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Introduction. The development of combinatorial adjuvants is a promising strategy to boost vaccination efficiency. Accumulating evidence indicates that manganese exerts strong immunocompetence and will become an enormous potential adjuvant. Here, we described a novel combination of Mn2+ plus aluminum hydroxide (AH) adjuvant that significantly exhibited the synergistic immune effect. Methodology. Initially, IsdB3 proteins as the immune-dominant fragment of IsdB proteins derived from Staphylococcus aureus (S. aureus) were prepared. IsdB3 proteins were identified by western blotting. Furthermore, we immunized C57/B6 mice with IsdB3 proteins plus Mn2+ and AH adjuvant. After the second immunization, the proliferation of lymphocytes was measured by the cell counting kit-8 (CCK-8) and the level of IFN-γ, IL-4, IL-10, and IL-17 cytokine from spleen lymphocytes in mice and generation of the antibodies against IsdB3 in serum was detected with ELISA, and the protective immune response was assessed through S. aureus challenge. Results. IsdB3 proteins plus Mn2+ and AH obviously stimulated the proliferation of spleen lymphocytes and increased the secretion of IFN-γ, IL-4, IL-10, and IL-17 cytokine in mice, markedly enhanced the generation of the antibodies against IsdB3 in serum, observably decreased bacterial load in organs, and greatly improved the survival rate of mice. Conclusion. These data showed that the combination of Mn2+ and AH significantly acted a synergistic effect, reinforced the immunogenicity of IsdB3, and offered a new strategy to increase vaccine efficiency. [ABSTRACT FROM AUTHOR]

Background: Virus infection can cause the changes of lncRNA expression levels to regulate the interaction between virus and host, but the relationship between BHV-1 infection and lncRNA has not been reported.Methods: In this study, in order to reveal the molecular mechanism of RNA in BoHV-1 infection, the Madin-Darby bovine kidney (MDBK) cells were infected with BoHV-1, transcriptome sequencing were performed by next-generation sequencing at 18 h or 24 h or 33 h of viral infection and then based on the competitive endogenous RNA (ceRNA) theory, lncRNA-miRNA-mRNA networks were constructed using these high-throughput sequencing data. The network analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for functional annotation and exploration of ncRNA ceRNAs in BoHV-1 infection.Results: The results showed that 48 lncRNAs, 123 mRNAs and 20 miRNAs as differentially expressed genes, and the mitogen activated protein kinase (MAPK) pathway and calcium signaling pathway were significantly enriched in the ceRNA network. Some differentially expressed lncRNA genes were randomly selected for verification by RT-qPCR, and the results showed that their expression trend was consistent with the results of transcriptome sequencing data.Conclusion: This study revealed that BoHV-1 infection can affect the expression of RNAs in MDBK cells and the regulation of ceRNA network to carry out corresponding biological functions in the host, but further experimental studies are still necessary to prove the hub genes function in ceRNA network and the molecular mechanism in BoHV-1 infection. [ABSTRACT FROM AUTHOR]

Introduction: Staphylococcus aureus seriously threatens human and animal health. IsdB137‐361 of the iron surface determinant B protein (IsdB) from S. aureus exhibits the strong immunogenicity, but its immunoprotective effect is still to be further promoted. Because PEI‐PLGA nanoparticles are generated by PEI conjugate with PLGA to develop great potential as a novel immune adjuvant, the immunogenicity of IsdB137‐361 is likely be strengthened by PEI‐PLGA. Methods: Here, PEI‐PLGA nanoparticles containing IsdB137‐361 proteins were prepared by optimizing the entrapment efficiency. Mice were immunized with IsdB137‐361‐PEI‐PLGA nanoparticles to assess their anti‐S. aureus effects. The level of IFN‐γ, IL‐4, IL‐17, and IL‐10 cytokines from spleen lymphocytes in mice and generation of the antibodies against IsdB137‐361 in serum was assessed by ELISA, the protective immune response was appraised by S. aureus challenge. Results: IsdB137‐361 proteins loaded by PEI‐PLGA were able to stimulate effectively the proliferation of spleen lymphocytes and increase the secretion of IFN‐γ, IL‐4, IL‐17, and IL‐10 cytokine from spleen lymphocytes, and significantly enhance generation of the antibodies against IsdB137‐361 in serum, reduce the level of bacterial load in liver, spleen and kidney, and greatly improve the survival rate of mice after challenge. Conclusion: These data showed that PEI‐PLGA nanoparticles can significantly enhance the immunogenicity of IsdB137‐361 proteins, and provide an important reference for the development of novel immune adjuvant. [ABSTRACT FROM AUTHOR]

