Gene Expression
Nebulas
Gene Expression NebulasSummary: SARS-CoV-2 primarily affects the respiratory system but extra-pulmonary manifestations in individuals with COVID-19 are commonly seen. All major organ systems have been reported to be affected by SARS-CoV-2 and complications arising from ensuing organ dysfunction significantly increase the mortality rate of COVID-19. Yet, despite the clinical importance of systemic involvement of SARS-CoV-2, little is known about the pathogenesis of extra-pulmonary complications of COVID-19. Here, we create a murine model of SARS-CoV-2 induced severe systemic toxicity and multi- organ involvement by expressing the human ACE2 transgene in multiple tissues using an adeno associated virus (serotype 9) followed by administration of the SARS-CoV-2 virus intra-peritoneally. The animals develop a profound phenotype within 7 days of SARS-CoV-2 infection with severe weight loss, morbidity and failure to thrive, that necessitated euthanasia. We demonstrate that following a robust anti-viral immune response, there is metabolic suppression of oxidative phosphorylation and the tri- carboxylic acid (TCA) cycle in multiple organs with neutrophilia, lymphopenia and splenic atrophy mirroring human COVID-19 phenotypes with adverse prognosis. Animals had a significantly lower heart rate and electron microscopy demonstrated myofibrillar disarray and myocardial edema, a common pathogenic cardiac phenotype in human COVID-19. An organ wide metabolic reprogramming consistent with depression of oxidative phosphorylation led to utilization of peripheral fat stores and gross accumulation of fat in the heart and other vital organs. We perform metabolomic profiling of peripheral blood and identify a panel of TCA cycle metabolites that serve as biomarkers of depressed oxidative phosphorylation, several of these markers been noted in human clinical studies to be associated with adverse prognosis. Finally, we observed that SARS-CoV-2 induces epigenetic changes with a significant number of differentially methylated sites in vital organs, across the whole host cell genome, that affects expression of immune response genes and could in part contribute to dysregulated gene expression in affected tissues. Our model suggests that SARS-CoV- 2 induced metabolic reprogramming and epigenetic changes in internal organs could contribute to systemic toxicity and lethality in COVID-19
Overall Design: Examination of expression changes of mouse organs with nCOV2 infection
| Strategy: |
|
| Species: |
|
| Tissue: |
|
| Healthy Condition: |
|
| Cell Type: |
|
| Cell Line: |
|
| Development Stage: |
|
| Growth Protocol: | All animal studies were approved by the Animal Research Committee, University of California, Los Angeles. Male C57Bl/6 mice (000664, Jackson Labs) (14-17 weeks) were housed in groups and fed standard chow diets. AAV9-CMV-hACE2 (AAV- 200183, Vector Biolabs) or control AAV9-CMV-eGFP viruses were purchased from Vector Biolabs. Animals were injected intravenously with 100-μl injection containing 2x1012 genomic copies of AAV-CMV-hACE2 or control (AAV-CMV-eGFP). Animals were housed in BSL-3 facility for the duration of the experiment (n=5/cage). Cage food weight and individual mouse body weight were recorded daily after SARS-CoV- 2 virus infection; All animal studies were approved by the Animal Research Committee, University of California, Los Angeles. Male C57Bl/6 mice (000664, Jackson Labs) (14-17 weeks) were housed in groups and fed standard chow diets. AAV9-CMV-hACE2 (AAV- 200183, Vector Biolabs) or control AAV9-CMV-eGFP viruses were purchased from Vector Biolabs. Animals were injected intravenously with 100-μl injection containing 2x1012 genomic copies of AAV-CMV-hACE2 or control (AAV-CMV-eGFP). Animals were housed in BSL-3 facility for the duration of the experiment (n=5/cage). Cage food weight and individual mouse body weight were recorded daily after SARS-CoV- 2 virus infection. |
| Treatment Protocol: | SARS-CoV-2, isolate USA-WA1/2020, was got from the Biodefense and Emerging Infections (BEI) Resources of the National Institute of Allergy and Infectious Diseases (NIAID). SARS-CoV-2 was passaged once in Vero-E6 cells (ATCC) and viral stocks were aliquoted and stored at -80 C. Virus titer was determined by plaque assay using Vero E6 Cells. 200μL of SARS-CoV2 (0.5 x 106 PFU/mL) was injected intraperitoneally. |
| Extract Protocol: | All samples were stored in RNA later stabilization solution and subsequently total RNA was extracted using RNA mini Kit (7326830, BioRad). cDNA was synthesized by using iScript cDNA Synthesis Kit (1708890, BioRad) and qPCR performed. |
| Library Construction Protocol: | For RNA-sequencing, libraries were prepared by the Technology Center for Genomics & Bioinformatics at UCLA using Illumina TruSeq Stranded Total RNA Sample Prep kit and sequenced with 50bp single end reads on an Illumina HiSeq3000. |
| Molecule Type: | rRNA- RNA |
| Library Source: | |
| Library Layout: | SINGLE |
| Library Strand: | Forward; Reverse |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 3000 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
|---|