Effects Of Amino Acid Changes At Hemagglutinin(HA) Protein Cleavage Site On Virulence Of H7N9 Subtype AIV

Human infections with low-pathogenic AIV(LPAIV)H7N9 were first reported in March 2013 in China.The virus showed no symptoms on poultry and was low pathogenicity(LP).Since then,the LP H7N9 subtype virus has continued to exist in China,causing five waves of epidemic.In mid-2016,a novel highly pathogenic(HP)H7N9 virus was isolated for the first time in China.,which possessed multiple amino acid(KRTA)insertions at the cleavage site of hemagglutinin(HA)protein.At the beginning of 2017,HP H7N9 virus began to spread in a large scale.The HP H7N9 virus not only caused large-scale deaths of poultry,but also led to a surge in human infections.According to statistics,764 cases of human infection were reported in the fifth wave of H7N9 epidemics,the number of human infections reported in the fifth epidemic was almost as many as reported during the previous four epidemics combined.The cleavage site of HP H7N9 HA protein is characterized by the joint insertion of basic and non-basic amino acids,and involves mutations of amino acid site in adjacent regions.In the early stage of H7N9 virus epidemic,HACS mainly existed in four motifs: PEV.PKRKRTAR/G,PEV.PKGKRTAR/G,PEV.PKGKRIAR/G and PEV.PKRKRAAR/G.Among them,PEV.PKRKRTAR/G were widely circulating in poultry.PEV.PKGKRTAR/G were isolated only in poultry,and there was only one human case virus strain in the latter two motifs.So far,the virulence effects of different HACS and non-basic amino acid insertion functions on H7N9 virus remain unclear.Therefore,in this study,the HA gene of HP H7N9 subtype AIV(A/chicken/HZ-3/2016)isolated was used as template,and different HA cleavage site motifs were assigned to HP H7N9 by site-directed mutation.Finally,six recombinant H7N9 viruses with different HA cleavage site regions were constructed by reverse genetics.We then characterized the virus in avian and mammalian cells and assessed the virulence in chickens and mice.Studies have shown that highly pathogenic H7N9 virus enhances virulence through enhanced replication and HA cleavage efficiency,as well as improved stability.The thermal stability of H7-VPKRKRTAR virus was increased after TA insertion,and the cleavage level was significantly increased after 338 mutation from glycine(G)to arginine(R).Meanwhile,the titer of H7-VPKRKRTAR in SPF chickens was slightly lower than that of other highly pathogenic strains,reflecting moderate virulence.This comprehensive characteristic is conducive to the prevalence of PEV.PKRKRTAR/G motif in viruses.The H7-VPKGKRIAR virus had a low level of replication in CEF cells,while the transmissibility in contact chickens was restricted.The virus carrying the PEV.PKGKRIAR/G motif was not well adapted to CEF cells and SPF chickens,demonstrating that the different non-basic amino acids inserted in H7N9 subtype HACS affected the adaptability of the virus to host species.Remarkably,the presence of proline at position 335 in the low pathogenic H7N9 virus enhanced the acid resistance of the virus,improved the level of replication in the cell and the transmission efficiency in SPF chickens.The results indicated that the evolution of H7N9 HPAIV from low pathogenic precursors involved adaptation in adjacent regions.Our results clarify the effect of the unique motif of the cleavage site on the virulence of H7N9 virus,and highlight the importance of non-basic amino acids.Therefore,comprehensive monitoring risk assessment of H7N9 subtype avian influenza virus should be strengthened and the biological characteristics of emerging viruses should be assessed in a timely manner.