Infection of cattle with bovine herpesvirus type 1 (BHV−1) can lead to upper respiratory tract disease, conjunctivitis, or genital disease and cause serious economic losses to the cattle industry worldwide. The role of long noncoding RNAs in BHV−1 infection is not well understood. To explore the role of lncRNA−MSTRG.16919.1 in bovine herpes virus type I (BHV−1) infected MDBK cells, the lncRNA−MSTRG.16919.1 gene was silenced and sequenced transcriptome and sequencing data were analyzed by Edge R software, Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and an interaction network of proteins. Real−time quantitative PCR (RT−qPCR) and Western blotting were used to verify the results of bioinformatic analyses. The results showed that 1151 differential genes were obtained in the siRNA−MSTRG.16919.1 group compared with an NC group. Compared with BHV−33 h, 6586 differentially expressed genes were obtained. A total of 498 differentially expressed genes were screened from the two groups. To verify the accuracy of the sequencing, six genes were randomly selected for RT−qPCR, and the results showed that the expression trend of selected genes was consistent with the sequencing results. GO enrichment analysis showed that the differential genes were related to such biological processes as nucleotide binding, enzyme binding, cell cycle, and glial macromolecule metabolism. KEGG analysis enriched 378 and 2634 signaling pathways, respectively, that were associated with virus infection, ubiquitin−mediated protein hydrolysis, phosphoinositol metabolism, apoptosis, and other metabolic pathways. The STRING protein interaction database was used to analyze the interaction network of proteins encoded by differential genes, and the degree algorithm in Cytoscape was used to screen the top 20 proteins. The results showed that SKIV2L2, JAK2, PIK3CB, and MAPK8 were related to virus infection. Western blot analysis of TNF, NF−κB, MAPK8, MAPK9, and MAPK10 proteins showed that lncRNA−MSTRG.16919.1 was involved in regulating the expression of these functional proteins. The results of this study provide basic information for exploring the function and regulatory mechanism of lncRNA−MSTRG.16919.1 in organisms and help for further studying the interaction between virus and host. [ABSTRACT FROM AUTHOR]

Introduction: Staphylococcus aureus (S. aureus) is a gram‐positive opportunistic pathogen, there are currently no high effective vaccine against S. aureus in humans and animals, the development of an efficient vaccine remains an important challenge to prevent S. aureus infection. Here, we prepared Als3‐Th‐cell‐epitope‐Target of RNAIII Activating Protein (TRAP) (ATT) proteins plus the novel combined adjuvants to develop a promising vaccine candidate against S. aureus. Methods: The recombinant pET‐28a (+)‐att plasmids were constructed, and the ATT proteins were expressed and obtained, then, ATT plus Freund's adjuvant or the novel combined adjuvants of cytosine‐phosphate‐guanosine oligodeoxynucleotides (CpG), muramyl dipeptides (MDP), and FIA were immunized in mice. After booster immunization, the levels of interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4), IL‐10 and IL‐17A cytokine were evaluated, the humoral immune responses against TRAP were detected in mice, and the survival rate of mice was confirmed by challenge assay. Results: The mice immunized with ATT plus Freund's adjuvant exhibited significantly higher level of IFN‐γ, IL‐4, IL‐10, and IL‐17A, and displayed the stronger humoral immune response against TRAP than control groups, importantly, the survival rate of these mice was significantly higher than control groups. In addition, compared with the control groups, ATT + CpG + MDP + FIA group was elicited significantly higher level of IFN‐γ, IL‐4, IL‐10, and IL‐17A and was triggered the stronger humoral immune responses against TRAP, moreover, generated the higher survival rate of mice. Conclusion: Als3 epitopes significantly enhanced TRAP immunogenicity. ATT plus the novel combined adjuvants of CpG, MDP, and FIA induced the strong immune response and protection against S. aureus, revealing the combination of CpG, MDP, and FIA adjuvant acts the synergistic effect. [ABSTRACT FROM AUTHOR]

