Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is an acute, febrile, and highly contagious disease that has led to significant economic losses in the global swine industry. Although the attenuated lapinized CSF vaccine (C-strain) has effectively controlled CSF outbreaks in China since the 1950s, it remains challenging to serologically differentiate infected from vaccinated animals (DIVA). Currently, the application of E2 subunit vaccines allows for DIVA by detecting antibodies against the E-rns protein. Therefore, this study aimed to develop a blocking ELISA for CSFV E-rns antibody detection using porcine monoclonal antibodies (mAbs) derived from single B cell technology. Peripheral blood mononuclear cells (PBMCs) were isolated from immunized pigs, and single CD21(+)IgM(-)E(rns)-His tag(+) B cells were sorted via flow cytometry. Using one-step PCR, full-length genes of porcine IgG heavy and light chains were amplified separately, yielding 11 porcine mAbs against the CSFV E-rns protein. Among these, three mAbs (E0S3, E0S5, and E0S10) exhibited broad reactivity, while two (E0S1, E0S4) showed no cross-reaction with bovine viral diarrhea virus (BVDV). Using mAb E0S4 as the blocking antibody, a blocking ELISA was established and optimized. The assay demonstrated a detection limit of 1:128, no cross-reactivity with other swine viruses or BVDV, and intra- and inter-assay coefficients of variation below 10%. ROC curve analysis determined an optimal cut-off value of 48.4%, with high sensitivity and specificity. In conclusion, the developed blocking ELISA provides a reliable tool for high-throughput serological surveillance, facilitating the DIVA strategy and contributing to CSF eradication programs.

Background/Objectives: African swine fever (ASF) causes massive global swine industry losses with no effective vaccine available. This study constructed T7 phages displaying key ASFV proteins to evaluate their potential as an ASF vaccine by assessing viral shedding and immune responses in pigs. Methods: Five ASFV proteins were displayed on T7 phages to form VLPs (ASFV-SC-T7 group), with soluble proteins (ASFV-SC group) and PBS as controls; 9 piglets were immunized, boosted at 28 days, challenged with virulent ASFV, and assessed via ELISA, flow cytometry, and real-time PCR. Results: ASFV-SC-T7 induced more high-titer antibodies and elevated monocytes/CD8+ T cells, but all groups developed ASF lesions, with ASFV-SC-T7 having higher lung/mesenteric lymph node viral loads and no survival improvement (only delayed fever). Conclusions: T7 phage-displayed ASFV proteins activate strong immunity, confirming T7 phages as a viable delivery platform, but failed to protect against virulent ASFV, requiring future optimization of antigens and regimens.

(1) Background: Contagious ecthyma, also known as orf, is an epitheliotropic zoonotic disease caused by the orf virus (ORFV), primarily affecting the skin and mucous membranes of ruminants such as goats and sheep, leading to the formation of papules and pustules. Vaccination is the most effective way to prevent this disease in susceptible animals; however, traditional attenuated vaccines carry the potential risk of reversion to virulence. Therefore, there is an urgent need to develop safe and effective vaccines for the prevention and control of orf. (2) Methods: In this study, building upon the previously constructed ORFV three-gene deletion strain rGS14-TrypMut, we employed homologous recombination to knock out the VIL-10 gene and successfully constructed a four-gene deletion strain, rGS14-QuadMut. We evaluated its in vitro growth characteristics, safety, and protective efficacy in a challenge model. (3) Results: The in vitro results show that rGS14-QuadMut had a replication ability similar to that of other two-gene deletion strains, with good genetic stability. In in vivo experiments, compared to rGS14-TrypMut, rGS14-QuadMut caused only mild redness and swelling at the inoculation site, with a faster healing rate, indicating better safety. Additionally, rGS14-QuadMut induced strong differentiation of CD4+ and CD8+ T cells, increased the CD4+/CD8+ ratio, and primarily stimulated a Th1-type immune response, with significant changes in cytokine levels, including IL-8, IFN-γ, and IL-2. In the challenge protection experiment, both rGS14-QuadMut and rGS14-TrypMut provided 100% protective efficacy. In conclusion, rGS14-QuadMut demonstrated enhanced safety without compromising immune protection efficacy and is a promising candidate for an orf live vaccine strain.

