Compatibility between haemagglutinin and neuraminidase drives the recent emergence of novel clade 2.3.4.4 H5Nx avian influenza viruses in China

Genetic reassortments between highly pathogenic avian influenza (HPAI) H5 subtype viruses with different neuraminidase (NA) subtypes have increased in prevalence since 2010 in wild birds and poultry from China. The HA gene slightly evolved from clade 2.3.4 to clade 2.3.4.4, raising the question of whether novel clade 2.3.4.4 HA broke the balance with N1 but is matched well with NAx to drive viral epidemics. To clarify the role of compatibility between HA and NA on the prevalence of H5Nx subtypes, we constructed 10 recombinant viruses in which the clade 2.3.4 or clade 2.3.4.4 HA genes were matched with different NA (N1, N2 and N8) genes and evaluated viral characteristics and pathogenicity. Combinations between clade 2.3.4 HA and N1 or between clade 2.3.4.4 HA and NAx, but not between clade 2.3.4.4 HA and N1, or between clade 2.3.4 HA and NAx, promoted viral growth, NA activity, thermostability, low-pH stability and pathogenicity in chicken and mice. These findings suggest that both clade 2.3.4 HA/N1 and clade 2.3.4.4 HA/NAx displayed a better match, which could promote the increased prevalence of clade 2.3.4 H5N1 AIV (prior to 2010) and clade 2.3.4.4 H5Nx AIV (since 2010) in China, respectively.