Here, we prepared the novel combined adjuvants, CTB as intra-molecular adjuvant, CpG and aluminum hydroxide (Alum) to strengthen the immunogenicity of clumping factor A221-550 of Staphylococcus aureus (S. aureus). The protein-immunoactive results showed CTB-ClfA221-550 elicited the strong immune responses to serum from mice immunized with CTB and ClfA221-550, respectively. The mice immunized with CTB-ClfA221-550 plus CpG and Alum adjuvant exhibited significantly stronger CD4+ T cell responses for IFN-γ, IL-2, IL-4, and IL-17 and displayed the higher proliferation response of splenic lymphocytes than the control groups, in addition, these mice generated the strongest humoral immune response against ClfA221-550 among all groups. Our results also showed CTB-ClfA221-550 plus CpG and Alum adjuvant obviously increased the survival percentage of the mice challenged by S. aureus. These data suggested that the novel combined adjuvants, CTB, CpG, and Alum, significantly enhance the immune responses triggered with ClfA221-550, and could provide a new approach against infection of S. aureus. CTB: Cholera Toxin B; CpG: Cytosine preceding Guanosine; ODN: Oligodeoxynucleotides; Alum: Aluminum hydroxide; TRAP: Target of RNAIII-activating Protein; TLR9: Toll-like Receptor 9; TMB: 3, 3ʹ, 5, 5ʹ-tetramethylbenzidine; mAbs: Monoclonal Antibodies; OD: Optical Densities; S. aureus: Staphylococcus aureus; ClfA: Clumping factor A; FnBPA: Fibronection-binding protein A; IsdB: Iron-regulated surface determinant B; SasA: Staphylococcus aureus Surface Protein A; GapC: Glycer-aldehyde-3-phosphate dehydrogenase-C The research outline of the combined adjuvants boosted the immunogenicity of clumping factor A221-550. [ABSTRACT FROM AUTHOR]

ATP-binding cassette transporters are ubiquitous in almost all organisms. The Escherichia coli genome is predicted to encode 69 ABC transporters. Eleven of the ABC transporters are presumed to be exporters, of which seven are possible drug export transporters. There has been minimal research on the function of YbhFSR, which is one of the putative drug resistance exporters. In this study, the ybhF gene of this transporter was characterized. Overexpression and knockout strains of ybhF were constructed. The ATPase activity of YbhF was studied using the malachite green assay, and the efflux abilities of knockout strains were demonstrated by using ethidium bromide (EB) as a substrate. The substrates of YbhFSR efflux, examined with the minimum inhibitory concentration (MIC), were determined to be tetracycline, oxytetracycline, chlortetracycline, doxycycline, EB, and Hoechst33342. Furthermore, tetracycline and EB efflux and accumulation experiments confirmed that the substrates of YbhFSR were tetracyclines and EB. The MIC assay and the fluorescence test results showed that tetracyclines are likely to be the major antibiotic substrate of YbhFSR. The existence of the signature NatA motif suggested that YbhFSR may also function as a Na+/H+ transporter. Overexpression of YbhF in E. coli KNabc lacking crucial Na+/H+ transporters conferred tolerance to NaCl, LiCl, and an alkaline pH. Together, the results showed that YbhFSR exhibited dual functions as a drug efflux pump and a Na+ (Li+)/H+ antiporter. [ABSTRACT FROM AUTHOR]

