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Additional file 3: Fig. S3. The percentage of IFN-γ-producing CD8+ T cells (represented by P5) in PBMCs from each immunized pig at 42 dpv after in vitro stimulation with inactivated ASFV, mediumor a cell activation cocktail. Group 1 and group 2 respectively represent pigs immunized with O-Ags-T formulation or Ags formulation.

Additional file 2: Fig. S2. The percentage of IFN-γ-producing CD4+ T cells (represented by P8) in PBMCs from each immunized pig at 42 dpv after in vitro stimulation with inactivated ASFV, mediumor a cell activation cocktail. Group 1 and group 2 respectively represent pigs immunized with O-Ags-T formulation or Ags formulation.

Marc-145 cells (monkey embryonic kidney epithelial cells) play a critical role in the biotechnology industry as certain virus host cells. To investigate the expression of enhanced green fluorescent protein (eGFP) gene as a foreign gene in Marc-145 cells, which we developed an approach of foreign gene site-specific knock-in into Marc-145 cells by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and putatively explored appropriate genomic recombination sites in Marc-145 cells. Our study demonstrated that the specific homologous recombination (HR) site between the Rac GTPase activating protein 1 (RACGAP1) and the acid-sensing ion channel subunit 1 (ASIC1) genes of the 11th chromosome could be used as the target site of Cas9 for the generation of target gene knock-in into Marc-145 cells, by the insertion of the eGFP cassette into the specific HR site and subsequent expression. Junction PCR, sequencing, Southern blot and fluorescence assay determined eGFP gene-specific knock-in HR site between the RACGAP1 and ASIC1 genes of the 11th chromosome, which was identified by the genomic safe harbours in Marc-145 cells. Our study encouraged a broader range of applications, such as Marc-145 cells development and engineering for virus adaption and yield increase in the vaccine biotechnology industry.

Background: Foot-and-mouth disease (FMD) is a devastating animal disease. Differentiation of infected from vaccinated animals (DIVA) is very important for confirming suspected cases, evaluating the prevalence of infection, certifying animals for trade and controlling the disease. Methods: In this study, a competitive chemiluminescence immunoassay (3B-cCLIA) for DIVA was developed for the rapid detection of antibodies against non-structural proteins (NSPs) in different species of livestock animals using monoclonal antibody 9E2 as a competitive antibody that recognizes NSP 3B.Results: The cut-off (50%), diagnostic sensitivity (97.20%, 95.71%, and 96.15%) and diagnostic specificity (99.51%, 99.43%, and 98.36) of the assay were estimated by testing a panel of known background sera from swine, cattle and sheep. The accuracy rate of 3B-cCLIA was further validated followed by its comparison with two commercial diagnostic kits. The early diagnostic performance showed that antibodies to NSPs occurred later (about 1–2 days) than antibodies to structural proteins. Furthermore, NSP antibodies present in animals vaccinated multiple times (false-positive), especially in cattle and sheep, were confirmed, and the false-positive rate increased with the number of vaccinations. Conclusions: These results indicated that 3B-cCLIA is suitable to rapidly detect antibodies against FMDV NSP 3B in a wide range of species for DIVA.

Foot‐and‐mouth disease (FMD) is a highly contagious disease that affects cloven‐hoof animals including cattle, swine, sheep, goats, and lots of wild species. Effectively control measures are urged needed. Here, we showed that homoharringtonine treatment exhibited a strong inhibitory effect against two different strains of FMDVs (O/MYA98/BY/2010 and A/GD/MM/2013) in swine kidney (IBRS‐2) cells. Further experiments demonstrated that homoharringtonine did not affect virus attachment or entry. Using time‐of‐addition assays, we found that the antiviral activity of homoharringtonine occurred primarily during the early stage of infection. These results demonstrated that homoharringtonine might be an effective anti‐FMDV drug. Further studies are required to explore the antiviral activity of homoharringtonine against FMDV replication in vivo., Highlight This is the first study to demonstrate the antiviral activity of homoharringtonine against FMDV, including two different serotypes of FMDV in cell culture.Homoharringtonine did not suppress the attachment and entry of FMDV into the cell.The antiviral effect of homoharringtonine occurred in the early phases of FMDV replication.

