characteristics and searching for key genes against SARS-CoV-2.