ABSTRACT
COVID-19 is caused by severe acute respiratory SARS-CoV-2. Regardless of the availability of treatment strategies for COVID-19, effective therapy will remain essential. A promising approach to tackle the SARS-CoV-2 could be small interfering (si) RNAs. Here we designed the small hairpin RNA (named as shRNA688) for targeting the prepared 813 bp Est of the S protein genes (Delta). The conserved and mutated regions of the S protein genes from the genomes of the SARS-CoV-2 variants in the public database were analyzed. A 813 bp fragment encoding the most part of the RBD and partial downstream RBD of the S protein was cloned into the upstream red florescent protein gene (RFP) as a fusing gene in the pCMV-S-Protein RBD-Est-RFP plasmid for expressing a potential target for RNAi. The double stranded of the DNA encoding for shRNA688 was constructed in the downstream human H1 promoter of the plasmid in which CMV promoter drives enhanced green fluorescent protein (EGFP) marker gene expression. These two kinds of the constructed plasmids were co-transfected into HEK293T via Lipofectamine 2000. The degradation of the transcripts of the SARS-CoV-2 S protein fusing gene expressed in the transfected HEK293T treated by RNAi was analyzed by RT-qPCR with a specific probe of the targeted SARS-CoV-2 S protein gene transcripts. Our results showed that shRNA688 targeting the conserved region of the S protein genes could effectively reduce the transcripts of the S protein genes. This study provides a cell model and technical support for the research and development of the broad-spectrum small nucleic acid RNAi drugs against SARS-CoV-2 or the RNAi drugs for the other hazard viruses which cause human diseases. Copyright © Weiwei Zhang, Linjia Huang, Jumei Huang, Xin Jiang, Xiaohong Ren, Xiaojie Shi, Ling Ye, Shuhui Bian, Jianhe Sun, Yufeng Gao, Zehua Hu, Lintin Guo, Suyan Chen, Jiahao Xu, Jie Wu, Jiwen Zhang, Daxiang Cui, and Fangping Dai.
ABSTRACT
Introduction: Obesity is a major risk factor for severe coronavirus disease, and clinical evidence now supports the GI tract, in addition to the respiratory system, as a potential route for SARS-CoV-2 infection. Expression of viral entry factors ACE2, TMPRSS2, and CTSL have been detected along the human GI tract including gastric, ileal and colonic mucosa. It is unclear whether obesity confers increased susceptibility to initial SARS-CoV-2 infection, or what gut mechanisms in obesity predispose to vulnerability to SARS-CoV-2. Thus, we aimed to investigate, by single cell RNA-sequencing (scRNA-Seq) of human colonic mucosa, whether patients with obesity may be more susceptible to SARS-CoV-2 infection, by virtue of enhanced expression of SARS-CoV2 entry cofactors followed protein assessment in colon biopsies. Methods: We studied 19 patients: 10 lean (age 33±3y, BMI 23±1kg/m2, 90% female), and 9 with obesity (age 43±3y, BMI 36±1kg/m2, 89% female). Human colonic biopsies from lean (n=4 scRNA-Seq;n=6 validation) and obesity (n=6 scRNASeq;n=3 validation) participants were obtained by sigmoidoscopy. Biopsies were dissociated, and viable cells were FACS-isolated. Chromium-10X Genomics was used for scRNA-Seq library prep, followed by Illumina HiSeq4000 sequencing. COVID-19 entry factors displaying significant differential expression between lean and obesity were then validated for gene, and protein expression in the validation cohort using Illumina TruSeq, and quantitative immunofluorescence confocal microscopy, respectively. Results: The initial dataset analysis revealed sequencing of 59,653 cells, 705 million reads, at 127,000 reads per cell. The human colonic mucosa partitioned into 20 cell subsets (Fig1A,B), and 15 of the 20 clusters displayed detectable expression of at least one of the COVID-19 entry factors: TMPRSS2, CTSL, or ACE2 (Fig1C,D,E). Goblet cell expression of TMPRSS2 was increased 4.6-fold (p<0.05), stromal cell expression of CTSL was increased 1.2-fold (p<0.0001), and ACE2 expression was increased 1.27-fold (p<0.001) in crypt-top (CT) colonocytes of obesity compared to lean controls (Fig2A). Colonic overexpression of TMPRSS2 mRNA (p<0.05) and protein (p<0.05), and CTSL (p<0.05) mRNA, but not ACE2 mRNA, in obesity was further validated in a second validation cohort (Fig2B-F). Conclusions: scRNA-Seq analysis of human colonic epithelium in obesity compared to healthy controls revealed multiple epithelial cell subsets (goblet cell, stromal, and colonocytes) with overexpression of COVID-19 entry factors TMPRSS2, CTSL, and ACE2, confirming the digestive system as a portal for infection by SARS-CoV-2.Furthermore goblet, stromal, and colonocyte-specific overexpression of TMPRSS2, CTSL, and ACE2 in obesity may play a significant role in increased initial susceptibility to COVID-19, and worse disease outcomes in human obesity.