Fig. 7: Spatial analysis of gene expression in primary and re-infected animals show altered inflammatory and type I IFN response and increased IFNγ response in re-infected animals.

C57BL/6 mice that were either naive or infected 30 days earlier with Influenza A virus (IAV-WSN), were infected with IAV-X31, their lungs harvested 2 days later, their epithelial cells sorted by FACS and their gene expression analyzed by Nanostring nCounter or lungs fixed and embedded in paraffin. A, B Heatmap and example boxplots showing Differentially Expressed Genes (DEG) from primary or re-infected animals in FACS sorted epithelial cells. DEG with a twofold change and an FDR q value < 0.05 in lung epithelial cells were considered statistically significant to take into account multiple comparisons. Primary group (n = 4) and re-challenge group (n = 7), data are from two independent experiments combined and each dot represents a mouse, the bounds of the boxes are the 25–75th percentiles, the lines in the boxes are at the median and the whiskers show the min and the max values. C RNAscope analysis for X31-IAV was performed on FFPE lung tissue; representative X31-IAV+ areas are shown in red and labeled with black arrows to highlight virus + bronchial epithelium. Bars, 200 micrometers. Virus-positive and negative airways from six primary and three re-infected mice were analyzed by GeoMX. D DEG between normal tissue and three areas: virus+ airway cells; virus negative cells within the same airway (Close); and adjacent virus negative airways (Further) were classified based on presence within MGI GO terms ‘Inflammation’ or ‘Type 1 IFN’. E Heatmap showing DEG between X31-IAV positive airways in primary versus re-challenge groups, DEG with a twofold change and an FDR q value < 0.05 in lung epithelial cells were considered statistically significant. F Enriched pathways found in DEG identified between viral+ airways in day 2 primary or re-infected C57BL/6 mice analyzed by GeoMx. The x axis represents the −log10 of the P value given during the analysis; (FDR < 0.05 to take into account multiple comparisons) with the number of DEG listed from each pathway. Source data for B, D, and F data are provided as a Source Data file.