Embryonated chicken eggs clear systemic H3N2 influenza without RIG-I: transcriptomic evidence for innate sufficiency and brain immune privilege

1 Abstract An apparent paradox drives this study: H3N2 influenza virus concentrates in the brain of infected 10-day chicken embryos while kidney and lung, which express the same viral entry receptors (ST3GAL3 and other sialic acid receptors), are essentially virus-free. Using mRNA-seq on brain, kidney, and lung from H3N2-infected 10-day chicken embryos, we resolve this paradox identifying immune privilege rather than neurotropism as the cause; circulating macrophages clear the virus from peripheral tissues but cannot cross the embryonic brain barrier. The innate response is robust despite lacking RIG-I: MDA5/IFIH1 and TLR3-TLR7-IRF signaling compensate fully, driving complete viral clearance in peripheral organs. At 48 h post-infection, kidney and lung are in a post-clearance M2 macrophage state; complement is activated but lacks both the H3N2-specific antibodies and the terminal C9 component for productive effect. These findings directly challenge the hypothesis that RIG-I loss renders chickens susceptible to influenza, and identify the embryonic brain as an immune-privileged viral sanctuary with implications for influenza neurological disease in young hosts. Competing Interest Statement The authors have declared no competing interest. Footnotes Glossary - APP - Acute phase proteins - BBB - Blood-brain barrier - CP - Ceruloplasmin; a copper-binding ferroxidase involved in iron oxidation and transport - CTL - Cytotoxic T Lymphocytes - DEG - Differentially expressed gene - ECE - Embryonated chicken egg - HA - Hemagglutination - IFN - Interferon - IRF - Interferon regulatory factor - ISG - Interferon-stimulated gene - MAC - Membrane attack complex - MDA5 - Melanoma differentiation-associated protein 5; officially IFIH1. Primary cytosolic RNA sensor in chickens, compensating for absent RIG-I - NAMPT - Nicotinamide phosphoribosyltransferase - NK - Natural Killer - NHA - No hemagglutination - PAMP - Pathogen-Associated Molecular Patterns - PRR - Pattern recognition receptor - RIG-I - DDX58;Retinoic acid-inducible gene I; recognizes viral RNA and initiates the innate antiviral response; absent in Galliformes - RLR - RIG-I-like receptor - SPF - Specific pathogen-free - TLR - Toll-like receptor