Oltean T, Maelfait J, Saelens X. Need for standardization of Influenza A virus-induced cell death in vivo to improve consistency of inter-laboratory research findings. Cell Death Discov. 2024 May 22;10(1):247
The involvement of necroptosis in the control of influenza A virus (IAV) infection has been reported in multiple studies. Downstream of the nucleic acid sensor ZBP1, RIPK3 kinase activity is critically involved in the induction of necroptotic cell death by phosphorylating MLKL, while RIPK3 as a scaffold can induce apoptosis. Paradoxically, RIPK3-deficiency of mice may result in increased or decreased susceptibility to IAV infection. Here, we critically review the published reports on the involvement of RIPK3 in IAV infection susceptibility and try to identify differences in experimental settings that could explain seemingly conflicting outcomes. Analysis of the experimental reports revealed differences in the IAV challenge dose, the IAV inoculum preparation, IAV titer assessment, as well as the route of inoculation between studies. Furthermore, differences were noticed in the inclusion of littermate controls, which show high variance in viral sensitivity. Our evaluation argues for a standardized setup for IAV infection experiments including the preparation of the IAV virus, the use of different IAV infectious doses description and the proper experimental genetic controls of the mouse strains to increase inter-laboratory consistency in this field. Workflow for IAV infection studies in vivo: Viral preparation and titer assessment should be as standardized as possible with the use of a universal repository (such as BEI resources). Infection studies in genetically modified mice and littermate controls should include dose-response experimentation, following a defined infection route and inoculation volume. Data are generated by consistent analysis methods.
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