Construction and Analysis of ceRNA Networks Reveal the Key Genes Associated with Bovine Herpesvirus Type 1 Infection.

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.

PEI-PLGA nanoparticles significantly enhanced the immunogenicity of IsdB137-361 proteins from Staphylococcus aureus.

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.

Immunogenicity of the Non-toxic Form of Staphylococcus Aureus α-Hemolysin Using a Chimeric Fusion in Mice.

Background: Staphylococcus aureus is an opportunistic pathogen responsible for various infections with diverse clinical presentation and severity. The α-hemolysin is a major virulence factor in the pathogenesis of S. aureus infections. Objective: To produce a chimeric fusion protein for hemolytic detection of the S. aureus isolates and as a component of a multi-antigen vaccine. Methods: The fused strategy employed a flexible linker to incorporate the possible B cell and T cell determinants into one chimera (HlaD). The humoral and cellular response to the HlaD in mice was assessed to reveal a non-significant difference compared with the full-length α-hemolysin mutant (Hla H35L). Results: The results of the protective effect, the mimetic lung cell injury, and bacterial clearness demonstrated that the mice vaccinated with the HlaD alleviated the severity of the infection of the S. aureus, and the HlaD could similarly function with Hla H35L. Conclusion: The chimeric fusion (HlaD) provided a diagnostic antigen for hemolysis of the S. aureus strains and a potential vaccine component.

ORFV can carry TRAP gene expression via intracellular CRISPR/Cas9 gene editing technology.

Orf is an acute and highly contracted human and animal infection caused by orf virus (ORFV), which mainly affects sheep, goats, and other species. Clinically, opportunistic or conditional pathogens such as Staphylococcus aureus (S. aureus) are often detected in cases of orf, which greatly increases the risk of disease progression and clinical death. It has been reported that TRAP gene products of S. aureus can broadly influence bacterial life and pathogenicity in vivo, and introduction of exogenous TRAP genes may help to inhibit the proliferation of bacteria. In order to achieve the combined control of ORFV and S. aureus, a novel approach to design a S. aureus TRAP gene vaccine using a live attenuated ORFV vector is proposed. In this study, CRISPR/Cas9 gene editing technology was used to disable vascular endothelial growth factor E of ORFV (VEGF-v) and introduced TRAP gene into this position. TRAP gene expression was detected in keratinocytes infected with recombinant virus. The construction and experimental verification of recombinant ORFV (ORFV-v/TRAP) will provide a reference for in-depth studies on the prevention and control of mixed infectious disease.

The cooperation between orf virus and Staphylococcus aureus leads to intractable lesions in skin infection.

A large amount of evidence shows that different kinds of microorganisms can jointly cope with environmental pressures including cell hosts. For example, in many cases, it has been found that secondary or mixed infection of animals caused by ORFV (an epitheliophilic Parapoxvirus) and bacteria (such as Staphylococcus aureus or Streptococcus) shows a mutual aid mode that indirectly leads to the deterioration of the disease. However, the lack of research on the co-pathogenic mechanism, including how to hijack and destroy the cell host in the pathological microenvironment, has hindered the in-depth understanding of the pathogenic process and consequences of this complex infection and the development of clinical treatment methods. Here, we summarized the current strategies of trapping cell hosts together, based on the previously defined ORFV-Host (O-H) system. The opportunistic invasion of S. aureus destroyed the delicate dynamic balance of the O-H, thus aggravating tissue damage through bacterial products (mediated by Agr), even causing sepsis or inducing cytokine storms. In fact, the virus products from its adaptive regulatory system (VARS) weaken the immune attacks and block molecular pathways, so that S. aureus can settle there more smoothly, and the toxins can penetrate into local tissues more quickly. This paper focuses on the main challenges faced by cell hosts in dealing with mixed infection, which provides a starting point for us to deal with this disease in the future.

The OH system: A panorama view of the PPV-host interaction.

