2.2. Influenza virus and heat inactivation

Mouse-adapted influenza A virus, A/WSN/1933 (H1N1), was sourced from BEI Resources, NIAID, NIH (catalog number NR-3688, titered in MDCK cells). The number of infectious units used (280 TCID50) was determined by pilot experiments, where rates of survival and ventriculomegaly were optimized following intracerebroventricular injections in P4 mice. To heat-inactivate A/WSN/1933 influenza virus, frozen virus was thawed, mixed, and incubated in a 70°C thermal block for 30-min. Virus inactivation was confirmed according to Jonges et al. (2010) by culturing inactivated samples on MDCK cells and looking at lack of cytopathic effects, as well as by quantitative reverse transcription PCR (RT-qPCR) analysis for lack of influenza virus mRNA production in infected MDCK cells.