A single-amino-acid mutation at position 225 in hemagglutinin attenuates H5N6 influenza virus in mice.

The highly pathogenic avian influenza H5N6 viruses are widely circulating in poultry and wild birds, and have caused 38 human infections including 21 deaths; however, the key genetic determinants of the pathogenicity of these viruses have yet to be fully investigated. Here, we characterized two H5N6 avian influenza viruses - A/duck/Guangdong/S1330/2016 (GD/330) and A/environment/Fujian/S1160/2016 (FJ/160) - that have similar viral genomes but differ markedly in their lethality in mice. GD/330 is highly pathogenic with a 50% mouse lethal dose (MLD50) of 2.5 log10 50% egg infectious doses (EID50), whereas FJ/160 exhibits low pathogenicity with an MLD50 of 7.4 log10 EID50. We explored the molecular basis for the difference in virulence between these two viruses. By using reverse genetics, we created a series of reassortants and mutants in the GD/330 background and assessed their virulence in mice. We found that the HA gene of FJ/160 substantially attenuated the virulence of GD/330 and that the mutation of glycine (G) to tryptophan (W) at position 225 (H3 numbering) in HA played a key role in this function. We further found that the amino acid mutation G225W in HA decreased the acid and thermal stability and increased the pH of HA activation, thereby attenuating the H5N6 virus in mice. Our study thus identifies a novel molecular determinant in the HA protein and provides a new target for the development of live attenuated vaccines and antiviral drugs against H5 influenza viruses.

H3N2 avian influenza viruses detected in live poultry markets in China bind to human-type receptors and transmit in guinea pigs and ferrets.

The H3N2 influenza viruses became widespread in humans during the 1968 H3N2 pandemic and have been a major cause of influenza epidemics ever since. Different lineages of H3N2 influenza viruses are also commonly found in animals. If a different lineage of H3N2 virus jumps to humans, a human influenza pandemic could occur with devastating consequences. Here, we studied the genetics, receptor-binding properties, and replication and transmission in mammals of 15 H3N2 avian influenza viruses detected in live poultry markets in China. We found that the H3N2 avian influenza viruses are complicated reassortants with distinct replication phenotypes in mice. Five viruses replicated efficiently in mice and bound to both human-type and avian-type receptors. These viruses transmitted efficiently to direct-contact guinea pigs, and three of them also transmitted among guinea pigs and ferrets via respiratory droplets. Moreover, ferret antiserum induced by human H3N2 viruses did not react with any of the H3N2 avian influenza viruses. Our study demonstrates that the H3N2 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H3N2 influenza viruses circulating in nature.