Establishing ex vivo infection to influenza A virus in mouse precision cut lung slices

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1. Precision cut lung slices (PCLS) offer a unique integrated experimental platform for investigating both inflammatory and immune responses to viral infection. 2. This study aimed to develop a protocol for robust infection of PCLS <i>ex vivo</i> with influenza A virus (IAV) with a view to its application for assessment of antiviral agents. 3. PCLS were prepared using agarose-inflated lungs from naïve C57Bl6 mice and infected with IAV (HKx31 mouse strain). Matched slices from each mouse at 3 IAV concentrations (1x10⁴-1x10⁶ PFU) were assessed 24 and 48 hr post infection (n=4-8). Viral loads were assessed by plaque assay of homogenised PCLS. Conditioned media was used to measure both cell death via LDH assay and inflammatory responses to infection via TNFα&nbsp;ELISA. 4. A dose-dependent increase in viral load was observed at 24 hr, with a further 3-fold increase in plaque numbers at 48 hr at the lowest IAV concentration only (1x10⁴PFU, p&lt;0.05, paired t-test).&nbsp;LDH was increased at 48 hr, irrespective of viral load. TNFα levels in conditioned media were increased 5-fold between 24 and 48 hr with 1x10⁴PFU IAV (57±11, 243± 56 pg/ml, p&lt;0.01). 5. Treatment of mouse PCLS with IAV <i>ex vivo</i> elicits dose- and time-dependent infection and release of inflammatory cytokines. Further studies assessing immune responses and sensitivity to viral treatment are required. PCLS&nbsp;may be a viable screening tool for pre-clinical assessment of mechanisms of infection and validation of new therapeutic targets.