Mass spectrometry-based proteomic analysis of potential infectious bursal disease virus VP3-interacting proteins in chicken embryo fibroblasts cells.

The structural protein VP3 of infectious bursal disease virus (IBDV) plays a critical role in viral assembly, replication, immune escape, and anti-apoptosis. Interaction between VP3 and host protein factors can affect stages in the viral replication cycle. In this study, 137 host proteins interacting with VP3 protein were screened through liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics approach. The functions and relevance of the proteins were obtained through bioinformatics analysis. Most VP3-interacting proteins were linked to binding, catalytic activity, and structural molecular activity, and performed functions in cell parts and cells. Biological functions of VP3-interacting proteins were mainly relevant to "Cytoskeleton", "Translation", and "Signal transduction mechanisms", involving ribosomes, "Tight junction", regulation of actin cytoskeleton, and other pathways. Six potential VP3-interacting proteins in host cells were knocked down, and vimentin, myosin-9, and annexin A2 were found to be related to IBDV replication. This study would help explore regulatory pathways and cellular mechanisms in IBDV-infected cells, and also provided clues for the in-depth study of VP3 biological functions and IBDV replication or pathogenesis.

Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China.

BACKGROUND: The porcine circovirus-like agent P1 is a newly discovered DNA virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type 2. P1 infection can cause symptoms resembling postweaning multisystemic wasting syndrome. This study aims to develop a rapid, sensitive and specific method to detect P1. RESULTS: A pair of primers was designed and used to amplify a 119 bp DNA fragment to generate a recombinant plasmid which was served as the standard. A SYBR I qPCR protocol was established using the P1 recombinant plasmid standard and the sensitivity, specificity and stability of this method was analyzed. The results demonstrate a strong correlation with P1 recombinant plasmid titers when virus DNA copy numbers fall in between 10(0) ~ 10(9) copies/µL. This method doesn't detect pseudo rabies, porcine parvovirus or porcine reproductive and respiratory syndrome virus; moreover it can distinguish porcine circovirus type 2 from P1 by melting temperature analysis. Coefficient of variation for each batch of reaction is less than 5%. The serum virus titers of P1 positive in this study were measured by this protocol to be 10(3) to 10(7) copies/mL. CONCLUSIONS: The established qPCR is sensitive, specific, and reliable, which could be a useful tool when applied to quantification of P1 in a variety of samples from infected pigs.

[Reversal Effect of NVP-BEZ235 on Doxorubicin-Resistance in Burkitt Lymphoma RAJI Cell Line].

OBJECTIVE: To study the reversal effect of NVP-BEZ235 on doxorubicin resistance in Burkitt lymphoma RAJI cell line. METHODS: The doxorubicin-resistant cell line was induced by treating RAJI cells with a concentration gradient of doxorubicin. The levels of Pgp, p-AKT, and p-mTOR in cells were detected by Western blot. Cell viability was detected by MTT assay. IC50 was computed by SPSS. RESULTS: The doxorubicin-resistant Burkitt lymphoma cell line, RAJI/DOX, was established successfully. The expression of Pgp and the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line were both higher than those in RAJI cell line. NVP-BEZ235 downregulated the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line. NVP-BEZ235 inhibited the proliferation of RAJI/DOX cell line, and the effect was obvious when it was cooperated with doxorubicin. CONCLUSION: The constitutive activation of PI3K/AKT/mTOR pathway of RAJI/DOX cell line was more serious than RAJI cell line. NVP-BEZ235 reversed doxorubicin resistance of RAJI/DOX cell line by inhibiting the PI3K/AKT/mTOR signal pathway.

gga-miR-142-5p attenuates IRF7 signaling and promotes replication of IBDV by directly targeting the chMDA5's 3' untranslated region.

