Molecular Characteristics And Pathogenicity Of Novel H5 Subtype Avian Influenza Virus

The newly reassortant H5 subtype avian influenza virus(AIV)has been reported to infect human beings,often causing severe illness,leading to high mortality and posing great threat to human beings.In 2014,Sichuan province of China witnessed the world's first human fatal case of H5N6 subtype avian influenza virus infection.Since the current epidemic situation of the newly reassortant H5 subtype AIV in birds is still not clear,and the mechanism of high mortality in humans is still unknown,the following study were carried out in this paper.In the first part,to study the epidemic situation of the newly reassortant H5 subtype AIV,from October 2013 to September 2016,we screened 7,200 poultry samples in Zhejiang province,isolated 37 strains of H5N6 virus and 6 strains of H5N8 virus and conducted whole-genome sequencing and evolutionary analysis of the virus.Analysis of HA showed that all H5 subtype AIV belong to the Eurasian lineage and were the currently prevalent clade 2.3.4.4,and the HA cleavage sites showed high pathogenicity pattern.Homologous analysis showed that some of the segments of poultry isolates from zhejiang province are highly homologous with corresponding segments of human isolates,which has great risk of human infection.In the second part,in order to evaluate the pathogenic characteristics of the newly reassortant H5 subtype AIV on mammals,representative strains 6D2(H5N6)and W24(H5N8)from Zhejiang province were selected for the mouse infection experiment.The results showed that 6D2 and W24 were hypolethal to mice.At the same time,in order to simulate the pathogenicity changes in the process of interspecies transmission,whole genome sequencing analysis was conducted through the lung-to-lung passage experiment in mice to find out the adaptive amino acid substitutions.Amino acid substitutions such as HA(A150V),PB2(E627K)and PA(A343T)occurred during the mouse passage from the 1st to the 10th generation.The pathogenicity of the mouse adaptive strain was significantly higher than that of the wild type,leading to the death of 100%of the mice.In addition,the second-generation sequencing found that HA(R239H)and PB1(T110M)are also of great significance for the adaptation process to mice.In the first 4 generations,amino acids had a higher mutation rate,and then the frequency of new mutations gradually flattened out.These results are of important significance for understanding the interspecies transmission law of AIV.In the third part,the recombinant 6D2 and W24 virus strains were constructed by reverse genetics to further verify the virulence enhancement mechanism of the above-mentioned mutation sites on the newly reassortant H5 subtype AIV.Combined with 3D structure analysis,it was confirmed in cell and animal models that HA(A150V)variation can enhance the binding ability to human receptors,and PA(A343T)and PB2(E627K)variation increased the polymerase activity of viral ribonucleoprotein,resulting in significantly increased virus replication and virulence.The results suggest that the above mutation loci are of great significance in the process of interspecies transmission and deserve high attention in the future clinical testing.To sum up,this study came to the following conclusions:1.The popular H5 strain in zhejiang province is mainly clade 2.3.4.4,Eurasian lineage,has the characteristics of highly pathogenic strain,and multiple recombination of H5 subtype AIV and other AIVs exists in zhejiang province.2.For the first time,HA(A150V),PA(A343T)and PB2(E627K)mutations were found and verified in the newly reassortant H5 subtype AIV to have great significance in the process of interspecies transmission.To conclude,the newly reassortant H5 subtype AIV appear constantly under the background of human infections,this study is based on the newly reassortant H5 subtype AIV systemic investigation and the research of interspecies transmission,preliminary understanding its prevalence in Zhejiang area,basically illustrates the mechanism of possible mutation the newly reassortant H5 subtype AIV may adopt in the process of mammal adaption and virulence enhancement.This study provides an important reference for the prevention and control of H5 subtype AIV in China and has important guiding significance for the research and development of vaccines and drugs.