Poxviruses are a family of specialized cytoplasm-parasitic DNA viruses that replicate and assembly in virus factory. In Parapoxvirus (PPV) genus, with the orf virus (ORFV) as a representative species of this genus, their behaviors are significantly different from that of Orthopoxvirus, and the plots of viral practical solutions for evading host immunity are intricate and fascinating, particularly to anti-host and host's antiviral mechanisms. In order to protect the virus factory from immune elimination caused by infection, PPVs attempt to interfere with multiple stress levels of host, mainly by modulating innate immunity response (IIR) and adaptive immunity response (AIR). Given that temporarily constructed by virus infection, ORFV-HOST (OH) system accompanied by viral strategies is carefully managed in the virus factory, thus directing many life-critical events once undergoing the IIR and AIR. Evolutionarily, to reduce the risk of system destruction, ORFV have evolved into a mild-looking mode to avoid overstimulation. Moreover, the current version of development also focus on recognizing and hijacking more than eight antiviral security mechanisms of host cells, such as the 2',5'-oligoadenylate synthetase (OAS)/RNase L and PKR systems, the ubiquitin protease system (UPS), and so on. In summary, this review assessed inescapable pathways as mentioned above, through which viruses compete with their hosts strategically. The OH system provides a panoramic view and a powerful platform for us to study the PPV-Host interaction, as well as the corresponding implications on a great application potential in anti-virus design.

Als3-Th-cell-epitopes plus the novel combined adjuvants of CpG, MDP, and FIA synergistically enhanced the immune response of recombinant TRAP derived from Staphylococcus aureus in mice.

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.

Immunogenicity of multi-epitope vaccines composed of epitopes from Streptococcus dysgalactiae GapC.

Glyceraldehyde-3-phosphate dehydrogenase C (GapC) of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce a protective immune response against S. dysgalactiae infection. To investigate the immune response and protective efficacy induced by epitope-vaccines against S. dysgalactiae infection, we constructed epitope-vaccines GTB1, GB1B2, and GTB1B2 using a T cell epitope (GapC63-77, abbreviated as GT) and two B cell epitopes (GapC30-36, abbreviated as GB1, and GapC97-103, abbreviated as GB2), which were identified in GapC1-150 of S. dysgalactiae in tandem by a GSGSGS linker. BALB/c mice were immunized via an intramuscular injection with the epitope vaccines. The levels of the cytokines, IFN-γ, IL-4, and IL-17, secreted by splenic lymphocytes and the antibody levels in the sera of the immunized mice were detected by ELISA. The immunized mice were subsequently challenged with S. dysgalactiae, and the bacterial colonization in the immunized-mouse organs was examined using the plate counting method. The results showed that the level of the cytokines induced by GTB1B2 was lower than that induced by GapC1-150, but higher than that induced by other epitope vaccines. The level of IgG induced by GTB1B2 was lower than that induced by GapC1-150, but higher than the levels induced by other epitope vaccines. The bacterial colonization numbers in the organs of the mice immunized with GTB1B2 were higher those of the mice immunized with GapC1-150, but significantly lower than those from the mice immunized with other epitope-vaccines. Our results demonstrated that the T cell and B cell epitopes in the epitope-vaccines worked synergistically against bacterial challenge. The multi-epitope vaccine, GTB1B2, could induce stronger cellular and humoral immune responses, and provide a better protective effect against S. dysgalactiae infection.

Laboratory Diagnosis of a NZ7-like Orf Virus Infection and Pathogen Genetic Characterization, Particularly in the VEGF Gene.

Orf is a widespread contagious epithelial viral disease found particularly in most sheep breeding countries in the world. Recently, an orf virus (ORFV) strain OV-HLJ05 was isolated from an outbreak in northeast China. Three genes of interest including ORFV011 (B2L), ORFV059 (F1L), and ORFV132 (VEGF) of ORFV, were recruited to identify and genetically characterize this newly isolated virus. Amino acid (aa) sequence compared with the ORFV references listed in GenBank, both B2L and F1L of OV-HLJ05 showed less microheterogeneity from their references. In contrast, the VEGF gene was included in the NZ7-VEGF like group as previously considered by Mercer in 2002. Unexpectedly, further multiple VEGF matches were made, using 34 published sequences from China and India, resulting in 27 strains of the NZ7 members. Based on Karki's report in 2020, NZ7-VEGF like viruses are emerging more and more frequently in these two countries, damaging the Asian sheep industry. Obvious heterogeneity with the NZ2, insertion of two oligopeptides TATI(L)QVVVAI(L) and SSSS(S) motif were found in the NZ7-like VEGF protein. These VEGFs are divided mainly into two types and a significant increase in the number of hydrogen bonds within the NZ7-like VEGF dimers was observed. The NZ7-like ORFV apparently favors the goat as a host and an emphasis on this in future epidemiological and pathological studies should be considered, focusing on the NZ7-like virus.

The novel combinations of CTB, CpG, and aluminum hydroxide significantly enhanced the immunogenicity of clumping factor A 221-550 of Staphylococcus aureus.

