IntroductionBy pioneering the use of an 80 MeV/u 12C6+ heavy-ion beam for mutagenesis, we have engineered a stably polarized BHK-21 cell model for FMDV replication.MethodsThis approach yielded two distinct clones: a highly antiviral line (BHK-5) and a highly proviral line (BHK-7). Multi-omics analyses were employed to investigate the mechanisms driving these divergent phenotypes.ResultsThe divergent phenotypes stem from a profound reprogramming of host transcriptional networks. The antiviral BHK-5 clone exhibits a pre-activated innate immune state, leveraging RIG-I/TLR signaling for a rapid interferon response and viral clearance via autophagy. In stark contrast, the proviral BHK-7 clone enhances glycolysis and activates the PI3K-Akt pathway to suppress TNF-mediated immunity and hijack the G2/M cell cycle phase, forming organized "virus factories." At the core of this reprogramming lies a systemic remodeling of transcription factor circuits, particularly within the Runt and C2H2 zinc-finger families.DiscussionOur work demonstrates that 12C6+ heavy-ion mutagenesis can rewire the host immunity-metabolism-cell cycle axis to dictate infection outcomes, providing a powerful framework and cellular toolkit for developing high-yield vaccine substrates and novel antiviral strategies.

BACKGROUND:African swine fever (ASF) is a highly contagious disease, and the core-shell protein p34 is an important antigen that can induce immune responses. The use of ferritin nanoparticles for the orderly and repetitive display of antigens on the particle surface can improve the immunogenicity of subunit vaccines. Here, we used the SpyCatcher/Spytag system to conjugate ferritin nanoparticles with the p34 protein (F-p34). RESULTS:The N-terminus of ferritin was conjugated to a truncated SpyCatcher (SC-ferritin). The antigen of ASFV p34 was linked to SpyTag (p34-ST). SC-Ferritin and p34-ST were expressed in E. coli and purified via CaptoTM Core 700 and Ni-NTA columns, respectively. Based on the isopeptide bonds formed between SpyCatcher and SpyTag, p34 linked to SpyTag was readily surface-displayed on SC-ferritin via in vitro covalent conjugation (F-p34). F-p34 can be taken up by bone marrow dendritic cells (BMDCs) and effectively stimulate BMDC maturation. Compared with the monomeric p34 protein, in vivo studies confirmed that the recombinant F-p34 nanoparticle can induce more robust T-cell responses and stronger specific IgG antibody responses against ASFV. Moreover, F-p34 can increase serum cytokines, which is also significantly greater than that of the p34 protein. These results indicate that the recombinant nanoparticles can induce not only humoral immune responses but also cellular immune responses. In addition, there were no significant pathological changes in the heart, liver, spleen, lung or kidney tissue sections of the mice immunized with F-p34, demonstrating that the recombinant nanoparticles exhibit favorable histocompatibility or safety. CONCLUSIONS:These results demonstrate that we successfully designed a recombinant plasmid and obtained a recombinant protein through the prokaryotic expression system of E. coli. The covalently coupled recombinant F-p34 nanoparticles significantly increased the antigenicity of p34 and contributed to research on African swine fever subunit vaccines.

FMD poses a significant threat to animal husbandry and public health security. This study aims to investigate an innovative method for producing FMD vaccines. Wild-type BHK-21 cells were subjected to heavy ion irradiation. Following the optimization of irradiation parameters, the mutant cell line BHK-7 was selected using the limited dilution method. The concentration of FMDV 146S in the BHK-7 cells was markedly elevated, significantly enhancing FMDV replication. The suspension culture and domestication experiments demonstrated that BHK-7 exhibited characteristics like those of the control BHK-21 cells, thereby improving production efficiency and reducing costs. The metabolic analysis of the BHK-7 suspension cultures indicated that glutamine (GLN) may play a crucial role in FMDV replication, with the addition of an appropriate amount of GLN enhancing viral replication levels. Ten successive generations of BHK-7 cells showed stability in FMDV replication post-domestication, indicating good genetic stability. In this study, we obtained a mutant somatic cell line, BHK-7, which promotes FMDV replication through heavy ion irradiation technology. Through suspension culture domestication and metabolic analysis, this study provides a novel approach and concept for FMD vaccine production, as well as a reference for the development of other vaccine cell lines.

Previously, nanobodies (Nbs) targeting ORF2 protein of novel goose astrovirus (nGAstV) were identified and tested for their specificity and binding affinity to nGAstV. However, their ability to neutralize and prevent the viral infection in goslings remained unexplored, especially those fused with Fc domain of chicken IgG (cFc). In the present study, using nGAstV ORF2 as target antigen, Nb-positive clones from already developed Nb phage library reacting with nGAstV were obtained through three rounds of panning and enrichment. Four Nbs based on sequence variation and solubility were selected, fused with cFc, and genetically engineered to produce chimeric nanobodies. The specific reactivity of these Nbs-Fc with nGAstV was assessed by IFA and WB analysis, the affinity and IC50 of Nbs-Fc were determined by bio-layer Interferometry tests and iELISA, respectively. And the neutralizing activity of one nanobody with good biological activity (Nb9-2-cFc) was assessed by cell infection and in vivo antiviral assays in goslings. All the four Nbs-Fc were successfully expressed and confirmed through SDS-PAGE. These Nbs exhibited strong binding affinity to nGAstV, as demonstrated by IFA and WB analysis. Among these, Nb9-2-cFc showed the highest affinity (Kd=59.6 pM) and neutralization activity (IC50=15.75ng/mL) against nGAstV. Further, In vitro assays revealed that this Nb could reduce virus proliferation by blocking the binding of ORF2 to its receptor. In vivo evaluation of prophylactic activity in goslings revealed that Nb9-2-cFc provided significant protection against nGAstV infection by reducing viral load and histopathological damage in target organs. These findings highlight Nb9-2-cFc as a promising candidate for emergency prevention and treatment against nGAstV infection in goslings.

