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 immunomodulatory functions and molecular mechanism of a new bursal heptapeptide (BP7) in immune responses and immature B cells.

The bursa of Fabricius (BF) is the acknowledged central humoural immune organ unique to birds and plays a vital role in B lymphocyte development. In addition, the unique molecular immune features of bursal-derived biological peptides involved in B cell development are rarely reported. In this paper, a novel bursal heptapeptide (BP7) with the sequence GGCDGAA was isolated from the BF and was shown to enhance the monoclonal antibody production of a hybridoma. A mouse immunization experiment showed that mice immunized with an AIV antigen and BP7 produced strong antibody responses and cell-mediated immune responses. Additionally, BP7 stimulated increased mRNA levels of sIgM in immature mouse WEHI-231 B cells. Gene microarray results confirmed that BP7 regulated 2465 differentially expressed genes in BP7-treated WEHI-231 cells and induced 13 signalling pathways and various immune-related functional processes. Furthermore, we found that BP7 stimulated WEHI-231 cell autophagy and AMPK-ULK1 phosphorylation and regulated Bcl-2 protein expression. Finally, chicken immunization showed that BP7 enhanced the potential antibody and cytokine responses to the AIV antigen. These results suggested that BP7 might be an active biological factor that functions as a potential immunopotentiator, which provided some novel insights into the molecular mechanisms of the effects of bursal peptides on immune functions and B cell differentiation.

Identification and characterization of novel immunomodulatory bursal-derived pentapeptide-II (BPP-II).

The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.

Enantioconvergent hydrolysis of racemic 1,2-epoxypentane and 1,2-epoxyhexane by an engineered Escherichia coli strain overexpressing a novel Streptomyces fradiae epoxide hydrolase.

In order to excavate microbial epoxide hydrolases (EHs) with desired catalytic properties, a novel EH, SfEH1, was identified based on the genome annotation of Streptomyces fradiae and sequence alignment analysis with local protein library. The SfEH1-encoding gene, sfeh1, was then cloned and over-expressed in soluble form in Escherichia coli/BL21(DE3). The optimal temperature and pH of recombinant SfEH1 (reSfEH1) and reSfEH1-expressing E. coli (E. coli/sfeh1) were both determined as 30 â„ƒ and 7.0, also indicating that the influences of temperature and pH on reSfEH1's activities were more obvious than those of E. coli/sfeh1 whole cells. Subsequently, using E. coli/sfeh1 as catalyst, its catalytic properties towards thirteen common mono-substituted epoxides were tested, in which E. coli/sfeh1 had the highest activity of 28.5 U/g dry cells for rac-1,2-epoxyoctane (rac-6a), and (R)-1,2-pentanediol ((R)-3b) (or (R)-1,2-hexanediol ((R)-4b)) with up to 92.5% (or 94.1%) eep was obtained at almost 100% conversion ratio. Regioselectivity coefficients (αS and ßR) displayed in the enantioconvergent hydrolysis of rac-3a (or rac-4a) were calculated to be 98.7% and 93.8% (or 95.2% and 98.9%). Finally, the reason of the high and complementary regioselectivity was confirmed by both kinetic parameter analysis and molecular docking simulations.

Molecular cloning, characterization, and expression of duck 2'-5'-oligoadenylate synthetase-like gene.

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein that exerts antiviral effects through the RNase L- or retinoic acid-inducible gene I (RIG-I)-dependent signalling pathway. In this study, we identified and cloned the OASL gene (named duOASL) from healthy adult Cherry Valley ducks. Full-length duOASL cDNA (1630bp) encoded a 504-amino acid polypeptide containing three conserved domains, namely, nucleotidyltransferase domain, 2'-5'-oligoadenylate synthetase domain, and two ubiquitin-like repeats. DuOASL mRNA expression was quantified by performing quantitative reverse transcription-PCR (qRT-PCR). Results of qRT-PCR showed that duOASL was broadly expressed in all examined tissues, with the highest mRNA expression in the large intestine. Antiviral activity of duOASL was measured by determining its effect on Duck Tembusu virus (DTMUV) replication in vitro. We found that duOASL overexpression slightly inhibited DTMUV replication, whereas duOASL knockdown by using a specific small interfering RNA increased DTMUV replication in DF-1 cells. Thus, we successfully cloned and characterized the antiviral protein duOASL from Cherry Valley ducks and found that it exerted antiviral effects against DTMUV. These results provide a solid foundation for performing further studies to determine the mechanism underlying the antiviral effect of duOASL at the cellular level.