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. ABBREVIATIONS: 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, 3', 5, 5'-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.

Identification of CD4+ T cell epitopes on glyceraldehyde-3-phosphate dehydrogenase-C of Staphylococcus aureus in Babl/c mice.

Glyceraldehyde-3-phosphate dehydrogenase-C (GapC) is a highly conserved surface protein of Staphylococcus aureus, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, which represents an excellent vaccine candidate antigen. It can induce protective immune responses to S. aureus infections. However, CD4+ T cell epitopes of GapC that induce CD4+ T cell immune responses are currently unclear. In this study, we used bioinformatics prediction algorithms to predict CD4+ T cell epitopes of GapC. Ten peptides were synthesized to investigate the candidate epitopes. Our results showed that the peptides, G4 (GapC 104-123) and G10 (GapC 314-333) were able to induce proliferation of CD4+ T cells and secrete high levels of interferon (IFN)-γ, respectively. In addition, they significantly reduced bacterial loads in tissue and induced immunoprotective effects. It is suggested that G4 and G10 are Th1-type epitopes of S. aureus GapC. This study provides the potential development of the design of epitope-based vaccine against S. aureus.

Structural Features of a Conformation-dependent Antigen Epitope on ORFV-B2L Recognized by the 2E4 mAb.

Previously, we successfully prepared a monoclonal antibody (mAb) named 2E4, that directly recognizes the major envelope protein B2L of the orf virus (ORFV), but there is little information about its epitope. Here, we meticulously mapped the 2E4 epitope through combinatorial programs and identified the functional binding domain and a key amino acid residue. Briefly, the simulated epitope peptide closely resembles 84VDVQSKDKDADELR97 located at the N-terminus of B2L, strongly suggesting that the epitope is conformationally or spatially structure-dependent. Subsequently, we combined these findings with the results from the antigenicity prediction of B2L to design three truncated fragments of B2L (F1, F2 and F3) selected using 2E4, and only the F1 fragment was found to be eligible for the advanced stage. Alanine-scanning mutagenesis suggested that the D94 residue is structurally crucial for the 2E4 epitope. The other participating residues, including K61, E62, and D92, together with D94 were responsible for enabling 2E4 binding and served as factors that synergistically enabled binding to the whole 2E4 epitope. In this paper, we describe, for the first time, the architecture of an ORFV conformational epitope, and it is also expected that mAb 2E4 and its epitope can be used for applications relating to orf control.

Evaluation of the immunogenicity of an omp A and staphylococcal target of RNAIII activating fusion protein displayed on the surface of Escherichia coli.

The purpose of this investigation was to construct a recombinant Escherichia coli strain displaying the Staphylococcus aureus target of RNAIII activating protein (TRAP) on its surface, and to investigate the strain for its immunogenicity. The lpp'ompA and lpp'ompA-TRAP genes were fused by the overlap polymerase chain reaction and then ligated into expression plasmid pQE30 producing pLO and pLO-TRAP. These two recombinant plasmids were transformed into E. coli XL1-Blue, resulting in XL1-Blue/pLO and XL1-Blue/pLO-TRAP, which were induced to express protein. The expressed TRAP protein was displayed on the surface of XL1-Blue as judged by whole cell ELISA, flow cytometric analysis, and laser scanning confocal microscopy using the lpp'ompA surface display system. ICR mice were intramuscularly immunized with recombinant strains XL1-Blue/pLO and XL1-Blue/pLO-TRAP as well as recombinant protein TRAP. Immunized mice were assessed for anti-TRAP antibody and lymphocytes for secreted IL-4 and IFN-γ by ELISPOT and secreted IL-17A by indirect ELISA. Immunized mice were challenged with S. aureus Newman and HLJ23-1 strains. The results showed both XL1-Blue/pLO-TRAP and TRAP protein immunized mice to produce better cellular and humoral immunity than XL1-Blue/pLO and PBS injected mice.

Oral pre-administration of Purslane polysaccharides enhance immune responses to inactivated foot-and-mouth disease vaccine in mice.