African swine fever (ASF) has caused a devastating pandemic among domestic and wild swine, leading to significant economic losses in the global swine industry. Recombinant live-attenuated vaccines are a potential option for the control of ASF. However, safe and effective vaccines against the ASF virus (ASFV) are not yet commercially available, and thus, additional vaccine candidates still need to be developed. In this study, we demonstrate that the simultaneous deletion of the ASFV genes CD2v and A137R from the highly virulent isolate ASFV HuB/HH/2019 substantially attenuated the virulence in swine. All pigs (4/4) infected with 105 TCID50 doses of the double gene deletion virus HuB/HH/2019ΔCD2vΔA137R (HuBΔCD2vΔA137R) remained healthy during a 27-day observation period, with no fevers (>40.5°C) observed. Cases of viremia were mild and cleared by 24 days post-inoculation (dpi). Antibodies against ASFV p30 were induced, and the inoculated pigs were well-protected against homologous challenges. All inoculated pigs survived after being challenged with 102 HAD50 ASFV HuB/HH/2019, and no fever or clinical symptoms developed during the 22-day monitoring period. In contrast, all control group pigs died before 11 dpi (2/2). Cytokine assays and RNA sequencing revealed mild responses elicited by HuBΔCD2vΔA137R. Successful innate and passive immune responses were also triggered. The findings suggest that deletion of the CD2v and A137R genes can attenuate ASFV, and the deletion mutant virus HuBΔCD2vΔA137R is a promising vaccine candidate.IMPORTANCEThe emergence of ASF has caused substantial economic losses in the global pig industry. In light of this, the development of a safe and effective vaccine is crucial to control the spread of ASF. In this study, a live-attenuated ASFV strain was developed by simultaneously knocking out the viral genes CD2v and A137R. The mutant virus exhibited weakened virulence in pigs, elicited robust humoral and cellular immunity, and conferred protection upon challenge with high doses of the parental virus. Notably, no fever or clinical symptoms were observed during a 27-day monitoring period. Consequently, our research presents a highly promising candidate strain for a live-attenuated vaccine, offering a significant step forward in the quest for effective prevention measures. Additionally, the identification of these specific gene targets opens up avenues for further exploration and refinement of strategies aimed at combating ASF outbreaks.

African swine fever (ASF) is a highly pathogenic and hemorrhagic swine infectious disease caused by the African swine fever virus (ASFV). It encodes over 150 proteins, among which the CD2v protein plays multiple roles throughout the infection process. Single B-cell antibody technology is a cutting-edge method for preparing monoclonal antibodies (mAbs), which has the advantages of rapid, efficient, and high yield in antibody production, while possessing natural conformations. In this study, by cloning and expressing antibody genes in vitro, 14 murine-derived mAbs were prepared using recombinant CD2v proteins as immunogenic sources, which brings sufficient enrichment and selectivity for the development of antibodies based on the single B-cell antibody technique. All 14 mAbs demonstrated reactivity with CD2v protein by indirect ELISA, whereas 8 mAbs successfully detected CD2v in ASFV-infected PAM cells by IFA, indicating the tested mAbs can effectively recognize and bind to ASFV CD2v. Finally, a blocking ELISA method for detecting CD2v antibodies using CD2v mAb C89 was established, which holds significant potential for broad application in the serological diagnosis of ASFV with determination of the CD2v-blocking ELISA specificity, sensitivity, reproducibility, and compliance rate. It could be used for the rapid clinical detection of ASFV CD2v protein to provide a powerful tool for the monitoring of epidemics.