Chicken melanoma differentiation-associated gene 5 (chMDA5) is a key pattern recognition receptor (PRR) that recognizes RNA viral infections and initiates an antiviral innate immune response in chickens. MicroRNAs (miRNAs) are involved in the regulation of chMDA5 to sense RNA virus infection, but how it exerts antiviral activity against infectious bursal disease virus (IBDV) infection and regulates chMDA5 in chicken cells is unclear. Thus, we measured the expression of chMDA5 in IBDV-infected DT40 cells and found it significantly increased. Overexpression of chMDA5 activated the IFN-ß and Mx promoters via IRF7-dependent pathways and inhibited replication of IBDV in DT40 cells. The opposite effect occurred after chMDA5 knockdown using siRNA. Also, gga-miR-142-5p regulated chMDA5 according to bioinformatic analysis and data from a dual-luciferase reporter system. Overexpression of gga-miR-142-5p reduced the expression of the chMDA5 protein, promoting IBDV replication, and decreased the activity of the IFN-ß and Mx promoters via an IRF7-dependent pathway; however, it had no effect on the NF-κB-dependent pathway in DT40 cells. Thus, gga-miR-142-5p is a negative regulator of chMDA5 and promotes IBDV replication in DT40 cells through an IRF7-dependent pathway.

[Expression and immunogenic analysis for the tandem-arranged multiple mimic epitope gene of infectious bursal disease virus].

Five mimic epitopes of Infectious bursal disease virus (IBDV) have been identified from a 12-mer phage-displayed peptide library by 5 monoclonal antibodies. Based on the sequences of the five epitopes, multiple epitope gene 5epis was constructed by the five epitopes being tandemly arranged and linked with 4-peptide GGGS. The expression plasmid pET-5epis was constructed and successfully expressed in E. coli. The resultant protein of 5epis was called r5EPIS. The results from SDS-PAGE analysis showed that the proportion of r5EPIS was 15% of the total bacterial proteins and the molecular weight of r5EPIS was 10kDa. By use of parallel immunoblotting test with corresponding monoclonal and polyclonal antibodies, the immunological specificity and reactivity of r5EPIS against IBDV have been verified. Rabbits were subcutaneously injected with r5EPIS (400 micro g per injection), twice with 7 days interval. The titers of the IBDV-specific antibody measured by indirect ELISA were up to 1:4000 at the 7th day after first immunization and 1:256000 at the 14th day after the second immunization. To determine the protective ability of r5EPIS to the challenge of IBDV, chickens were injected intramuscularly with r5EPIS in adjuvant twice with 7 days interval (501g per injection) and the resultant antibody titer was up to 1:12800 at the 7'h day after the second immunonization. After challenge with 200ELD50 of virulent IBDV GX8/99 strain, all the chicken in r5EPIS-immunized group were survived in contrast to the mortality of 86.7%(13/15) in adjuvant control group, suggesting that r5EPIS had a potent ability to generate protective immune response and it implied that the constructed gene 5epis is a prospective candidate for the development of epitope-based IBD vaccine.

gga-miR-2127 downregulates the translation of chicken p53 and attenuates chp53-mediated innate immune response against IBDV infection.

Infectious bursal disease (IBD) is characterized by the immune suppression of infected birds. The molecular mechanism by which IBD virus (IBDV) suppresses the host immune system remains to be elucidated. The tumor suppressor protein p53 can inhibit the replication of various viruses, but its effect on IBDV remains unknown. This study established an in vitro infection model based on DF-1 cells (chicken embryo fibroblast cell line) to investigate the antiviral effects of chicken p53 (chp53) on IBDV infection. The expression level and activity of chp53 remarkably increased in IBDV-infected DF-1 cells. The overexpression of chp53 inhibited IBDV replication and upregulated the expression of multiple chicken antiviral innate immunity genes (IPS-1, IRF3, PKR, OAS, and Mx), whereas the suppression of chp53 led to the opposite effect. This result indicates that chp53 activates the antiviral innate immune response of chickens to IBDV infection. Bioinformatics analysis and dual-luciferase reporter assay showed that gga-miR-2127 targeted the 3'UTR of chp53. qRT-PCR and western blot revealed that gga-miR-2127 overexpression in DF-1 cells not only downregulated the expression levels of chp53 and of the antiviral innate immunity genes in chickens but also promoted IBDV replication. Our results suggest that gga-miR-2127 downregulates chp53 mRNA translation by targeting its 3'UTR and attenuates chp53-mediated antiviral innate immune response against IBDV.

gga-miR-9* inhibits IFN production in antiviral innate immunity by targeting interferon regulatory factor 2 to promote IBDV replication.

