Comparative Proteome Analysis Of Chicken Trachea In Response To IBV, NDV, And AIV H9Subtype Virus Infections And The Mechanism Of Chicken Galectin CG-1B During NDV Infection

Newcastle disease virus (NDV), infectious bronchitis coronavirus (IBV) and avian influenzavirus (AIV) H9subtype are the primary cause of morbidity and mortality worldwide in the poultryindustry which can cause acute, highly contagious poultry respiratory tract disease and result inheavy economic losses to the poultry industry throughout the world.The tracheal epithelium of chicken is not only the primary target for replication and infectionof avian respiratory viruses, but also the first line of defense in the innate immune response. Theavian respiratory viruses, including IBV, NDV and AIV H9, can infect tracheal epithelial cells andinduce damage to tracheal epithelium resulting in respiratory disease. However, the severity andoutcome of the clinical illness caused by these different viruses are different. To better understandthe molecular basis of pathogenesis and specific virus-host interactions of IBV, NDV and AIV H9,comparative proteomic analysis was performed to investigate the proteome changes of primarytarget organ (trachea) during IBV, NDV and AIV H9infections, using2D-DIGE followedMALDI-TOF/TOF-MS. In total,44,39,41and48differentially expressed proteins wereidentified in the tracheal tissues of the chickens inoculated with IBV (ck/CH/LDL/97I, H120),NDV (La Sota), and AIV H9, and38proteins differentially expressed between different IBVsitains ck/CH/LDL/97I and H120infected groups. Transcriptional alteration analysis of EIF5A2,GNB2L1, PRDX1, PRDX3, SOD1, LGALS1, ANXA2, ANXA1, ERP29and HSPB1wasperformed by utilizing Real time RT-PCR, the results showed that the trends in the change inmRNA abundance of these differentially expressed proteins were similar to the change patterns oftheir corresponding proteins showed by2D-DIGE. Western blot further validated the expressionchange of ANXA1, ANXA2, PRDX1and HSPB1and the results were consistent with2D-DIGE.In2D-DIGE analysis, HSPB1was indentified from several unique spots migrated at almost thesame Mr value but different pI values, suggesting that IBV, NDV and AIV H9infections mayinduce phosphorylation of HSPB1.Western blot analyses with specific antibodies to phospho-HSPB1(pSer82) showed that up-regulation of phosphorylated HSPB1was induced by IBV(ck/CH/LDL/97I, H120), NDV (La Sota) and AIV H9infections. Bioinformatics analysis showedthat IBV, NDV and AIV H9induced similar core host responses involved in biosynthetic,catabolic, metabolic, signal transduction, transport, cytoskeleton organization, macromolecularcomplex assembly, cell death, response to stress and immune system process. Comparativeanalysis of host response induced by different viruses indicated differences in protein expressionchanges induced by IBV, NDV, and AIV H9may be responsible for the specific pathogenesis ofthese different viruses. The present study using the same virus of different strains (IBV virulentck/CH/LDL/97I and H120vaccine) and different viruses (IBV, NDV and AIV H9) might providemore significant information on specific aspects of molecular pathogenesis and virus-hostinteractions of these different viruses. Furthermore, these findings might also provide useful cluesfor the development of novel prevention or therapeutics strategies against IBV, NDV and AIV H9. Our comparative proteomics analysis showed the abundance of chicken galectin (chickenbeta-galactoside-binding lectin, CG-1B) was up-regulated in trachea of NDV La Sota-infectedchickens. Mammalian galectins can promote or inhibit the adorption and infection of virus viabinding to the viral envelope glycoproteins; however, the potential roles of avian galectins in thecourse of viral infection have not yet been reported. In order to explore the potential roles of CG-1B during NDV infection, CG-1B protein was expressed and purified in vitro, hemagglutinationtest showed that CG-1B can inhibit the hemagglutination activity of NDV. A capture ELISA assaywas developed to demonstrate the direct binding between CG-1B and NDV virion; the resultsshowed that CG-1B can bind to NDV in a dose-dependent manner. In order to identify the NDVproteins which interact with CG-1B, microscale affinity chromatography and co-immunoprecipitation were applied to capture the NDV proteins binding to CG-1B, the resultsshowed that CG-1B can interact with a70kD protein from NDV which identified ashemagglutinin-neuraminidase protein through LC-MS/MS. In our in vitro cell infection model, weshowed that CG-1B can inhibit the adsorption of NDV virions to cells, and cells transfected withan expression vector encoding CG-1B produced lower levels of viral titers after NDV infectioncompared to those transfected with a control vector. Furthermore, confocal microscopy confirmedthat CG-1B colocalized with the NDV HN protein in the cytoplasm and near the cellularmembrane. This study provides first evidences that CG-1B can inhibit adsorption of NDV virionsto cells and reduce NDV production in NDV-infected cells through the interaction with HNglycoprotein of NDV, demonstrating the antiviral activity of CG-1B. These results could facilitatethe studies on the pathogenesis of NDV and virus–host interaction in the future.