Effects Of Different Evolutionary Patterns Of 80-84 Amino Acid At NS1 Protein Of H5 Avian Influenza Virus On Pathogenicity And Innate Immune Response In Mammals

H5 highly pathogenic avian influenza(HPAI)is an important zoonotic disease,which seriously threatens human health.Dendritic cells(DCs),as antigen presenting cells,are the key of the regulation of the host’s innate immune response.Non-structrual protein 1(NS 1),as an important virulence factor,is a non-structural protein of influenza virus,which can influence the virulence of virus through regulating innate immune response in the host.In recent years,some new evolutionary patterns of 80-84 amino acid at NS1 of H5 AIV emerged.However,how the new evolutionary patterns regulate the innate immune response and how it influence the pathogenicity,which are remain unclear.Here,we firstly analysed the evolutionary rule of NS1 protein 80-84 amino acids of H5 subtype AIV in recent two decades.Next,three recombinant viruses were generated based on reverse genetic technology according main patterns of 80-84 amino acid at NS1(NIASV,TIASV,and TVASS)in recent five years.And differences of pathogenicity for recombinant viruses in mammals were determined in vivo and in vitro.Finally,we evaluated the effect of recombinant viruses on the innate immune response induced by DCs in mice.This study will provide a basis for the pandemic alert and the pathogenesis of H5 subtype AIV.1.Generation and biological characteristics of H5 subtype AIV with different reversion patterns of 80-84 amino acid at NS1This study aligned and analyzed the sequence of H5 subtype AIV from the global influenza database.H5 subtype AIV with different evolutionary patterns of 80-84 amino acid at NS1 from 2000 to 2019 were analyzed.The results showed that the 8084 reversion pattern with five amino acids has become the main pattern since 2015.Next,we selected H5 subtype AIV with main 80-84 reversion patterns(NIASV,TIASV,and TVASS)isolated from avians and mammals in recent five years.Therefore,three recombinant virues with different patterns at 80-84 position of NS1,including NIASV,TVASS,and TIASV,were generated based on reverse genetic technology by using the skeleton of H5 AIV BYG strain(A/Goose/Baoying/20160916).Next,the biological characteristics including 50%embryo infective dose(EID50),50%tissue culture infective dose(TCID50)and the viral replication level in A549 cells were detected.In the end,the expression of interferon(IFN)and IFN-related downstream genes in A549 cells which were infected by four recombinant viruses were detected by quantitative reverse transcription-PCR(qRT-PCR).The results showed that the EID50 of four recombinant viruses were 108.5-108.33 EID50/mL and the TCID50 were 106.66-107.5 TCID50/mL in MDCK cells.TVASS strain showed a strong replication ability and enhanced the transcriptional level of IFN-β while reduced the transcriptional level of Mxl and ISG56 in A549 mammal cells in vitro.2.Effects of different reversion patterns of 80-84 amino acid at NS1 protein of H5 avian influenza virus on pathogenicity in miceIn this study,Six-week-old BALB/c mice were infected intranasally with 106 EID50/50 μL of each virus and PBS control group was seted.Mice infected by virus were observed continuously for 14 days,the weight change and survival of the mice were recorded.The nasal cavity,trachea,lung,brain,and spleen of mice were collected at 3 days,5 days,and 7 days post infection(dpi)respectively for making pathological slices and detecting the level of virus replication and cytokines by qRT-PCR.The results showed that the group of TVASS strain showed 60%of mortality rate,while other groups all survived.The lung lesions of mice in TVASS group were serious,with a mass of inflammatory cell infiltration and severe exfoliation of epithelial cells in the trachea.The lungs and trachea of mice in other groups also had certain lesions.Virus load of nasal cavity,trachea,lung,brain,and spleen show that TVASS group had the higher virus replication level in the nasal cavity,trachea,and lung compared with the BYG group.In addition,the expression of cytokines including TNF-α,IL-6,and IP-10 in lungs were significantly increased while the expression of IFN downstream protein Mx1,ISG15,ISG56 were decreased compared with the BYG group.Therefore,it is concluded that TVASS at position 80-84 of NS1 enhanced the pathogenicity of H5 subtype AIV in mice through inducing cytokine storm and inhibitting the expression of interferon(IFN)downstream protein.3.Effects of H5 subtype AIV with different reversion patterns of 80-84 amino acid at NS1 on innate immune response in murine DCsFirstly,DCs were cultured successfully from murine bone marrow.Next,four recombinant virues(NIASV,TVASS,TIASV,and BYG)were purified by virus purification technology.DCs were infected by four recombinant virues at a dose of 0.1 MOI.The infection ability of virus,maturation phenotypic markers,activation marker,migration marker of DCs,and mixed lymphocyte reaction were determined by flow cytometry,and the cytokine expression of DC supernatant was measured by ELISA.The results showed that the TVASS strain showed a strong ability to infect DCs,and the expression of maturation phenotype markers(MHCII,CD40,and CD80),activation marker(CD69),and migration marker(CCR7)were significantly increased compared with the BYG group.The results of migration test also showed that TVASS strain significantly upregulated DCs migration ability and induced DCs to secrete cytokines including TNF-α,IL-6,IL-1β,IL-12p70,and IL-10 compared with the BYG group.The result of mixed lymphocyte reaction between DCs and CD4+T lymphocytes showed that the TVASS strain significantly stimulated the proliferation of CD4+T cells compared with the BYG group.These results suggested that TVASS of 80-84 amino acid at NS1 activated the innnate immune response of DCs and induced downsteam immune cells for cascade amplification of immune response.This may be the underlying mechanism of the "cytokine storm" and the strong virulence of viruses in manmals.