Interferon-γ (IFN-γ), a cytokine produced by activated natural killer cells and T lymphocytes, is an important regulator of innate and adaptive immunity. Interleukin (IL)-18, also known as IFN-γ-inducing factor, is a cytokine that induces T and natural killer cells to produce IFN-γ. In this study, the chicken IL-18 (ChIL-18) and chicken IFN-γ (ChIFN-γ) genes were inserted into the pET28a prokaryotic expression vector, resulting in pET28a-IL-18 and pET28a-IFN-γ, respectively. These plasmids were transformed into Escherichia coli strain BL21, and the ChIL-18 and ChIFN-γ proteins were expressed and purified. To determine their antiviral activities, 200 ng/mL of ChIL-18 and/or ChIFN-γ were inoculated into chicken embryonic fibroblast cells. After 24 h, one 50% tissue culture infective dose (TCID50) of infectious bursal disease virus (IBDV) was inoculated into the chicken embryonic fibroblast cells. The results showed that the antiviral effect of ChIL-18 and ChIFN-γ in combination was better than that of ChIL-18 or ChIFN-γ alone. Next, 14-day-old chicken were injected with 200 µg of ChIL-18 and/or ChIFN-γ and then were challenged with 10³ TCID50 of IBDV via intraperitoneal injection. The results showed that the proliferation of IBDV was inhibited by the injection of the recombinant proteins, especially the combination of ChIL-18 and ChIFN-γ, as evidenced by cytokine detection, quantitative PCR, and pathology analyses. These results indicate that ChIL-18 and ChIFN-γ could inhibit IBDV infection and the combination of ChIL-18 and ChIFN-γ has a better inhibitory effect than either cytokine alone. [ABSTRACT FROM AUTHOR]

The GapC of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce protective humoral immune response in animals. However, B-cell epitopes on the S. dysgalactiae GapC have not been well identified. In this study, a monoclonal antibody (mAb5B7) against the GapC1-150 protein was prepared. After passive transfer, mAb5B7 could partially protect mice against S. dysgalactiae infection. Eleven positive phage clones recognized by mAb5B7 were identified by screening phage-displayed random 12-peptide library, most of which matched the consensus motif DTTQGRFD. The motif sequence exactly matches amino acids 48-55 of the S. dysgalactiae GapC protein. In addition, the motif 48DTTQGRFD55 shows high homology among various streptococcus species. Site-directed mutagenic analysis further confirmed that residues D48, T50, Q51, G52 and F54 formed the core motif of 48DTTQGRFD55. This motif was the minimal determinant of the B-cell epitope recognized by the mAb5B7. As expected, epitope-peptide evoked protective immune response against S. dysgalactiae infection in immunized mice. Taken together, this identified conserved B-cell epitope within S. dysgalactiae GapC could provide very valuable insights for vaccine design against S. dysgalactiae infection. [ABSTRACT FROM AUTHOR]

The aim of this article was to develop an indirect enzyme-linked immunosorbent assay (ELISA) for efficient detection of the infection ofE. coliin cattle. OmpT, a highly conserved protease in allE. colistrains, was successfully expressed inE. coliXL-1-Blue strain with PET32a vector. Molecular weight of recombinant protein was identified by analyzing SDS-PAGE and the immunogenicity of OmpT was confirmed by Western Blotting. The recombinant OmpT was then employed as capture antigen in the ELISA. The antigen concentration and serum dilution were determined using a checkerboard titration. Results showed that the optimal concentration of coated antigen was 1 μg/ml at a serum dilution of 1:640 and the cut-off value of the assay was 0.335. In addition, the cross-reactivity assay showed that the OmpT wasE. colispecific and the reproducibility experiments displayed good repeatability of the assay. Three hundred and forty cattle serum samples were tested by rOmpT-ELISA and sera coagulation tests. The ELISA has showed relative sensitivity of 100% and specificity of 96.47%. Results of these experiments indicated that the rOmpT-ELISA is a simple, rapid, and convenient method for detection the infection ofE. coliwith different serotype strains. [ABSTRACT FROM PUBLISHER]

