Fig.4: MHV-1 infection causes diffuse alveolar damage (DAD) in the lungs of C57BL/6N and C3H/HeJ mice.
Lungs of MHV-1 and mock-infected C3H/HeJ were collected for histopathological analysis. H&E-stained images were captured at 20X, scale bar = 100 um. Semi-quantitative histopathology scores showed significant inflammation of the septa and alveoli, along with the presence of type II pneumocyte hyperplasia, in MHV-1 infected compared to mock-infected C57BL/6N (A-C) and C3H/HeJ (D-F) mice. Statistical analysis was carried out by 2way ANOVA. (G) Representative lung histopathology images from C3H/HeJ mice depicting diffuse alveolar lung damage (DAD). Lungs of MHV-1 infected mice show alveolar and septal inflammation, along with type II pneumocyte hyperplasia compared to mock-infected mice. The level of significance is represented as follows: p < 0.05 (*); p < 0.001 (**); p = 0.001 to 0.0001 (***); p < 0.0001 (****), ns = non-significant. The error bars indicate SD of the mean.
Pathways and genes altered in obese C3H/HeJ mice exposed to MHV-1 are similar to those identified in severe COVID-19 patients.COVID-19 is a complex multisystem disorder involving vast interactions of immune and inflammatory pathways55. Identifying potential disease-associated biomarkers may be useful to predict the outcome of COVID-19 in humans to assess risk levels to enable timely medical intervention. In this study, we hypothesized that blood would be an easily accessible clinical sample to monitor the complex transcriptional changes occurring early during the course of COVID-19. Therefore, we carried out RNAseq on blood from MHV-1 and mock-infected lean and obese C3H/HeJ mice at 2 dpi.
First, we carried out principal component analysis (PCA) to assess the clustering of the samples. We observed a clear separation between MHV-1 and mock-infected groups in lean and obese C3H/HeJ mice; in contrast, we observed no obvious clustering for lean and obese groups (Fig.5A), suggesting that infection, but not diet, contributes more significantly to gene expression changes between groups. Differential gene expression (DEG) analysis revealed 2635 upregulated and 863 downregulated genes in MHV-1 vs mock-infected lean mice (Supplementary Fig.S1-A) and 2089 upregulated and 681 downregulated genes in MHV-1 vs mock-infected obese mice (Supplementary Fig.S1-B). Pathway analysis was initially carried out by Gene Set Enrichment Analysis (GSEA) using Kyoto Encyclopedia of Genes and Genomes (KEGG) for identifying canonical pathways that were up or downregulated in MHV-1 infected vs mock lean (Supplementary Fig.S1-C) and obese mice (Supplementary Fig.S1-D). To account for the effect of mock infection on gene expression, we removed the genes expressed in mock-infected lean or obese mice from their MHV-1 infected counterparts and then identified unique DEGs in MHV-1 infected obese vs lean mice. There were 177 upregulated and 334 downregulated genes that were unique to MHV-1 infected obese mice compared to the infected lean mice, after adjusting for the DEG expression in their respective mocks and this list was used for generating pathways using the ShinyGO 0.77 analysis tool. These pathways were then compared to those identified in severe vs mild cases of COVID-19 (generated from data obtained from Wang et al.56) and we found numerous upregulated (Fig.5B) and downregulated (Fig.5C) pathways shared between MHV-1 infected obese vs lean C3H/HeJ mice and human patients with severe vs mild COVID-19. The common upregulated pathways included Tumor necrosis factor (TNF) signaling, NOD-like receptor (NLR) signaling, platelet activation, Neutrophil extracellular trap (NET) formation, and Rap1 signaling pathways; while the common downregulated pathways included T cell receptor signaling pathway, Th1, Th2, Th17 cell-differentiation, inflammatory bowel disease, hematopoietic cell lineage, Natural Killer (NK) cell mediated cytotoxicity and primary immunodeficiency pathways.