BackgroundLumpy skin disease (LSD), caused by infection with the lumpy skin disease virus (LSDV), is a highly infectious disease that poses a notable challenge to the cattle industry worldwide. To conduct epidemiological monitoring of LSDV infection in cattle and evaluate the immune efficacy of LSDV vaccines, it is essential to develop a rapid, sensitive, and specific ELISA-based antibody detection method.ResultsWe utilized the LSDV P32 protein, stably expressed in a CHO-K1 suspension cell system, as a coating antigen to develop an indirect ELISA for Capripoxvirus (CaPV) antibody detection. This method specifically recognizes CaPV-positive sera without cross-reactivity with sera positive for bovine viral diarrhea virus, bovine rotavirus, infectious bovine rhinotracheitis virus, and Brucella antibodies. The method demonstrated a maximum serum dilution detection capacity of 1:3200, with intra- and inter-assay variation coefficients below 10%. Comparison with a commercially available kit showed an agreement of 95.7%.ConclusionThe indirect ELISA antibody detection method established exhibited excellent specificity, sensitivity, and reproducibility, providing a reliable tool for clinical detection and epidemiological surveys of LSDV. This method offers significant potential for the prevention and control of LSD outbreaks.

African swine fever virus (ASFV), a highly contagious pathogen characterized by a complex structure and a variety of immunosuppression proteins, causes hemorrhagic, acute, and aggressive infectious disease that severely injures the pork products and industry. However, there is no effective vaccine or treatment. The main reasons are not only the complex mechanisms that lead to immunosuppression but also the unknown functions of various proteins. This review summarizes the interaction between ASFV and the host immune system, along with the involvement of virulence-related genes and proteins, as well as the corresponding molecular mechanism of immunosuppression of ASFV, encompassing pathways such as cGAS-STING, nuclear factor kappa–light-chain-enhancer of activated B cells (NF-κB), Janus Kinase (JAK) and JAK Signal Transducers and Activators of Transcription (STAT), apoptosis, and other modulation. The aim is to summarize the dynamic process during ASFV infection and entry into the host cell, provide a rational insight into development of a vaccine, and provide a better clear knowledge of how ASFV impacts the host.

Single B-cell antibody generation technology is an advanced method that offers several advantages, including rapid production, high efficiency, and high yield. The antibodies generated via this technique retain their natural conformation and are well suited for applications in pathogen diagnosis, disease treatment, and investigations of virus cross-species transmission mechanisms. Our study aimed to establish a platform for generating single B-cell antibodies specifically targeting the foot-and-mouth disease virus (FMDV) 146S antigen in mice. Female BALB/c mice were immunized with inactivated O-type FMDV 146S antigen, and spleen cells were collected for further analysis. Flow cytometric sorting was performed using a biotin-labeled O-type FMDV 146S antigen as a decoy to identify and select CD19 + /CD21/35 + /CD43-/IgM-/Biotin + antigen-specific individual B cells. The gene sequences encoding the variable regions of the heavy and light chains of the murine IgG antibodies were obtained via single-cell nested PCR amplification. Separate constructs were created for the heavy and light chain plasmids to ensure the proper expression of intact IgG antibodies. These plasmids were cotransfected into human embryonic kidney 293T (HEK293T) cells, leading to the successful production and purification of 15 specific monoclonal antibodies (mAbs), 10 which exhibited activity in ELISA tests, and six antibodies that displayed activity in IFA tests. These findings highlight the successful development of a method for generating mouse-derived single B-cell antibodies that target FMDV. This achievement provides a solid foundation for diagnostic techniques and the analysis of antigenic structural variations.

