The molecular evolutionary characteristics of new isolated H9N2 AIV from East China and the function of vimentin on virus replication in MDCK cells.

BACKGROUND: The low pathogenic H9N2 AIV caused the serious impact on the poultry industry and public safety. Our purpose was to investigate the molecular evolutionary characteristics of the new isolated H9N2 virus and investigate the intracellular target protein of H9N2 AIV replication in sensitive cells. METHODS: AIV A/chicken/Shandong/LY1/2017 (H9N2) was isolated from the cloaca of the healthy chicken in Shandong, and the full-length eight gene segments of this isolated H9N2 AIV were amplified by RT-PCR and analyzed. MDCK cells were used as the target cell model, and VOPBA assay and LC-MS/MS were carried out to identify the virus-binding protein of H9N2 AIV. MDCK cells were pre-treated with the special antibody and siRNA, and treated with H9N2 AIV to detect the virus replication. Additionally, Vimentin-pcDNA3.0 was successfully constructed, and transinfected into MDCK cells, and then H9N2 AIV mRNA was detected with RT-PCR. RESULTS: Phylogenetic analysis revealed that HA, NA, PB2, PB1, PA, NP and M seven genes of the isolated H9N2 AIV were derived from A/Chicken/Shanghai/F/98, while NS gene was derived from A/Duck/Hong Kong/Y439/97. The cleavage site sequence of HA gene of the isolated H9N2 AIV was a PARSSR G pattern, and the left side sequence (224 ~ 229) of receptor binding site was NGQQGR pattern, which were similar to that of A/Chicken/Shanghai/F/98. Following VOPBA assay, we found one protein of about 50KDa binding to H9N2 AIV, and the results of LC-MS/MS analysis proved that vimentin was the vital protein binding to H9N2 AIV. The pre-incubation of the specific antibody and siRNA decreased the viral RNA level in MDCK cells treated with H9N2 AIV. Furthermore, we found that over-expressed vimentin increased H9N2 AIV replication in MDCK cells. CONCLUSIONS: These findings suggested that the isolated H9N2 AIV might be a recent clinical common H9N2 strain, and vimentin protein might be one vital factor for H9N2 AIV replication in MDCK cells, which might be a novel target for design and development of antiviral drug.

The Functions and Mechanism of a New Oligopeptide BP9 from Avian Bursa on Antibody Responses, Immature B Cell, and Autophagy.

The bursa of Fabricius is an acknowledged central humoral immune organ unique to birds, which is vital to B cell differentiation and antibody production. However, the function and mechanism of the biological active peptide isolated from bursa on B cell development and autophagy were less reported. In this study, we isolated a new oligopeptide with nine amino acids Leu-Met-Thr-Phe-Arg-Asn-Glu-Gly-Thr from avian bursa following RP-HPLC, MODIL-TOP-MS, and MS/MS, which was named after BP9. The results of immunization experiments showed that mice injected with 0.01 and 0.05 mg/mL BP9 plus JEV vaccine generated the significant increased antibody levels, compared to those injected with JEV vaccine only. The microarray analysis on the molecular basis of BP9-treated immature B cell showed that vast genes were involved in various immune-related biological processes in BP9-treated WEHI-231 cells, among which the regulation of cytokine production and T cell activation were both major immune-related processes in WEHI-231 cells with BP9 treatment following network analysis. Also, the differentially regulated genes were found to be involved in four significantly enriched pathways in BP9-treated WEHI-231 cells. Finally, we proved that BP9 induced the autophagy formation, regulated the gene and protein expressions related to autophagy in immature B cell, and stimulated AMPK-ULK1 phosphorylation expression. These results suggested that BP9 might be a strong bursal-derived active peptide on antibody response, B cell differentiation, and autophagy in immature B cells, which provided the linking among humoral immunity, B cell differentiation, and autophagy and offered the important reference for the effective immunotherapeutic strategies and immune improvement.

