Screening Host Proteins Required For Bacterial Adherence After H9N2 Virus Infection And The Preliminary Functional Verification

The H9N2 subtype low pathogenic avian influenza virus(LPAIV)is extensively distributes worldwide.The virus infects humans and lower mammals.Chickens clinically infected with H9N2 virus often show symptoms of growth retardation of broilers and decrease egg production of hens.Notably,the secondary Escherichia coli(E.coli)infection caused by the H9N2 virus is the main cause of the large increase in poultry casualty rate,causing great economic losses in the poultry farming.Tissue damages caused by virus infection provide an opportunity for bacteria invasion,but this mechanism is not sufficient for low virulence strains.The secondary infection mechanism of low virulence strains is epithelial cells in the lower respiratory tract will change such that receptors are exposed and activated,thereby facilitating the adhesion of bacteria to host cells.The study used co-infected H9N2 avian influenza virus and E.coli from clinical isolation to study the relationship between H9N2 virus and secondary bacterial infection.This study was divided into three parts:1.Influences of H9N2 AIV on adhesion of E.coli to hostCEF and DF-1 cells were cultured on three 6-well cluster plates,respectively.Three cell wells were inoculated with H9N2 AIV in each cell plate,and the other three wells were uninfected with virus as control groups.All cell wells were infected with APEC every 24 h and incubated at 4 °C for 1.5 h.The serial dilutions of the cell suspension were plated onto LB agar for the quantitation of colony-forming unit(CFU).We found that the amount of E.coli that adhered to both virus-infected cells were significantly higher than those adhered to the normal cells,and the bacterial numbers on virus-infected cells were more than twofold higher than those on uninfected cells up to 72 h post infection(hpi).Wright's staining also confirmed this result visually.In addition,thirty 5-weeks-old SPF chickens were kept in the sterile isolator in vivo experiment.Fifteen chickens were inoculated with H9N2 AIV through the nasal cavity.All chickens were infected with APEC every 24 h as experimental group.The other fifteen chickens were uninfected with virus as control groups.Immunofluorescence histochemical staining and hematoxylin-eosin(HE)staining were performed in experimental and control groups to examine the virus infection and pathological damages,respectively.Record the number of E.coli in the experimental and control groups.In vivo infection testshowed that the amount of E.coli that adhered to the virus-infected lungs were remarkably higher than those that adhered to the uninfected lungs at 72 hpi,and the CFU counts increased in a time-dependent manner.Immunofluorescence histochemical staining results showed that chicken lungs had high viral loads.However,no significant pathological difference was found between the virus-infected and normal lung,as indicated by the HE staining results.Both in vitro and in vivo experiments demonstrated that H9N2 virus can facilitate the adhesion of E.coli to the host,and the adhesion ability of the bacteria increases in a time-dependent manner.However,no necessary link was observed between increased adhesion and pathologic damages caused by viral infection.2.Proteomics iTRAQ analysisThe study constructed a late chicken embryo infection model and used proteomics techniques to analyze the expression of proteins associated with bacterial adhesion after H9N2 AIV infection.After iTRAQ coupled with LC-MS/MS analysis,279 differentially expressed proteins were obtained,of which 233 proteins were up-regulated and 46 proteins were down-regulated.Up-regulated proteins contain TGF-?1 protein associated with bacterial adhesion.KEGG enrichment analysis results indicated that differentially expressed proteins were enriched in pathogenicity-related signaling pathways,cell proliferation,differentiation and apoptosis,and host innate immunity.3.Expression and preliminary functional verification of the TGF-?1 proteinDesign specific primers based on the gene sequence of chicken TGF-?1 in GenBank.Reverse transcription products of RNA from lungs of chicken infected with H9N2 virus as template,then gene was amplified by PCR.The amplification product was linked to the pMD18-T vector,and pMD18-T-TGF-?1 vector was double-digested with Xho I and EcoR I enzyme.The target fragment was cloned into pPIC9 plasmid.Then the recombinant pPIC9-TGF-?1 plasmid and negative control pPIC9 plasmid were transformed into Pichia pastoris(P.pastoris).The methanol-induced expression of positive transformants and bank plasmid transformants in P.pastoris GS115 was performed.A protein band corresponding to44.5 kDa in the culture supernatant of the recombinant pPIC9-TGF-?1 transformants were determined through SDS-PAGE and Western blot analysis.This band was not found in the culture supernatant of blank pPIC9 plasmid transformants.The expressed protein was addedto CEF and DF-1 cells in vitro experiment,then infected with APEC.The result showed that the amount of APEC that adhered to cells having TGF-?1 protein were remarkably higher than those that adhered to the cells having no TGF-?1 protein,and the CFU counts increased in a dose-dependent manner.These results indicate that H9N2 AIV infection is conducive to APEC adhesion to the host,and TGF-?1 host protein is associated with APEC adhesion.