Viral Expression And Inflammation Response In The Brain Of Chickens Artificial Infected With H9N2 Avian Influenza Virus

Avian Influenza(AI)is a comprehensive infection disease caused by Avian Influenza Virus(AIV)belonged to sticky virus,influenza virus genera.H9N2 AIV belonged to low pathogenic avian influenza virus(LPAIV),and was extracted from the North American Turkey in 1966.Since the separation of H9N2 AIV from chickens in Guangdong in 1994,H9N2 subtype avian influenza has been the main type of avian influenza epidemic in China.It is widely found in most areas of China as one of the most important diseases threatening poultry breeding industry.Many studies and our laboratory studies have confirmed that H9N2 subtype AIV mainly affects the function of respiratory system,digestive system and reproductive system in poultry.There are studies about the neurological symptoms caused by influenza virus,the mouse as a model,found that influenza virus could infect in the brain tissue,and the studies about the nervous system pathogenicity in chickens after infected H9N2 AIV are rarely reported.In order to reveal the mechanism of the neurological disorders in chickens after infected with H9N2 AIV,and to understand the immune response of chicken responsing to influenza virus,we carried out this test.In this study,we infected 70 day-old chickens by intranasal infection artificially,and we detected the distribution of NP protein,the mRNA expression changes of immune-related genes and the infiltration of CD3 + CD4 + and CD3 + CD8α + lymphocytes at different time points in chickens brain tissue after infected with H9N2 AIV by direct immunofluorescence,real-time quantitative PCR and immunofluorescence double labeling.Get the following main test results:1 The establishment of chicken model about artificial infection H9N2 subtype AIV 25 SPF chickens were infected by H9N2 AIV artificial,and each inoculated 3mL virus stock solution by nasal,while 5 chickens were the healthy control group.The chickens were sacrificed by cervical bleeding at each time point 2 d,4 d,6 d,8 d and 15 d,and we randomly selected 5 chickens at each time point and immediately cut the brain tissue.The CT value of M gene of H9N2 AIV was detected by absolute quantification,and the viral load of brain was calculated according to the standard curve established in the laboratory.We carried out the distribution of NP protein in the chicken brain tissue.The study found that the virus can be copied in the cerebrum and cerebellum,and the highest in the fourth day,of which the cerebrum is 1269.685 copies / 0.1g,and the cerebellum is 804.67 copies / 0.1g.The chicken restored health at 15 dpi,and we didn’t detect viral load in the healthy control group.In the cerebellum,NP protein is mainly distributed in the neurons of particle layer molecular layer,and most of which are expressed in the cytoplasm.The number of NP-positive cells in the brain was the highest at the 4 dpi,which was consistent with the expression trend of viral load.Those shown that chicken model artificial infected H9N2 AIV is established successful and the virus could be replicated in brain tissue.2 The mRNA expression of immune-related genes in chicken brain tissue infected with H9N2 AIV The mRNA content of Toll-like receptors(TLR3 / TLR4 / TLR2),MDA-5,cytokines(IL-2 / IL-6 /IFN-α/IFN-β),chemokines and receptor(CXCLi1 / CXCLi2 / XCL1 / XCR1 / CCR5)and anti-inflammatory cytokine(IL-4 / IL-10 / TGF-β1 / TGF-β2)were detected by Real-time PCR.The results showed that the TLR3 / 4/7/21 and MDA5 genes were up-regulated in the brain tissue at 2 dpi(P <0.05),and the TLR21 gene was the most significant compared with the control group(P <0.001).IL-2 gene was the most significant up-regulated in brain tissue at 4 dpi(P <0.001),while IL-6 / IFN-α / IFN-β gene expression was up-regulated(P﹥0.05),but it was not significant compared with the control group at the early infection and was down-regulated at 6 dpi.CXCLi1,CXCLi2,XCL1,XCR1 and CCR5 were up-regulated in the cerebrum and cerebellum at the early infection,and the uptake of XCL1 and XCR1 was the most significant compared with the control group(P <0.001).IL-4 was significantly up-regulated at 2 dpi and decreased at 4 dpi(P <0.001).TGF was slightly down-regulated in brain tissue,but it was not significant compared with the control group(P﹥0.05).The expression of genes in the cerebrum was more regularity than that in the cerebellum,and the expression of genes presented different trends followed by infected time.3 The infiltration of CD3 + CD4 + and CD3 + CD8α + lymphocyte in chicken brain tissue infected with H9N2 AIV The expression of CD3 + CD4 + and CD3 + CD8α + in cerebrum and cerebellum increased with the increase of infection time after infection with H9N2 AIV,and the number of infected group was significantly higher than that of healthy control group.The number reached the most on the 4 dpi and 6 dpi.The distribution of CD3 + CD4 + and CD3 + CD8α + lymphocytes in the cerebrum was scattered,and the two lymphocytes in the cerebellum were mainly concentrated in the granule layer.Both lymphocytes quantity differences in brain tissue,and CD3 + CD8α + lymphocytes are more distributed than CD3 + CD4 +,indicating that CD3 + CD8α + lymphocytes may play a major role in the occurrence of inflammation and virus clearance.The results showed that the H9N2 subtype AIV could infect the brain tissue,and the infection time and site correlation could induce the expression of immune-related gene and the infiltration CD3 + CD4 + and CD3 + CD8α + lymphocytes.This study provided a theoretical basis for revealing the neurological symptoms,pathological changes and immune response mechanism by H9N2 AIV infecting chicken brain tissue.