The Functions of Bursal Hexapeptide (BHP) on Immune Response and the Molecular Mechanism on Immature B Cell.

BACKGROUND: The bursa of Fabricius (BF) is an acknowledged central immune organ, and is important to B cell differentiation. Bursal hexapeptide (BHP) is the recently reported bursalderived peptide, while its inducing function on immune response is uncertain. OBJECTIVES: The main objective of this study was to analyze the immune responses to JEV vaccine in mice induced by BHP plus JEV vaccine, and to detect the signal and biological functions of BHP on immature B cells. METHODS: Mice were immunized with Japanese encephalitis virus (JEV) vaccine and BHP from 0.01 mg/mL to 0.25 mg/mL to detect antibody response and cellular immune response, respectively. The production of IgG, IgG1 and IgG2a specific to JEV in serum from immunized mice were measured by ELISA, and T cell subpopulation from immunized mice were detected with using fluorochrome conjugated mAbs of the corresponding PE-Cys/FITC/PE by flow cytometry. Spleen cells from all immunized mice were harvested after one week of second immunization for lymphocyte proliferation assay. Mouse immature B cell WEHI-231 cell was treated with 0.01µg/mL BHP for 4h, and analyzed the involved biological function and pathway of differentially expressed genes with gene microarray. RESULTS: BHP co-immunization with JEV vaccine generated significant increased antibody levels, neutralizing antibody titers and spleen lymphocyte viability, compared to that of vaccine control. The subpopulations of T cells in spleen lymphocytes were significantly modified in the mice coimmunized with JEV vaccine and BHP. The analysis results of gene expression profiles of WEHI- 231 mouse immature B cells with BHP treatment showed that the regulated genes with BHP treatment were involved various immune related biological functions, including proliferation and activation of lymphocyte and T cell, T cell mediated immunity and regulation of adaptive immune response. Furthermore, BHP stimulated three significant enriched pathways, including amphetamine addiction, long-term potentiation, and RIG-I-like receptor signaling pathway. CONCLUSION: Our results indicated BHP induced significant humoral and cellular immunity to JEV vaccine, and regulated various biological processes and signalling related to immune activation in immature B cells. These results proposed the immunomodulatory function and mechanism of BHP on immune induction, which provided the novel insight on the candidate reagent for immune improvement.

The Regulatory Functions of a New Tetrapeptide from the Bursa of Fabricius on AIV Vaccine Immunization and Antibody Production.

BACKGROUND: Understanding the regulatory functions of the biological peptide from the humoral central immune organ bursa of Fabricius on vaccine immune responses and antibody production is of vital importance. OBJECTIVES: Here we thoroughly verified the immunomodulatory functions of the new tetrapeptide BP4 from the bursa of Fabricius on vaccine immune responses in mice and chicken immunizaiton model, and on potential intracellular signaling during antibody production. METHOD: BP4 was isolated and identified by Reverse Phase High Performance Liquid Chromatography and matrix-assisted laser desorption ionization time of flight mass spectrometry. immunomodulatory functions of BP4 was verified by AIV vaccine immunization on mice and chickens regarding roles in vivo, by monitoring the impact of signalling inhibitors in hybridoma cells on antibody production in vitro. RESULTS: Our investigation revealed the strong inducing roles of new isolated BP4 on immune responses in mice immunization, the immunomodulatory effects in the immunized chicken, four potential key intracellular signaling during antibody production in hybrdoma cells. CONCLUSION: The new bursal-derived peptide BP4 was isolated and identified, and the immunomodulatory effects on antigen-specific immune responses in vivo and in vitro were verified, suggesting BP4 might be highly relevant to the humoral immune responses, and PI3K/Akt, p38 MAPK, NF-κB and tyrosine phosphorylation signaling might be the key activated intracellular signaling during antibody production during BP4 stimulation, which provided a novel potential adjuvant candidate for vaccine immunization improvement and precaution on animal epidemic disease.