Adaptation of avian influenza H5N1 virus in humans: The role of the neuraminidase

As a member of the family Orthomyxoviridae, the avian influenza viruses (AIV) contain a segmented RNA genome with negative strand orientation. The two surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) enable the differentiation into 16 HA and 9 NA antigenic subtypes. The spread and endemicity of H5Nx Goose/Guangdong AIV pose a continuous zoonotic and pandemic threat. Here, after analysis of the NA sequence of human-origin H5N1 viruses, we studied the role of mutations in residues 46, 204, 219 and 430 for virus fitness. Although H5N1 AIV with avian- or human-like NA had similar replication efficiency in avian cells, human-like NA enhanced replication in human airway epithelia and reduced NA activity conferred by an L204M mutation. This mutation consistently reduced NA activity in nine other influenza viruses, indicating a broad effect. Viruses with reduced NA activity had lower NA expression levels due to reduced viral RNA replication and NA transcription. They demonstrated increased accumulation of NA at the cell membrane and enhanced cell-to-cell spread. Furthermore, NA mutations A46D, S319F and 430G increased virus binding to human-type receptors. While not affecting the high virulence of H5N1 in chickens, NA mutations in human-origin viruses modulated virulence and replication in mice and ferrets. Together, we describe novel mutations in the NA of human-origin H5N1 viruses and studied the underlying mechanism for potential adaptation of H5N1 to mammals.