The Mechanism Of Difference In The IP-10 Expression Induced By H7N9 Subtype Avian Influenza Virus In Mouse And Cell Model

Avian influenza virus(AIV)of the H7N9 subtype is a zoonotic pathogen,which was first reported in China in 2013.Early genetic evolution analysis results showed that H7N9 subtype AIV is a ternary reassortant virus of duck origin H7N3,chicken origin H10N9 and poultry origin H9N2.The internal genes are all derived from H9N2 virus which has been the dominant genotype since 2010.Studies have shown that the epidemic peak and epidemic location of avian H7N9 are quite similar to those of human H7N9,further confirming the highly homology of human H7N9 and avian H7N9 viruses.The high mortality rate of H7N9 cases is often related to lung damage caused by cytokine storm,among which some cytokines or chemokines,such as interferon-inducible protein-10(IP-10),may be the biomarker as severity of lung damage in the acute infection phase.The phylogenetic and clinical features of H7N9 virus are clear,but the underlying molecular mechanism of its high pathogenicity in hosts(mammals)is still unknown.Therefore,it is of great significance to research and reveal the pathogenicity of H7N9 AIVs to mammals for the early warning and prediction of the impact of H7N9 AIVs on the breeding industry and public health.This study analyzed the expression level of IP-10 induced by H7N9 strains with different pathogenic to mice,and the effect of IP-10 expression level on its pathogenicity;analyzed the key polymerase subunit of the model viruses that caused the differential expression of IP-10 on A549 cells.1.Effect of chemokine IP-10 on the pathogenicity of H7N9 AIVs in miceWe selected H7N9 wild type strain JTC4,which is low pathogenic to mice,and its mouse-adapted strain JTC4M11 as the model virus from thirteen of H7N9 viruses with different pathogenicity and different resource.We determined its biological characteristics such as EID50,TICD50,growth curve,etc,and weight change and death rate of BABL/c mice infected with two viruses.Genomic sequence analysis showed that there were only six amino acid differences in the whole genome of the two strains of virus.The viruses could effectively replicate on MDCK cells with titer of 7 log10TCID50/0.1mL.EID50 of two viruses in chiken embryos reached about 8 log10EID50/0.1mL.The results of animal experiment showed that JTC4M11 is highly pathogenic to mice(MLD50=102.68EID50),and JTC4 is low pathogenic to mice(MLD50=106.5EID50).The results of the experiment of BABL/c mice infected with viruses showed that the body weight of the mice in JTC4M11 group significantly decreased at 3 dpi compared with the mice in the wild-type-JTC4 group.Pathological changes showed that obvious edema with inflammatory cell infiltration was observed in the lung tissue of the mice.JTC4M11 strain replicated at the viral load of 5.67 log10TCID50/mg significantly higher than JTC4 group at 1.93 log10TCID50/mg.IP-10 expression level in JTC4M11 group increased by 400 times than JTC4 group.In JTC4M11 challenge group administrated simultaneously with the IP-10 by nasal drops(JTC4M11+IP-10 group),the weight had a significantly loss and the lethality had a significantly increased in mice.During the 72 h-84 h post-infection,the mortality rate of JTC4M11+IP-10 group is as high as 57%(4/7),and the mortality rate of the JTC4M11 group is about 14.3%(1/7).It was deduced that IP-10 could increase the pathogenicity of JTC4M11.There was no significant difference in IP-10 expression level in A549 cell infected with viruses JTC4 and JTC4M11,respectively.It indicated that there was difference on IP-10 expression between mouse model and human-origin cell infected with H7N9 virus.2.Effect of polymerase protein on the expression level of IP-10 in A549 cells induced by H7N9 AIVsIn order to analyze the molecular mechanism of zoonotic nature of H7N9 virus,we selected A549 cell as research model.We analyzed IP-10 expression levels induced in A549 cell infected with thirteen of H7N9 AIVs.Two strains of JTC4 and SDL 124 with significant differences in IP-10 expression level were selected as model viruses.Genomic sequence analysis showed that there were 24 amino acid substitution through the whole genome,of which 14 amino acid substitutions located in polymerase proteins(PB2,PB1,and PA),nine amino acid substitutions are in glycoprotein HA and NA.Growth curves showed that two viruses could effectively replicate on MDCK cells.SDL 124 replicated slightly stronger than JTC4,and its maximum titer was about 5 log10TCID50/mL.However,SDL 124 had weaker replication ability than JTC4 on A549 cells,and its titer was stable at 2.5 log10TCID50/mL from 24 h post infection.In order to screen out the influence of viral polymerase gene PB2/PB1/PA on the expression of IP-10 in A549 cell,we constructed recombinant viruses in which single polymerase gene of JTC4 virus was replaced with the corresponding gene of SDL 124 viruse,and successfully rescued the recombinant virus:rJTC4-SDL124PB2,rJTC4-SDL124PB1,rJTC4-SDL124PA.IP-10 expression and biological characteristics were analyzed in A549 cells infected with the recombinant virus.The results showed that when IP-10 expression significantly reduced in A549 cells infected with rJTC4-SDL124 PB2 compared with the parent viruses,recombinant viruses rJTC4-SDL124PB1 and rJTC4-SDL124PA.In this study,we proved that H7N9 viruses induced different expression level of IP-10 in mice,and IP-10 could increase the pathogenicity of the mouse-adapted strain JTC4M11.Difference of IP-10 expression level in A549 infected with H7N9 model viruses was determined on PB2 protein.