Project description:Age-matched K18-hACE2 transgenic mice were infected intranasally with different SARS-CoV-2 viruses, including (1) USA-WA1/2020 (WA) of lineage A, (2) New York-PV09158/2020 (NY) of lineage B.1.3, (3) USA/CA_CDC_5574/2020 (CA) of lineage B.1.1.7 and (4) hCoV-19/South Africa/KRISP-EC-K005321/2020 (SA) of lineage B.1.351. Mouse lungs were harvested on 3 days post infection (dpi). Total RNA was extracted using QIAgen RNeasy Plus Mini Kit and was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Resultant cDNA was used as the template along with RT2 SYBR Green ROX qPCR Mastermix (Qiagen) to perform RT² Profiler™ PCR Array Mouse Hypoxia Signaling Pathway (Qiagen) real-time PCR in Stratagene MX3000p qPCR system.
Project description:Clonal hematopoiesis (CH) results from enhanced fitness of a mutant hematopoietic stem and progenitor cell (HSPC), but how such clones expand is unclear. Here, we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH-associated genes, like asxl1, promoted clonal dominance. Single-cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti-inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator, nr4a1, abrogated the ability of asxl1-mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny.
Project description:Clonal hematopoiesis (CH) results from enhanced fitness of a mutant hematopoietic stem and progenitor cell (HSPC), but how such clones expand is unclear. Here, we developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in CH-associated genes, like asxl1, promoted clonal dominance. Single-cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti-inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator, nr4a1, abrogated the ability of asxl1-mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny.
Project description:A new phase of the COVID-19 pandemic has started as SARS-CoV-2 variants with increased transmissibility are emerging globally, calling for the development of reliable platforms to rapidly phenotype viruses. Currently, no rapid phenotyping system exists that both accurately models human biology and can be standardized properly. Organoids accurately model viral target cells in vivo, can be established from many tissues, passaged indefinitely, biobanked and shared. We found that the British variant (clade B.1.1.7), compared to an ancestral 614G-containing clade B virus, produced higher levels of infectious virus late in infection and had a higher replicative fitness in human airway, alveolar and intestinal organoid models. Our findings unveil extended shedding as a correlate of fitness for SARS-CoV-2, possibly explaining the rapid emergence of SARS-CoV-2 B.1.1.7. Combined with genomic surveillance systems, virus phenotyping using standardized organoid models may be implemented into public health decision making.
Project description:hACE2 transgenic mice were infected with the original SARS-CoV-2 strain (B.1) and the Beta (B.1.351) variant. Lung and spleen samples were collected 1 day post infection (DPI), 3 DPI and 5 DPI, and mRNA was sequenced.