The whole-genome sequence of an African swine fever virus (ASFV) strain (HuB/HH/2019) isolated from Hubei, China, was highly similar to that of the Georgia 2007/1 strain ASFV. After infection with strong strains, domestic pigs show typical symptoms of infection, including fever, depression, reddening of the skin, hemorrhagic swelling of various tissues, and dysfunction. The earliest detoxification occurred in pharyngeal swabs at 4 days post-infection. The viral load in the blood was extremely high, and ASFV was detected in multiple tissues, with the highest viral loads in the spleen and lungs. An imbalance between pro- and anti-inflammatory factors in the serum leads to an excessive inflammatory response in the body. Immune factor expression is suppressed without effectively eliciting an immune defense. Antibodies against p30 were not detected in acutely dead domestic pigs. Sequencing of the peripheral blood mononuclear cell transcriptome revealed elevated transcription of genes associated with immunity, defense, and stress. The massive reduction in lymphocyte counts in the blood collapses the body’s immune system. An excessive inflammatory response with a massive reduction in the lymphocyte count may be an important cause of mortality in domestic pigs. These two reasons have inspired researchers to reduce excessive inflammatory responses and stimulate effective immune responses for future vaccine development.

Background Originating in Africa, African swine fever (ASF) was introduced to China in 2018. This acute and highly virulent infectious disease affects domestic pigs. The World Organization for Animal Health has listed it as a statutory reportable disease, and China has listed it as a category A infectious disease. Methods Primers and probes were designed for four ASFV genes ( B646L , EP402R , MGF505-3R , and A137R ). The primers/probes were highly conserved compared with the gene sequences of 21 ASFV strains. Results After optimization, the calibration curve showed good linearity (R 2 > 0.99), the minimum concentration of positive plasmids that could be detected was 50 copies/µL, and the minimum viral load detection limit was 10 2 HAD 50 /mL. Furthermore, quadruple quantitative polymerase chain reaction (qPCR) with nucleic acids from three porcine-derived DNA viruses and cDNAs from eight RNA viruses did not show amplification curves, indicating that the method was specific. In addition, 1 × 10 6 , 1 × 10 5 , and 1 × 10 4 copies/µL of mixed plasmids were used for the quadruple qPCR; the coefficient of variation for triplicate determination between groups was < 2%, indicating the method was reproducible. Conclusions The results obtained by testing clinical samples containing detectable EP402R , MGF505-3R , and A137R strains with different combinations of gene deletions were as expected. Therefore, the established quadruple qPCR method was validated for the molecular diagnosis of ASF using gene-deleted ASFV strains.

单克隆抗体在疾病预防、诊断和治疗方面具有重要意义,尤其在免疫机制研究方面具有突出贡献.随着单克隆抗体制备技术的发展,单个B细胞抗体制备技术成为新一代快速制备单克隆抗体的方法.该技术利用每个B细胞仅可产生一种特异性抗体的特性,直接筛选出分泌特异性抗体的B细胞,对其表达抗体重链和轻链的基因进行克隆和体外表达,从而获得特异性单克隆抗体.相较传统抗体制备技术,该技术具有快速、高效、产量高等优点,且表达的抗体具有天然构象,不仅可用于病原微生物相关抗原的抗体开发、病毒跨种传播机制等研究,还在抗肿瘤治疗、抗自身免疫病等方面发挥重要作用.本文主要对单个B细胞抗体制备过程和应用现状进行综述,以期为单克隆抗体制备技术的发展和完善提供参考,促进其更好地应用于相关领域.

