Mouse infections and health monitoring
Mice were transferred to disposable cages from Innovive (San Diego, CA, USA) and moved to a separate room, 3-4 days before MHV-1 infection. MHV-1 was a kind gift from Dr. Susan Compton and was propagated in 17Cl1 cells; infectious titers were determined by plaque assays on L2 Percy cells. Mice were anesthetized with ketamine and xylazine (90 mg/kg and 5 mg/kg respectively) intraperitoneally and then inoculated intranasally with 3x104 plaque-forming units (PFUs) of the virus in 50 µL of Roswell Park Memorial Institute (RPMI)-1640 media with no additives or only 50 µL of RPMI-1640 (mock-infection). Mice were monitored for visual symptoms of disease like inactivity, weight loss and mortality. They were euthanized at 8 dpi or when they crossed the threshold of weight loss (20% of weights at 0 dpi), by injecting ketamine and xylazine (90 mg/kg and 10 mg/kg respectively) intraperitoneally followed by cervical dislocation. For histopathology, the left lung lobe was perfused with and stored in 4% formaldehyde for at least a week. We also collected sections of the liver, spleen, and small intestine for histopathological analyses. The Virginia Tech Animal Laboratory Services (ViTALS) performed paraffin embedding, sectioning and hematoxylin-eosin staining, and a board-certified pathologist scored the slides in a blinded manner.