Increased Zoonotic Risk Posed by Emerging Haemagglutinin Variants of Clade 2.3.4.4b A(H5Nx) High Pathogenicity Avian Influenza Virus

High pathogenicity avian influenza viruses (HPAIVs) remain a major threat to animal and human health. The clade 2.3.4.4b H5Nx panzootic has been marked by increasing cross-species adaptation, including outbreaks in dairy cattle. The surge in mammalian infections in both wild and captive populations heighten concerns about zoonotic and pandemic potential. Here, we analyse haemagglutinin (HA) evolution in clade 2.3.4.4b H5Nx viruses from 2020 to 2025, focusing on key residues within the receptor-binding domain. We identify HA E185K (mature H5 HA numbering; E189K in H3 numbering) as a novel immune escape mutation that confers increased binding to avian- and human-type sialic acid receptors. Strikingly, this mutation is increasingly prevalent in cattle-derived H5N1 viruses and enhances binding to human-like receptors. However, ferret studies demonstrated that whilst pathogenicity of the E185K virus was comparable to the cattle derived isolate, the E185K mutation did not confer transmission to direct or indirect contact ferrets. Our results reveal a mechanism linking immune escape with expanded receptor specificity, highlighting a potential pathway for mammalian adaptation. These findings underscore the need for continued surveillance of evolving H5Nx viruses to mitigate emerging zoonotic threats.