Foot-and-mouth disease (FMD) is highly contagious and affects the economy of many countries worldwide. Serotype O is the most prevalent and is present in many regions of Asia. Lineages O/SEA/Mya-98, O/Middle East-South Asia (ME-SA)/PanAsia, O/Cathay and O/ME-SA/Ind-2001 have been circulating in Asian countries. Low antigenic matching between O/Cathay strains and current vaccine strains makes the disease difficult to control, therefore, analyzing the molecular evolution, diversity, and host tropisms of FMDV Serotype O in Asia may be helpful. Our results indicate that Cathay, ME-SA, and SEA are the predominant topotypes of FMDV serotype O circulating in Asia in recent years. Cathay topotype FMDV evolves at a higher rate compared with ME-SA and SEA topotypes. From 2011 onwards, the genetic diversity of the Cathay topotype has increased substantially, while large reductions were found in the genetic diversity of both ME-SA and SEA topotypes, suggesting a trend that infections sustained by the Cathay topotype were becoming a more severe epidemic in recent years. Analyzing the distributions of host species through time in the dataset, we found that the O/Cathay topotype was characterized by a highly swine-adapted tropism in contrast with a distinct host preference for O/ME-SA. The O/SEA topotype strains identified in Asia were isolated mainly from cattle until 2010. It is worth noting that there may be a fine-tuned tropism of the SEA topotype viruses for host species. To further explore the potential molecular mechanism of host tropism divergence, we analyzed the distribution of structure variations on the whole genome. Our findings suggest that deletions in the PK region may reflect a common pattern of altering the host range of serotype O FMDVs. In addition, the divergence of host tropism may be due to accumulated structural variations across the viral genome, rather than a single indel mutation.

病毒进入宿主细胞的途径和方式决定了病毒的趋向性和发病机制,病毒感染细胞包括吸附、穿入、脱壳、复制、组装及子代病毒颗粒的释放等步骤,对病毒入侵机制研究可以为开发有针对性的预防策略提供思路.非洲猪瘟病毒作为一种有囊膜双链DNA病毒,其入侵宿主细胞的机制与其他病毒有一定的相似之处,同样也有其特殊性.目前,人们对非洲猪瘟病毒与宿主细胞相互作用,特别是入侵机制的了解仍然非常有限.为此,本文就ASFV入侵细胞的有关研究进展进行了综述,以期为非洲猪瘟病毒致病机制相关研究提供参考.

口蹄疫是危害我国畜牧业的主要传染病之一,主要感染猪、牛、羊等偶蹄动物,其中猪口蹄疫不同于牛、羊口蹄疫,有其独特特点.本文聚焦猪口蹄疫,从其特点、全球流行情况以及我国疫苗使用现状等方面进行了阐述和分析,以期为我国猪口蹄疫防控提供参考.

This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×106 (±4.34%) Da and 2.40×106 (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×106 (±2.94%) Da and 2.88×106 (±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×106 (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all < 1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R2 of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.

猪瘟疫苗免疫是我国猪瘟防控的核心,猪瘟病毒抗体ELISA检测是进行免疫效果评价的最常用手段.中国兽医药品监察所国家/OIE猪瘟参考实验室(以下简称:参考实验室)承担了国家认证认可监督管理委员会(以下简称“认监委”)组织的A类能力验证项目“猪瘟诊断检测技术”(CNCA-19-A01),全面考察和评价我国动物疫病检测机构对猪瘟病毒抗体的检测能力.参考实验室负责制备检测样品,并进行随机编号.向每个参试实验室发放4份待检盲样,由其采用ELISA方法对各盲样进行定性判断,并向参考实验室提供检验结果及原始检验报告.出现“不满意”结果的单位可参加补测1次,若补测结果仍为“不满意”,则表明该实验室暂不具备该参数的检测能力.参加本次能力验证猪瘟病毒抗体检测的实验室共有103家,初测满意率为92.23％,初测和补测的总体满意率为100％.此次能力验证结果表明,参加此次能力比对试验的猪瘟检测机构均能够提供正确的检测结果,可以满足猪瘟病毒抗体监测的需求.通过本次能力验证项目查找出了存在的问题,参试单位可以及时纠正错误,进一步提升猪瘟抗体的检测能力.