(Figure Presented) Single-Cell RNA-Seq Profiling of Human Colonic Epithelium in Obesity. A) t-SNE plot of single-cell RNA-seq profiles of native human colonic epithelial cells, colored by cluster number and identity, listed by largest to smallest population, and annotated by cluster identity, determined by highest ranking gene marker for the colonic cells clustered and profiled between lean and obesity, where dotted blue, red, and green circles represents goblet cells, stromal cells, and CT colonocytes, respectively. B) Proposed cluster identities based on conserved expression of known markers for annotated cell types. Clusters identified displayed expression of at least one of the COVID-19 entry factors: TMPRSS2, CTSL, or ACE2. The average proportion of cells in annotated clusters expressing, C) TMPRSS2, D) CTSL, and E) ACE2 among all studied participants.
ABSTRACT
Objective: SARS-CoV-2 causes the coronavirus disease 2019 (COVID-19) and has spawned a global health crisis. Virus infection can lead to elevated markers of cardiac injury and inflammation associated with a higher risk of mortality. However, it is so far unclear whether cardiovascular damage is caused by direct virus infection or is mainly secondary due to inflammation. Recently, additional novel SARS-CoV-2 variants have emerged accounting for more than 70% of all cases in Germany. To what extend these variants differ from the original strain in their pathology remains to be elucidated. Here, we investigated the effect of the novel SARS-CoV-2 variants on cardiovascular cells. Results: To study whether cardiovascular cells are permissive for SARSCoV-2, we inoculated human iPS-derived cardiomyocytes and endothelial cells from five different origins, including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells, in vitro with SARS-CoV-2 isolates (G614 (original strain), B.1.1.7 (British variant), B.1.351 (South African variant) and P.1 (Brazilian variant)). While the original virus strain infected iPS-cardiomyocytes and induced cell toxicity 96h post infection (290±10 cells vs. 130±10 cells;p=0.00045), preliminary data suggest a more severe infection by the novel variants. To what extend the response to the novel variants differ from the original strain is currently investigated by phosphoproteom analysis. Of the five endothelial cells studied, only human coronary artery EC took up the original virus strain, without showing viral replication and cell toxicity. Spike protein was only detected in the perinuclear region and was co-localized with calnexin-positive endosomes, which was accompanied by elevated ER-stress marker genes, such as EDEM1 (1.5±0.2-fold change;p=0.04). Infection with the novel SARS-CoV-2 variants resulted in significant higher levels of viral spike compared to the current strain. Surprisingly, viral up-take was also seen in other endothelial cell types (e.g. HUVEC). Although no viral replication was observed (850±158 viral RNA copies at day 0 vs. 197±43 viral RNA copies at day 3;p=0.01), the British SARS-CoV-2 variant B.1.1.7 reduced endothelial cell numbers (0.63±0.03-fold change;p=0.0001). Conclusion: Endothelial cells and cardiomyocytes showed a distinct response to SARS-CoV-2. Whereas cardiomyocytes were permissively infected, endothelial cells took up the virus, but were resistant to viral replication. However, both cell types showed signs of increased toxicity induced by the British SARS-CoV-2 variant. These data suggest that cardiac complications observed in COVID-19 patients might at least in part be based on direct infection of cardiovascular cells. The more severe cytotoxic effects of the novel variants implicate that patients infected with the new variants should be even more closely monitored.