Molecular Basis For Virulence Difference Of H5N6 Avian Influenza Viruses In Mice

The H5N6 avian influenza viruses（AIVs）are widely circulating in wild birds and domestic poultry in many countries,and have caused 52 human infections including 26deaths since the first case in 2014.A continuous study conducted by the National Avian Influenza Reference Laboratory indicates that the virulence of H5N6 AIVs varies and some viruses display high lethality in mammals.Therefore,it is imperative for us to explore the detailed molecular determinants of pathogenicity of these H5N6 viruses in mammals,which may provide valuable information for the development of effective disease control and prevention strategies and for anticipating the emergence of potential influenza pandemic threats in the human population.In the present study,we selected 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 display strikingly different virulence in mice.GD/330 is highly pathogenic with a 50%mouse lethal dose(MLD₅₀)of 2.5 log₁₀50%egg infectious doses(EID₅₀),whereas FJ/160 exhibits low pathogenicity with an MLD₅₀ of 7.4log₁₀EID₅₀.To explore the molecular basis for the difference in virulence between these two viruses,we conducted the following study:By using reverse genetics,we created a series of reassortants and mutants in the GD/330 background and assessed their virulence in mice.The virus carrying the HA gene of FJ/160（GD/330-FJ160HA）was substantially attenuated by 1000-fold in mice compared with r GD/330.These results demonstrated that HA played a key role in attenuating the virulence of GD/330.Further study demonstrated that the virulence of GD/330-HA-G225W was reduced by 631-fold in comparison with that of r GD/330.These results indicated that the amino acid at position 225 in the HA protein contributed to the different pathogenicity between these two H5N6 viruses in mice.The multicycle replication of r GD/330,r FJ/160,and GD/330-HA-G225W in A549cells demonstrated that the titers of GD/330-HA-G225W were significantly lower than those of r GD/330 at all three timepoints post infection.These results indicated that the amino acid mutation of G225W in HA impaired the replication of H5N6 virus in mammalian cells.Syncytia formation assay demonstrated that the p H of HA activation of r GD/330,r FJ/160,and GD/330-HA-G225W was 5.5,5.9,and 5.7,respectively.These results indicated that the G225W mutation in HA increased the p H of HA activation.The acid and thermal stability test demonstrated that the stability of GD/330-HA-G225W differed markedly from that of r GD/330.These results indicated that the G225W mutation in HA reduced the acid and thermal stability of H5N6 influenza viruses.The structure prediction of the HA protein demonstrated that the smallest amino acid G has a single hydrogen atom as its side chain,while the largest amino acid W has an indole as its side chain.Therefore,the steric hindrance may have contributed to the observed functional difference of HA in the GD/330 and GD/330-HA-G225W viruses.In summary,our study found that the amino acid at position 225 in HA played an essential role in the virulence difference between these two H5N6 viruses in mice.Our study further demonstrated that the G225W mutation in HA increased the p H of HA activation and decreased the acid and thermal stability of GD/330,thereby attenuating the H5N6 virus in mice.Our study provides references for the development of live attenuated vaccines and antiviral drugs against H5 influenza viruses.