目的 考察灭活口蹄疫病毒(foot-and-mouth disease virus,FMDV)粒子146S抗原标准品的稳定性.方法 采用高效液相体积排阻色谱法(size-exclusion high-performance liquid chromatography,SE-HPLC)检测灭活FMDV粒子146S抗原标准品在不同保存条件下(-80及4℃保存0～180 d;4℃振荡保存0～7 d;-80℃保存0～150 d,每30 d冻融1次,共5次)的抗原含量变化,采用直线拟合法对监测数据进行统计学分析,考察稳定性.结果 获得溯源146S抗原标准品标准曲线方程为y=65 010 x-29 389,R2=0.998 8;不同保存条件下的灭活FMDV粒子146S抗原标准品含量变化分析显示,-80℃保存0～180 d、4℃振荡保存0～7 d、-80℃反复冻融5次,拟合直线方程分别为y=-0.159 5 x+63.215、y=-0.079 1 x+63.697和y=-0.635 7 x+63.881,| β1 |均＜t0.95.(n-2).s(β1),斜率不显著,未观测到不稳定性;4℃保存0～180 d,拟合直线方程为y=-2.278 2x+64.801,|β1|＞t0.95.(n-2).s(β1),斜率显著,观测到不稳定性.结论 制备的灭活FMDV粒子146S抗原标准品于-80℃条件下能满足长期贮存和冻融使用,于4℃条件下能满足短期冷藏运输.本文为146S抗原标准品的研制提供了技术数据支持.

Background Zoonotic hepatitis E virus (HEV) infection emerged as a serious threat in the industrialized countries. The aim of this study is exploring a new approach for the control of zoonotic HEV in its main host (swine) through the design and development of an economically interesting chimeric vaccine against HEV and against a devastating swine infection: the foot-and-mouth disease virus (FMDV) infection. Results First, we adopted a computational approach for rational and effective screening of the different HEV-FMDV chimeric proteins. Next, we further expressed and purified the selected chimeric immunogens in Escherichia coli ( E. coli ) using molecular cloning techniques. Finally, we assessed the antigenicity and immunogenicity profiles of the chimeric vaccine candidates. Following this methodology, we designed and successfully produced an HEV-FMDV chimeric vaccine candidate (Seq 8-P222) that was highly over-expressed in E. coli as a soluble protein and could self-assemble into virus-like particles. Moreover, the vaccine candidate was thermo-stable and exhibited optimal antigenicity and immunogenicity properties. Conclusion This study provides new insights into the vaccine development technology by using bioinformatics for the selection of the best candidates from larger sets prior to experimentation. It also presents the first HEV-FMDV chimeric protein produced in E. coli as a promising chimeric vaccine candidate that could participate in reducing the transmission of zoonotic HEV to humans while preventing the highly contagious foot-and-mouth disease in swine.

介绍了WHO 2004年公布的最新的国际及其他生物制品标准物质制备、鉴定和建立的指导原则,旨在引进科学的、具有指导性意义的国际生物制品标准物质建立程序和技术标准,为我国兽用生物制品标准物质制备程序的建立提供依据.

采用杆状病毒表达系统表达并纯化猪瘟病毒Erns蛋白,并对其反应原性进行鉴定.将猪瘟病毒的Erns基因克隆至pFastbac-HT-A载体上,构建了pFA-Erns重组质粒;经转座、转染后构建重组杆状病毒rBac-Erns;通过优化接毒时间和接毒量等参数,最终确定表达条件.将表达产物采用亲和层析法纯化后,采用Western Blotting鉴定Erns蛋白的反应性,并采用间接ELISA方法对其应用进行了初步探索.结果 显示,获得了重组杆状病毒rBac-Erns,经Western Blotting和间接免疫荧光(IFA)鉴定,该重组病毒能够与猪瘟阳性血清和Erns单抗发生特异性反应.纯化后的Erns蛋白纯度较好,浓度为0.2 mg/mL,经Western Blotting方法鉴定证明,纯化后的蛋白与Erns单抗和猪瘟阳性血清能够发生特异性反应;初步建立了间接ELISA方法检测猪瘟抗体,证明表达的蛋白可以区分猪瘟阳性血清和阴性血清.表明本研究利用杆状病毒表达系统成功表达并纯化了猪瘟病毒Erns蛋白,纯化后的Erns蛋白具有良好的反应性,可以作为开发鉴别诊断试剂的备选蛋白以及用于Erns蛋白的结构和生物学功能研究.