ABSTRACT The pathogen Staphylococcus aureus causes a wide range of serious infections, necessitating urgent development of a vaccine against this organism. However, currently developed vaccines are relatively ineffective because of the limited antigenic component that is contained in the vaccine formulations. To develop an effective S. aureus candidate vaccine, overlapping PCR was used to add the truncated immunodominant antigen iron-regulated surface determinant B (IsdB)(N126-P361) (tIsdB) to the N-terminal of intact antigen target of RNAIII activating protein (TRAP) and thus construct a tIsdB-TRAP chimera. The humoral and cellular immune responses against tIsdB-TRAP were compared with those against single or combined formulations. tIsdB-TRAP elicited significantly stronger humoral responses in mice ( P < 0.05). As to cellular immune responses in mice, the tIsdB-TRAP group resulted in a greater IL-4 response than did other groups ( P < 0.05). Greater amounts of IL-2 and IFN-γ were found in the tIsdB-TRAP group. Mouse challenge also showed that tIsdB-TRAP provided better protection against S. aureus than did the control groups. These results suggest that this chimeric protein may be a promising pathogen target for further vaccine development. [ABSTRACT FROM AUTHOR]

• PEDV ORF3 is a transmembrane protein that localizes at endoplasmic reticulum to induce ER stress response. • PEDV ORF3 protein has no effect on apoptosis, but induces autophagy dependent on ER stress response. • This work provides some new findings for the biological function of the PEDV ORF3 protein. Porcine epidemic diarrhea virus (PEDV), the causative agent of PED, is an enveloped, positive-stranded RNA virus in the genus Alphacoronavirus , family Coronaviridae , order Nidovirales. PEDV non-structural accessory protein ORF3 is an ion channel related to viral infectivity and pathogenicity. Our previous study showed that PEDV ORF3 has expression characteristic of aggregation in cytoplasm, but its biological function remains elusive. Thus in this study, we initiated the construction of various vectors to express ORF3, and found ORF3 localized in the cytoplasm in the aggregation manner. Subsequently, confocal microscopy analysis showed that the aggregated ORF3 localized in endoplasmic reticulum (ER) to trigger ER stress response via up-regulation of GRP78 protein expression and activation of PERK-eIF2α signaling pathway. In addition, our results showed that PEDV ORF3 could induce the autophagy through inducing conversion of LC3-I to LC3-II, but couldn't influence the apoptosis. In contrast, conversion of LC3-I/LC3-II could be significantly inhibited by 4-PBA, an ER stress inhibitor, indicating that ORF3-induced autophagy is dependent on ER stress response. This work not only provides some new findings for the biological function of the PEDV ORF3 protein, but also help us for the further understanding the molecular interaction between PEDV ORF3 protein and cells. [ABSTRACT FROM AUTHOR]

