Proteomic Analysis Of Chicken PBMCs Infected By NDV F48E9Strain And The Impact Of ILK And CyPA On NDV Replication And Proliferation

Newcastle Disease (ND) is an acute and highly pathogenic avian infectiousdisease caused by New Castle Disease virus (NDV), continuously posing threats toworld-wide poultry industry. Peripheral blood mononuclear cells (PBMCs) areimportant immunological effector cells, which could not only monitor the infectionand disease status, but also are an important integral part of innate and acquiredimmunity. Previous studies revealed that PBMCs are the primary targets of NDV thatNDV infection may lead to apoptosis, thereby resulting in immunosuppression andreduced avian resistance.In this study, high-throughput method based on2-DE and MALDI-TOF/TOF-MS proteomics was used for compare proteomic analysis of chickenPBMCs infected by NDV F48E9strain. It was identified13kinds of proteins whichshowed persistent up-regulation or down-regulation trend in the three phases, priorinfection NDV,3days after infection,5days after infection. According differentiallyfunctions they can be divided into five functional groups which contains thebiosynthesis of macromolecules, cytoskeleton, energy metabolism, stress responseand signal transduction. These differentially expressed proteins to cash the overallchanges in PBMC in NDV infected environment, and further study of thesedifferentially expressed proteins’ function, will promote understanding of themechanism of immune suppression induced by NDV virulent strains.Study the impact of ILK on NDV replication and proliferation usingup-expression and down-regulated method by transient transfection pFLAG-CMV-2-ILK and siILK respectively. F48E9NDV strains were inoculated withtransfected transfected group (transfected pFLAG-CMV-2-ILK or siILK), DF-1group(no treat) and MOCK group (transfected with empty vector or siScr). Using real-timePCR and TCID50detect the NDV genomic RNA copy number and viral infectioustiter in culture supernatants. The results showed that when the ILK up-regulated, DF-1 /ILK group culture supernatant virus titers and NDV genomic RNA copy numberafter inoculation,36h,48h,60h to72h were significantly higher than DF-1cells groupand MOCK group. When the ILK down-regulated, DF-1/siILK group culturesupernatant virus titers and NDV genomic RNA copy number after inoculation,36h,48h,60h to72h were significantly lower DF-1cells group and MOCK group. It isshowed that the up-regulation of ILK can promote the replication and proliferation ofNDV, and down-regulation of ILK can reduce the replication and proliferation ofNDV, so that, ILK has a positive regulatory role for the replication and proliferationof NDV. Combining the results of first chapter proteomics, ILK showeddown-regulated following NDV infection, indicating that ILK plays a negativeregulatory role in NDV infection.Study the impact of CyPAon NDV replication and proliferation usingup-expression by transient transfection pFLAG-CMV-2-CyPA. F48E9NDV strainswere inoculated DF-1group (no treat), DF-1/CyPA group (transfectedpFLAG-CMV-2-CyPA plasmid) and MOCK group (transfection siScr), Usingreal-time PCR and TCID50detect the NDV genomic RNA copy number and viralinfectious titer in culture supernatants. The results showed that when the CyPAup-regulated, DF-1/CyPA group culture supernatant virus titers and NDV genomicRNA copy number after inoculation,48h,60h to72h were significantly lower thanDF-1cells group and MOCK group. It is revealed that the up-regulation of CyPAexpression inhibits the replication and proliferation of NDV. Combining the results offirst chapter proteomics, CyPA showed up-regulated following NDV infection,indicating that CyPA plays a negative regulatory role in NDV infection.The results obtained in this study may help understand response to NDVinfection of target cells in vivo at the molecular level, which will help to clarify themolecular pathogenesis of NDV.