BACKGROUND: Foot-and-mouth disease (FMD) is one of the greatest disease threats to animal husbandry worldwide. Though various vaccines against foot-and-mouth disease virus (FMDV) have been developed, vaccine effectiveness is still not satisfactory. In this work, we studied the potential ability of Purslane polysaccharide (POL-P3b) as a nutrient food additive to enhance immune responses to FMD vaccination in mice. RESULTS: Our results demonstrated that oral administration of POL-P3b at mid- and high-doses significantly enhanced the FMDV-specific cellular and humoral immune responses in mice and increased the concentration of Ca2+ in lymphocytes. Importantly, POL-P3b could promote intestinal DC maturation and stimulate the secretion of intestinal SIgA in a dose-dependent manner. Moreover, the acute toxicity study showed that POL-P3b was non-toxic and safe in mice. CONCLUSION: Our findings provided solid evidence that POL-P3b might be a novel immunostimulator and a boosting agent for increasing the efficacy of FMD vaccination, and the mechanism was related to stimulating the intestinal mucosal immune function that subsequently enhanced the efficacy of FMD vaccination through pre-administration of oral POL-P3b.

Identification of a conserved linear B-cell epitope in the Staphylococcus aureus GapC protein.

The GapC protein of Staphylococcus aureus (S. aureus) is a surface protein that is highly conserved among Staphylococcus strains, and it can induce protective humoral immune responses. However, B-cell epitopes in S. aureus GapC have not been reported. In this study, we generated a monoclonal antibody (mAb2A9) targeting S. aureus GapC. Through a passive immunity test, mAb2A9 was shown to partially protect mice against S. aureus infection. We screened the motif 236PVATGSLTE243 that is recognized by mAb2A9 using a phage-display system. The motif sequence exactly matched amino acids 236-243 of the S. aureus GapC protein. Then, we identified the key amino acids in the motif using site-directed mutagenesis. Site-directed mutagenesis revealed that residues P236, G240, L242, and T243 formed the core of the 236PVATGSLT243 motif. In addition, this epitope was proven to be located on the surface of S. aureus, and it induced a protective humoral immune response against S. aureus infection in immunized mice. Overall, our results characterized a conserved B-cell epitope, which will be an attractive target for designing effective epitope-based vaccines against S. aureus infection.

Identification of a novel linear B-cell epitope as a vaccine candidate in the N2N3 subdomain of Staphylococcus aureus fibronectin-binding protein A.

PURPOSE: To explore an epitope-based vaccine against Staphylococcus aureus, we screened the epitopes in the N2N3 subdomain of fibronectin-binding protein A (FnBPA) as a surface component of S. aureus. METHODOLOGY: We expressed N2N3 proteins and prepared monoclonal antibodies (mAbs) against N2N3 by the hybridoma technique, before screening the B-cell epitopes in N2N3 using a phage-displayed random 12-mer peptide library with these mAbs against N2N3. Finally, we analysed the characters of the screened epitopes using immunofluorescence and an S. aureus infection assay. RESULTS: In this paper, we identified a linear B-cell epitope in N2N3 through screening a phage-displayed peptide library with a 3C3 mAb against the N2N3. The 3C3 mAb recognized the 159IETFNKANNRFSH171 sequence of the N2N3 subdomain. Subsequently, site-directed mutagenic analysis demonstrated that residues F162, K164, N167, R168 and F169 formed the core of 159IETFNKANNRFSH171, and this core motif was the minimal determinant of the B-cell epitope recognized by the 3C3 mAb. The epitope 159IETFNKANNRFSH171 showed high homology among different S. aureus strains. Moreover, this epitope was exposed on the surface of the S. aureus by using an enzyme-linked immunosorbent assay (ELISA) assay and an indirect immunofluorescence assay. As expected, the epitope peptide evoked a protective immune response against S. aureus infection in immunized mice. CONCLUSION: We identified a novel linear B-cell epitope, 159IETFNKANNRFSH171, in the N2N3 subdomain of S. aureus fibronectin-binding protein A that is recognized by 3C3 mAb, which will contribute to the further study of an epitope-based vaccine candidate against S. aureus.

The double adjuvants LTB and CpG significantly enhanced the immuno-protective effects of recombinant GIT derived from Staphylococcus aureus and Streptococcus in mice.