• PEDV E protein inhibits the poly(I:C)-induced IFN-β and ISGs production. • PEDV E protein suppresses the expression of RIG-I signaling-associated molecules. • PEDV E interacts IRF3 and further interferes its nuclear translocation. • PEDV E protein acts as an IFN-β antagonist through blockage of the RIG-I/ IRF3 signaling. Porcine epidemic diarrhea virus (PEDV) encodes many multifunctional proteins that inhibit host innate immune response during virus infection. As one of important structural proteins, PEDV E protein has been found to block the production of type I interferon (IFN) in virus life cycle, but little is known about this process that E protein subverts host innate immune. Thus, in this present study, we initiated the construction of eukaryotic expression vectors to express PEDV E protein. Subsequently, cellular localization analysis was performed and the results showed that the majority of PEDV E protein distributed at cytoplasm and localized in endoplasmic reticulum (ER). Over-expression of PEDV E protein significantly inhibited poly(I:C)-induced IFN-β and IFN-stimulated genes (ISGs) productions. We also found that PEDV E protein remarkably suppressed the protein expression of RIG-I signaling-associated molecules, but all their corresponding mRNA levels remained unaffected and unchanged. Furthermore, PEDV E protein obviously interfered with the translocation of IRF3 from cytoplasm to nucleus through direct interaction with IRF3, which is crucial for the IFN-β production induced by poly(I:C). Taken together, our results suggested that PEDV E protein acts as an IFN-β antagonist through suppression of the RIG-I-mediated signaling. This study will pave the way for the further investigation into the molecular mechanisms by which PEDV E protein evades host innate immune response. [ABSTRACT FROM AUTHOR]

• Lipid rafts are required for activation of PI3K/Akt signaling both at early and late stages of CA16 infection. • Lipid raft-associated PI3K/Akt plays critical roles in mediating SREBP1 maturation during late stage of CA16 infection. • This finding of lipid raft-associated PI3K/Akt/SREBP1 will deepen our understanding mechanism of CA16 infection. Coxsackievirus A16 (CA16) is one of predominant Enterovirus that possesses high pathogenicity. Lipid rafts, as cholesterol - and sphingolipid - enriched membrane nanodomains, are involved into many aspects of the virus life cycle. Our previous study found that lipid rafts integrity was essential for CA16 replication, but how lipid rafts regulate CA16 replication through activating downstream signaling remains largely unknown. Thus, in this study, we revealed that lipid rafts were required for activation of PI3K/Akt signaling at early stage of CA16 infection. Treatment with wortmannin significantly reduced the expression of virus protein, indicating PI3K/Akt signaling was beneficial for early stage of virus infection. In addition, lipid rafts integrity was also indispensable for PI3K/Akt activation during the late stage of CA16 infection, which played critical functions in mediating sterol regulatory element-binding proteins 1 (SREBP1) maturation. Whereas, over-expression of SREBP1 exhibited inhibition on virus replication, suggesting that PI3K/Akt signaling and SREBP1 might positively and negatively influence virus replication in two different stages of infection, respectively. Taken together, our study demonstrates an important role of the lipid raft-associated PI3K/Akt/SREBP1 signaling in modulating CA16 replication, which will deepen our understanding mechanism of CA16 infection. [ABSTRACT FROM AUTHOR]

• PEDV ORF3 inhibits the productions of pro-inflammatory cytokines IL-6 and IL-8. • PEDV ORF3 inhibits NF-κB activation through blockage of p65 activation. • Our study links PEDV ORF3 to inhibition of pro-inflammatory cytokines. Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded positive-sense RNA virus that belongs to a porcine entero-pathogenic alphacoronavirus , causing lethal watery diarrhea in piglets. Despite existing study reports the sole accessory protein ORF3 identified as NF-κB antagonist, the contribution of PEDV ORF3 to production of the pro-inflammatory cytokines mediated by NF-κB signaling remains largely unknown. Thus in this present study, we showed that PEDV ORF3 protein significantly inhibited the productions of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8. The phosphorylation of IκBα was inhibited by ORF3 protein, but no degradation of IκBα was induced in ORF3-expressing cells. Furthermore, PEDV ORF3 inhibited NF-κB activation through preventing nuclear factor p65 phosphorylation and down-regulating p65 expression level, as well as interfering nuclear translocation of p65, eventually resulting into the inhibition of IL-6 and IL-8 production. Our study definitely links PEDV ORF3 to inhibition of pro-inflammatory cytokines production, which will provide new insight into the molecular mechanisms of NF-κB activity inhibited by PEDV proteins to facilitate virus evasion of host innate immune. [ABSTRACT FROM AUTHOR]