MicroRNAs (miRNAs) are small non-coding RNAs that contribute to the repertoire of host-pathogen interactions during viral infections. In the current study, miRNA analysis showed that a panel of microRNAs, including gga-miR-9*, were markedly upregulated in specific-pathogen-free (SPF) chickens upon infection with infectious bursal disease virus (IBDV); however, the biological function of gga-miR-9* during viral infection remains unknown. Using a TCID50 assay, it was found that ectopic expression of gga-miR-9* significantly promoted IBDV replication. In turn, gga-miR-9* negatively regulated IBDV-triggered type I IFN production, thus promoting IBDV replication in DF-1 cells. Bioinformatics analysis indicates that the 3' untranslated region (UTR) of interferon regulatory factor 2 (IRF2) has two putative binding sites for gga-miR-9*. Targeting of IRF2 3'UTR by gga-miR-9* was determined by luciferase assay. Functional overexpression of gga-miR-9*, using gga-miR-9* mimics, inhibited IRF2 mRNA and protein expression. Transfection of the gga-miR-9* inhibitor abolished the suppression of IRF2 protein expression. Furthermore, IRF2 knockdown mediated the enhancing effect of gga-miR-9* on the type I IFN-mediated antiviral response. These findings indicate that inducible gga-miR-9* feedback negatively regulates the host antiviral innate immune response by suppressing type I IFN production via targeting IRF2.

Reduction of infectious bursal disease virus replication by shRNAs targeting the VP1 and VP2 genes driven by chicken U6 promoter.

Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease in young chickens and results in considerable economic losses for the poultry industry. To suppress the replication of IBDV, two short hairpin RNAs (shRNAs) were designed for targeting the VP1 and VP2 genes of IBDV. Recombinant plasmids carrying each shRNA or two shRNAs were constructed based on vector pSilencer2.1-U6 in which the human U6 promoter was replaced with chicken U6 promoter. In chicken embryo fibroblasts, transfection with these shRNA plasmids 24h before infection with IBDV B87 reduced 50% tissue culture infectious doses (TCID(50)) from 10(8.75) TCID(50)/0.1 mL to 10(3.75)-10(1.0) TCID(50)/0.1 mL. In 10-day old specific pathogen-free (SPF) chicken embryos, incubation with a mixture of IBDV B87 and a shRNA plasmid via the allantoic cavity resulted in 100% mortality and high IBDV virus titer in the control group but 25-0% mortality and near normal embryo development in the specific shRNA groups; additionally, IBDV VP1 and VP2 mRNA levels were reduced by 72-95% in the shRNA groups as compared with the control groups. When challenged with a virulent strain IBDV GX8/99, 14-day-old chickens pre-treated with the single shRNA plasmids or the dual shRNA plasmid showed approximately 70% or 90% survival at 5 days post-challenge while those pre-treated with control plasmid or saline had less than 5% survival. The current study suggests that two IBDV shRNAs expressed by a plasmid under chicken U6 promoter could effectively and synergistically reduce IBDV replication in vitro and in vivo.

Overexpression of microRNA gga-miR-21 in chicken fibroblasts suppresses replication of infectious bursal disease virus through inhibiting VP1 translation.