PURPOSE: In this study, we prepared GapC1-150-IsdB126-361-TRAP (GIT) proteins plus heat-labile enterotoxin B (LTB) as an intra-molecular adjuvant, together with CpG to further enhance its immunogenicity. METHODOLOGY: Initially, the target genes were acquired and inserted into pET-32a (+) vectors to express LTB-GIT protein. LTB-GIT expression was confirmed by Western blotting and its immunocompetence was estimated through ELISA. Further, we immunized BALB/c mice with the LTB-GIT plus CpG adjuvant. After the second immunization, the antigen-specific CD4+ cell responses for IFN-γ, IL-2, IL-4 and IL-10 were monitored by intracellular cytokine staining (ICS) assay. After the third immunization, the level of IgG antibodies in the serum from immunized groups was assessed by ELISA, and the protective immune response was appraised by Staphylococcus aureus and Streptococcus dysgalactiae challenge. RESULTS: The ELISA results showed that the OD450nm value of the LTB-GIT group was significantly higher than that of the BSA group. The group immunized with LTB-GIT plus CpG exhibited significantly stronger CD4+ T cell responses for IFN-γ, IL-2, IL-4 and IL-10 compared to the group immunized with LTB-GIT, GIT alone orLTB-GIT plus CpG. In addition, the group immunized with LTB-GIT plus CpG generated the highest level of IgG antibodies against GIT among all of the groups, and our results also showed that LTB-GIT plus CpG markedly improved the survival percentage of mice compared to other groups. CONCLUSION: We confirmed that the novel double adjuvants, LTB and CpG, are able to significantly improve GIT-induced immune responses. This formula could be a promising strategy for enhancing the immune efficacy of multi-subunit vaccines against Staphylococcus aureus and streptococcal infection.

Characterization of an orf virus isolate from an outbreak in Heilongjiang province, China.

Contagious ecthyma, caused by orf virus (ORFV), is an epitheliotrophic contagious disease with zoonotic implications that mainly affects sheep, goats, wild ruminants, and humans. Recently, a novel ORFV strain, OV/HLJ/04, was successfully isolated from the skin and mucosal lesions of a goat with severe clinical sore mouth symptoms in Heilongjiang province of China. The OV/HLJ/04 isolate was characterized by electron microscopy, serological tests, and experimental reproduction of disease. The purified virions exhibited a typical ovoid shape when observed by electron microscopy. Moreover, experimental reproduction of disease showed that a lamb developed typical clinical signs of contagious ecthyma, such as severe vascular proliferation, when inoculated with the virus. Subsequently, amplification of ORFV011 (B2L) gene fragments of viral DNA by polymerase chain reaction (PCR) and gene sequencing were performed. Phylogenetic analysis of the B2L protein gene revealed that this strain clusters with ORFV strains from epidemic-stricken areas worldwide, including recent mainland China isolates. Analysis using ClustalW MegAlign in DNAStar indicated that OV/HLJ/04 (GenBank: KU523790.1) was genetically closely related to the isolates Gansu (JQ904789), with 99.7% identity; NZ2 (DQ184476), with 97.4% identity; and Xinjiang (KF666560), with 90.6% identity. These results may provide insights into the genotype of the etiological agent responsible for the orf outbreak in Heilongjiang Province.

Identification of CD4+ T-cell epitopes on iron-regulated surface determinant B of Staphylococcus aureus.

Iron-regulated surface determinant B (IsdB) of Staphylococcus aureus (S. aureus) is a highly conserved surface protein that can induce protective CD4(+) T-cell immune response. A pivotal role of CD4(+) T-cells in effective immunity against S. aureus infection has been proved, but CD4(+) T-cell epitopes on the S. aureus IsdB have not been well identified. In this study, MHC binding assay was firstly used to predict CD4(+) T-cell epitopes on S. aureus IsdB protein, and six peptides were synthesized to validate the probable epitopes. Two novel IsdB CD4(+) T-cell epitopes, P1 (residues 159-178) and P4 (residues 287-306), were for the first time identified using CD4(+) T-cells obtained from IsdB-immunized C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice spleen based on cell proliferation and cytokines response. The results showed that P1 and P4 emulsified in Freund's adjuvant (FA) induced much higher cell proliferation compared with PBS emulsified in FA. CD4(+) T-cells stimulated with peptides P1 and P4 secreted significantly higher levels of IFN-γ and IL-17A. However, the level of the cytokine IL-4 almost remained unchanged, suggesting that P1 and P4 preferentially elicited polarized Th1-type responses. In addition, BALB/c mice just respond to P4 not P1, while C57BL/6 mice respond to P1 not P4, implying that epitope P1 and P4 were determined as H-2(b) and H-2(d) restricted epitope, respectively. Taken together, our data may provide an explanation of the IsdB-induced protection against S. aureus and highlight the possibility of developing the epitope-based vaccine against the S. aureus.

Identification of a Conserved Linear B-Cell Epitope of Streptococcus dysgalactiae GapC Protein by Screening Phage-Displayed Random Peptide Library.

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.