Foot-and-mouth disease virus (FMDV) has led to serious losses in animal husbandry worldwide. Seromonitoring of FMDV postvaccination is important for the control and eradication of foot-and-mouth disease (FMD) in regions and countries where vaccination is widespread. However, many commercial kits present high false-positive rates. In this study, a multiepitope-based indirect chemiluminescence immunoassay (ME-CLIA) was developed for specifically detecting antibodies against FMDV serotype O in swine sera. The developed method presented high diagnostic sensitivity and excellent diagnostic specificity, and it could detect a broad spectrum of antibodies against FMDV serotype O. The diagnostic performance, accuracy rate, and analytical sensitivity of ME-CLIA were compared with those of three commercial kits. The immune protection value of multiple-epitope recombinant vaccine detected using ME-CLIA was preliminarily determined by observation of clinical symptoms postimmunization challenge, the results of which indicated that the ME-CLIA can be employed as a matching detection method for evaluating multiple-epitope recombinant vaccine. The percent positive values of ME-CLIA determined using swine vaccinated with inactivated vaccine were significantly positively correlated with the titers of liquid-phase-blocking enzyme-linked immunosorbent assay (ELISA) (LBPE) (r = 0.8361; P

Additional file 2: Table S1 Detailed information of serum samples used in the study.

Background: African swine fever(ASF) is an acute, severe and highly fatal infectious disease of pigs. The disease spreads rapidly, causing huge economic losses to the pig industry in infected areas. The structural proteins p30 and p54 in African swine fever virus(ASFV) have been verified as diagnostic antigens.Methods: In this study, we constructed a novel multi-epitope fusion antigen gene based on P30 and P54 proteins, induced expression in a prokaryotic expression system and analyzed the reactivity of the recombinant fusion protein. The purified recombinant protein m35 was used as the coating antigen to establish an indirect enzyme-linked immunosorbent assay (ELISA) detection method for ASFV. 116 serum samples and positive sera of other swine diseases were detected by indirect ELISA.Results: Our results indicate that the m35 gene fragment with a length of 558bp was successfully constructed. SDS-PAGE and Western Blotting analysis showed that the protein had a band at 22kDa, proving its good reactogenicity. ROC analysis was performed to validate the assay, the area under the ROC curve is 0.9738 (95% confidence interval, 0.9336 to 1.014), and does not cross-react with other swine diseases.Conclusion: Our results show that its sensitivity and specificity were highly accurate. It is feasible to use this recombinant protein as a diagnostic antigen to distinguish ASFV infection.

African swine fever (ASF) is a highly fatal swine disease threatening the global pig industry. Currently, vaccine is not commercially available for ASF. Hence, it is desirable to develop effective subunit vaccines against ASF. Here, we expressed and purified two recombinant fusion proteins comprising ASFV proteins p30 and p54 fused to a novel cell-penetrating peptide Z12, which were labeled as ZPM (Z12-p30-modified p54) and ZPMT (Z12-p30-modified p54-T cell epitope). Purified recombinant p30 and modified p54 expressed alone or fused served as controls. The transduction capacity of these recombinant proteins was assessed in RAW264.7 cells. Both ZPM and ZPMT exhibited higher transduction efficiency than the other proteins. Subsequently, humoral and cellular immune responses elicited by these proteins were evaluated in mice. ZPMT elicited the highest levels of antigen-specific IgG responses, cytokines (interleukin-2, interferon-γ, and tumor necrosis factor-α) and lymphocyte proliferation. Importantly, sera from mice immunized with ZPM or ZPMT neutralized greater than 85% of ASFV in vitro. Our results indicate that ZPMT induces potent neutralizing antibody responses and cellular immunity in mice. Therefore, ZPMT may be a suitable candidate to elicit immune responses in swine, providing valuable information for the development of subunit vaccines against ASF.