Previously we have identified a series of cellular miRNA molecules up- or down-regulated in infectious bursal disease virus (IBDV) infected chicken embryo fibroblasts and Bursa of Fabricius with gene microarray analysis. Here we studied in detail a relatively well studied miRNA, gga-miR-21, for better understanding miRNAs involvement in IBDV-host interactions. Chicken pri-gga-miRNA-21 and a control miRNA Caenorhabditis elegans pri-cel-lin-4 gene were cloned into a lentiviral vector, respectively. The resulting recombinant lentiviruses were used to infect chicken fibroblast cell line DF-1, and two stable cell lines, DF-miR-21 (overexpressing gga-miR-21) and DF-lin-4 (overexpressing cel-lin-4), were selected. Replication of IBDV in DF-miR-21, DF-lin-4 and DF-1 cells were compared and molecular mechanism of IBDV replication alteration was explored using bioinformatics, reporter gene system, qRT-PCR and Western blot analysis. IBDV replication was markedly lower in DF-miR-21 than in DF-lin-4 or DF-1 cells. A gga-miR-21 target sequence was identified within IBDV VP1 gene (1713-1734bp). Fusion of a 520nt long partial IBDV VP1 gene containing the target with a luciferase gene resulted in significantly lower transient luciferase activity in DF-miR-21 cells as compared to that in DF-lin-4 or DF-1 cell. Following IBDV infection of the cell lines, VP1 protein level in DF-miR-21 cells was dramatically lower than that in DF-lin-4 or DF-1 cells but VP1 mRNA level was not different. The finding indicated that gga-miR-21 could suppress IBDV replication through down regulating IBDV VP1 expression at translational level.

Virus-like particles of hepatitis B virus core protein containing five mimotopes of infectious bursal disease virus (IBDV) protect chickens against IBDV.

Current infectious bursal disease virus (IBDV) vaccines suffer from maternal antibody interference and mimotope vaccines might be an alternative. Previously we demonstrated an IBDV VP2 five-mimotope polypeptide, 5EPIS, elicited protective immunity in chickens. In the current study, the 5epis gene was inserted into a plasmid carrying human hepatitis B virus core protein (HBc) gene at its major immunodominant region site. The recombinant gene was efficiently expressed in Escherichia coli to produce chimeric protein HBc-5EPIS which self-assembles to virus-like particles (VLP). Two-week old specific-pathogen-free chickens were immunized intramuscularly with HBc-5EPIS VLP or 5EPIS polypeptide without adjuvant (50 µg/injection) on day 0, 7, 14 and 21. Anti-5EPIS antibody was first detected on day 7 and day 21 in HBc-5EPIS and 5EPIS groups, respectively; on day 28, anti-5EPIS titers reached 12,800 or 1600 by ELISA, and 3200 or 800 by virus neutralization assay in HBc-5EPIS and 5EPIS groups, respectively. No anti-5EPIS antibody was detected in the buffer control group throughout the experiment. Challenge on day 28 with a virulent IBDV strain (GX8/99) resulted in 100%, 40.0% and 26.7% survival for chickens immunized with HBc-5EPIS, 5EPIS and buffer, respectively. These data suggest epitope presentation on chimeric VLP is a promising approach for improving mimotope vaccines for IBDV.

Development of a multi-mimotope peptide as a vaccine immunogen for infectious bursal disease virus.

To explore the mimotope vaccine approach against infectious bursal disease virus (IBDV), five IBDV-specific monoclonal antibodies (mAbs) were prepared and their binding peptides were screened against a phage-displayed 12-mer peptide library. After three rounds of biopanning, 12 phages were selected for each mAbs and their specificity to IBDV was verified by sandwich and competitive inhibition ELISAs. Seven phages per mAb were sequenced and their amino acid sequences were deduced. The five representative sequences of mimotopes corresponding mAbs were determined. An artificial gene, designated 5epis (5 epitopes) and consisting of the five mimotopes arranged in tandem (F1-F7-B34-2B1-2G8) with four GGGS spacers, was chemically synthesized and cloned into a prokaryotic expression plasmid pET28b. The protein, designated r5EPIS, was efficiently expressed in Escherichia coli and showed a size of 10kDa in SDS-PAGE. The r5EPIS protein reacted with anti-IBDV mAbs and polyclonal antibodies in Western blot immunoassays. Immunization of SPF chickens with r5EPIS protein (with Freund adjuvant, 50mug per injection on day 0 and 14) evoked high levels of antibody (12,800 by ELISA/1600 by virus neutralizing assay at day 21) and protected 100% of the chickens against a challenge of 200 ELD(50) of IBDV GX8/99 strain, which sharply contrasted with the, respectively, 13.3% and 6.6% survival rate in the adjuvant group and the untreated group. The multi-mimotope protein r5EPIS promises to be a novel subunit vaccine candidate for IBDV.