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

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 establish and propose a resolution to this paradox, by identifying immune privilege, rather than neurotropism, as the more likely explanation; circulating macrophages likely clear the virus from peripheral tissues but seemingly cannot cross the embryonic brain barrier. The innate response is robust despite lacking RIG-I: MDA5/IFIH1 and TLR3-TLR7-IRF signaling compensate robustly, likely driving complete viral clearance in peripheral organs. At 48 h post-infection, macrophages in kidney and lung are in a post-clearance M2 state; complement is activated but lacks both the H3N2-specific anti-bodies and the terminal C9 component for productive effect. These findings challenge the hypothesis that RIG-I loss necessarily renders chickens more susceptible to influenza: complete peripheral clearance of a productively infecting H3N2 strain in the absence of RIG-I is difficult to reconcile with an obligate role for RIG-I in this host’s antiviral defense. We also identify the embryonic brain as a potentially immune-privileged viral sanctuary with implications for influenza neurological disease in young hosts.