Recently, many countries, including China, have experienced a series of type A and O foot-and-mouth disease virus (FMDV) epidemics, causing serious economic losses. Although concerns about the safety of inactivated FMD vaccines have been raised, the development of a safe and effective subunit vaccine is necessary. We constructed two chimeric virus-like particles (VLPs; rHBc/AO and rHBc/AOT VLPs) displaying tandem repeats of B cell epitopes (VP1 residue 134-161 and 200-213) derived from type A and O FMDV and one T cell epitope (3 A residue 21-35) using the truncated hepatitis B virus core (HBc) carrier. Our results indicate that the chimeric HBc can self-assemble into VLPs with these FMDV epitopes displayed on the surface. Immunization with the chimeric VLPs induced specific IgG and neutralization antibodies against type A and O FMDV in mice. Compared with the commercial type A/O FMDV bivalent inactivated vaccine, rHBc/AO and rHBc/AOT VLPs significantly stimulated the production of Th1 type cytokines (IFN-γ and IL-2), whereas Th2 cytokine production (IL-4 and IL-10) was decreased. Compared with rHBc/AO, rHBc/AOT induced increased Th2 cytokine and specific IgG production. These results demonstrate that the VLPs constructed in the current study induced both humoral and cellular immune responses and may represent potential bivalent VLP vaccines targeting both FMDV type A and O strains.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which has significant economic consequences in affected countries. As the currently available vaccines against FMD provide no protection until 4–7 days post-vaccination, the only alternative method to control the spread of FMD virus (FMDV) during outbreaks is the application of antiviral agents. Hence, it is important to identify effective antiviral agents against FMDV infection. In this study, we found that mizoribine has potent antiviral activity against FMDV replication in IBRS-2 cells. A time-of-drug-addition assay demonstrated that mizoribine functions at the early stage of replication. Moreover, mizoribine also showed antiviral effect on FMDV in vivo. In summary, these results revealed that mizoribine could be a potential antiviral drug against FMDV.

In order to promote the synergistic interaction between source network and storage, and enhance the load control capability, the adjustable load measurement technology based on blockchain has become the focus of research at home and abroad. This paper reviews the current research status of block chain technology and its application in declaration and issuance, market supervision, market settlement, load measurement, enhancing mutual trust of market transactions and information security. Distributed ledger from block chain under containerized edge service engine, improved directed and acyclic chart distributed ledger structure and adjustable load execution score three based on distributed ledger data. In this respect, the key and difficult points of technology are summarized. Combined with the current situation of power development demand, power market, electricity price mode, demand response, the possible problems and development trend of adjustable load measurement technology based on block chain are discussed in detail.

As a substitute for the traditional generator units, the demand side resources can participate in peak-cutting and valley-filling in the power system by changing their operating power, which is called demand response (DR). The DR market mechanism will play a significant role in guiding power users to actively participate in DR. For a long time in the past, Chinese DR practice mainly relied on administrative methods. These methods have adapted to the monopoly power market environment, but it is difficult to adapt to the current state of the power system reform. Based on this background, this paper first summarizes the widely- used price-based DR market mechanism and incentive-based DR market mechanism. Besides, the point-based market mechanism recently proposed is analysed. Then, based on the theoretical research of the market mechanism, the DR engineering practice in the United States, Europe and China is introduced. The point-based market mechanism practice in Jiangsu Province is focused on. The advantages and disadvantages of each market mechanism are analysed. Finally, according to the current development situation of China’s power market, the suggestions about the DR mechanism are put forward for China.

Several studies have shown that epitope vaccines exhibit substantial advantages over conventional vaccines. However, epitope vaccines are associated with limited immunity, which can be overcome by conjugating antigenic epitopes with built-in adjuvants (e.g., some carrier proteins or new biomaterials) with special properties, including immunologic specificity, good biosecurity and biocompatibility, and the ability to vastly improve the immune response of epitope vaccines. When designing epitope vaccines, the following types of built-in adjuvants are typically considered: (1) pattern recognition receptor ligands (i.e., toll-like receptors); (2) virus-like particle carrier platforms; (3) bacterial toxin proteins; and (4) novel potential delivery systems (e.g., self-assembled peptide nanoparticles, lipid core peptides, and polymeric or inorganic nanoparticles). This review primarily discusses the current and prospective applications of these built-in adjuvants (i.e., biological carriers) to provide some references for the future design of epitope-based vaccines.