Evolutionary Monitoring Of H7N9 Avian Influenza Viruses And Molecular Mechanism Of Key Biological Phenotypes

In 2013,the case of human infections and deaths with H7N9 low pathogenic AIVs(LPAIVs)were first reported in China.The H7N9 LPAIVs evolved into highly pathogenic AIVs(HPAIVs)and showed high virulence to chickens in 2017.In view of this,China has fully implemented the vaccine immunization policy in poultry since September 2017.Vaccine immunization effectively reduced the prevalence of H7N9 virus in poultry and saved tens of billions of economic losses for the poultry industry every year.More importantly,the vaccination of chickens played an important role in preventing human infections.However,the H7N9 AIV has not been eradicated from poultry in China,and its evolution and biological characteristics still need to be further tracked and studied.In this study,we isolated and systematically analyzed 19 H7N9 AIVs during surveillance and diagnosis from February 2018 to December 2019.The HA,NA,and M genes of the 19 H7N9 viruses share 97%?100%,97.6%?100%,and 97.1%?100%identity at the nucleotide level,respectively;they cluster in phylogenetic trees with the H7N9 HPAIVs that were previously detected in 2017.The PB2,PB1,PA,NP and NS genes of 18 of the 19 viruses are closely related,sharing 97.6%?100%,98%?100%,98%?100%,98%?100%,and 97.7%?100%identity at the nucleotide level,respectively,but they are quite different from the DK/FJ/SE0377/18 virus,The PB2,PB1,PA,NP,and NS of DK/FJ/SE0377/18 and the other 18 viruses locate in different groups in the phylogenetic trees,sharing only85.7%?86.2%,88.8%?89.4%,90.5%?91.4%,93.7%?94.6%,and69.1%?69.7%identity,respectively.According to the 95%homology of nucleotides,the 19 H7N9 HPAIVs in this study were divided into two genotypes.18 of the19 viruses belong to the genotype 2 that was reported previously,whereas DK/FJ/SE0377/18 formed a new genotype 10.Animal studies indicated that the H7N9 viruses are highly lethal to chicken,cause mild infection in ducks,but have distinct pathotypes in mice.All viruses replicated effectively in the turbinates and lungs of all three mice tested,while some of the viruses also detected in the kidney and brain.Except for CK/NX/SD007/2018,CK/LN/SD004/2019 and CK/HEB/S1118/2019,all the other viruses caused weight loss in mice to different degrees.The MLD₅₀ of CK/IM/SD010/2019 is 4.8 log₁₀EID₅₀.Receptor-binding analysis showed that the currently circulating H7N9 AIVs bound to avian-type receptors with high affinity,but gradually lost their ability to bind to human-type receptors.Antigenic analyses showed these viruses formed two different antigenic groups:the first group(antigenic group I)contained the vaccine seed virus H7-Re2,CK/GX/SD098/17,and 11 viruses isolated in 2018,and the second group(antigenic group II)contained one virus isolated in 2018 and seven viruses isolated in 2019.These results indicate that the H7N9 viruses isolated in 2019 are antigenically different from the H7-Re2 vaccine strain.To investigate the molecular basis of H7N9 virus antigenic drift,we compared the HA gene and identified eight amino acids in the HA1 protein that were conserved in most of the viruses in antigenic group I,but mutated in viruses in antigenic group II.We rescued a series of point mutant viruses,whereas T135V and T160A mutations enhanced reactivity to the Re-2 antiserum and altered reactivity with monoclonal antibodies.Two amino acid mutation at positions 135 and 160 in the HA protein add two glycosylation sites,appear to contribute to this antigenic drift.The above findings were of great significance for us to understand the biological characteristics of the H7N9 subtype avian influenza virus after application of the H7N9 poultry vaccine in China.Our study provides important insights into H7N9 virus evolution and control.