The potential molecular effects of bursal septpeptide II on immune induction and antitumor activity.

The bursa of Fabricius (BF) is the acknowledged central humoral immune organ in birds. Bursal septpeptide II (BSP-II) is an immunomodulatory bioactive peptide isolated from BF. To understand the effects of BSP-II on immune induction, gene expression profiles of hybridoma cells treated with BSP-II were evaluated. Pathway analysis showed that regulated genes were involved in cytokine-cytokine receptor interactions, T cell receptor signaling pathway, and pathway in cancer. It was observed that BSP-II reduced tumor cells proliferation and stimulated p53 expression. These results indicate potential mechanisms underlying the effects of the humoral immune system on immune induction, including antitumor activities. Our study has provided a novel insight into immunotherapeutic strategies for treating human tumors.

Characterization and immunomodulatory function comparison of various bursal-derived peptides isolated from the humoral central immune organ.

The bursa of Fabricius (BF) is the acknowledged central immune organ, which is important to the B cell differentiation and antibody production. However, due to difficult purification, the immunomodulatory peptides from BF were little reported. In this study, the extract samples of BF were taken to a chromatographic analysis by RP-HPLC. Five novel low molecular weight peptides were isolated from BF, with amino acid sequences of YEYAY, RMYEE, GPPAT, AGCCNG, and RRL, and named as Bursal pentapeptide (BPP)-III, -IV, -V, and Bursal hexapeptide (BHP), and Bursal tripeptide (BTP), respectively. BSP-I, BSP-II, BPP-I and BPP-II are recently reported to be the bursal-derived bioactive peptides. In this paper, we analyzed the chemical formula and characteristics of these nine bursal-derived peptides. The immunization comparative experiment verified the different immunomodulatory activity of these nine bursal peptides on antibody and cytokine productions. Furthermore, the results showed that at reachable concentrations, BPP-II and BPP-I induced antibody productions, lymphocyte viabilities and cytokine responses in different dose-dependent manner in the immunized mice model, respectively. These results provided important orientations for the comprehensively understanding and study of the humoral central immune system of human, and provided a novel insight on the treatment of serious disease and immune improvement of human.

Immunomodulatory roles and functional analysis of pre-B lymphocyte DT40 cells with the bursal-derived BSP-II treatment.

The bursa of Fabricius, the acknowledged central humoral immune organ, is vital to B cell differentiation. However, the regulatory function of the bursal-derived peptide on avian B cell proliferation has not been reported. BSP-II is a recently reported bursal-derived bioactive peptide. In this paper, 75 days-old chicks were twice subcutaneously immunized with BSP-II and inactivated avian influenza virus (AIV, H(9)N(2) strain). It was proved that BSP-II induced a strongly AIV-specific HI antibody production in the immunized chicks. Also, BSP-II could enhance avian pre-B lymphocyte DT40 cell viability. To investigate the global patterns of gene expression in DT40 cells after BSP-II treatment, gene microarray was carried out. It was identified that the differentially expressed genes were involved in various pathways, of which six pathways were associated with signaling transductions, including ErbB signaling, MAPK signaling, Toll-like receptor signaling, Notch signaling, mTOR signaling, and Wnt signaling. Finally, RT-qPCR was used to confirm the microarray expression data. These results indicated the molecular basis of pre-B lymphocyte viability with BSP-II treatment, which provided a potential mechanism of the bursa of Fabricius on pre-B lymphocyte viability, differentiation, and development. These results are valid for the mechanism of the bursa of Fabricius on B lymphocytes development.