Foot-and-mouth disease (FMD) is an infectious disease in animals caused by the foot-and-mouth disease virus (FMDV). However, the mechanism of FMDV infection in host cells remains unclear. In this study, we utilized 12C6 heavy ion irradiation technology to process BHK-21 cells and systematically screened and evaluated cell lines with distinct virus replication characteristics. We subsequently employed proteomics to detect the differences between these cell lines and the control BHK-21 cell line following 12C6 heavy ion irradiation. Both cell lines exhibited common downregulation of cell adhesion molecules but also exhibited distinct upregulation pathways. In terms of immune and metabolic responses, BHK-5 infection triggered an immune response, including the upregulation of cytokine–cytokine receptor signaling pathways and lysosome-related pathways, while the upregulation of drug metabolism pathways enhanced the ability to metabolize exogenous substances. Conversely, BHK-7 infection tended to promote metabolic pathway changes that favor virus replication, such as the upregulation of folate biosynthesis, polysaccharide degradation, and linolenic acid metabolism pathways. Additionally, we observed significant downregulation of Cbr3 in cell lines that promoted virus replication and significant upregulation in those that inhibited virus replication. Upon validating the results in Cbr3 knockout cells, we found that knocking out Cbr3 could increase FMDV replication by increasing the cellular content of prostaglandin E2 (PGE2), suggesting a close relationship between FMDV replication and PGE2 levels. This method can increase the production efficiency of FMDV vaccines while reducing manufacturing costs. This study innovatively employed 12C6 heavy ion irradiation technology to induce cell transformation and explored its impact on FMDV, offering a new perspective for understanding virus replication mechanisms and potentially providing a target and idea for developing novel antiviral strategies.

To develop a novel water-in-oil-in-water (W/O/W) adjuvant and evaluate the effect on foot-and-mouth disease (FMD) inactivated vaccine, in this study, we prepared the novel nano-emulsion adjuvant based on QS-21 (BEA) which is composed of the mixture of mineral oil Marcol52, surfactant Tween80, oleate polyoxyethylene ether ester, polyoxyethylene palmitic acid ester and span80, cosurfactant polyethylene glycol and QS-21. The two-step emulsification method formed the W/O/W nano-emulsion with two films and three-phase structures. The effective particle diameter of the BEA was about 184 nm, and it has good thermal stability. Then, BEA was emulsified as an adjuvant to prepare for the inactivated FMDV vaccine, and BALB/c mice and pigs were immunized to evaluate its safety and immunization effect. The results showed that the inactivated BEA-FMDV vaccine significantly increased BALB/c mice and pigs' antibodies and cytokine IFN-gamma in serum. Meanwhile, the pig-neutralizing antibodies were higher than control group. Safety tests found no symptoms of FMD or significant toxic reactions. After 28 days of immunization, the protection rate can reach 93.3%. The BEA vaccine had good stability at 4 degrees C, no stratification after 180 days, and the content of 146S in the vaccine did not decrease. In conclusion, the BEA prepared in this study is suitable for FMDV inactivated vaccine and is an effective adjuvant.

整合应激反应(ISR)是一种适应性信号传导途径,可响应不同应激而被激活,例如未折叠和错误折叠蛋白质的积累,缺氧,氨基酸饥饿,病毒感染和紫外线照射等.病毒感染可激活ISR,但是ISR在病毒感染过程中的作用仍不清楚.在某些情况下,ISR可保护宿主细胞免受病毒感染,而在另一些情况下,ISR可能会被病毒劫持以促进其复制.论文介绍了宿主细胞被病毒感染后诱导ISR信号通路的最新进展,讨论了 ISR调节病毒复制的分子机制,以及病毒如何对抗由ISR引起的这种细胞应激反应,为阐明ISR在病毒感染中的作用及病毒与宿主相互作用的分子机制提供理论依据.

A型塞内卡病毒(Senecavirus A,SVA)是一种新型的小RNA病毒,严重危害我国养猪业的发展.目前尚无有效的SVA疫苗,为探究SVA新型亚单位疫苗,本研究以SVA的病毒结构蛋白2(viral structural protein 2,VP2)基因序列为基础,选取利用生物信息学方法预测的B细胞表位序列及已报道的口蹄疫病毒(Foot-and-mouth disease virus,FMDV)3A蛋白的T细胞表位基因,密码子优化后通过柔性连接肽进行串联连接,分别构建重组表达质粒pET28a-rSVP2-B和pET28a-rSVP2-BT.将原核可溶性表达并纯化的重组串联表位蛋白rSVP2-B和rSVP2-BT,经SDS-PAGE和Western blot鉴定后,免疫BALB/c小鼠(Mus musculus),评价其免疫原性.结果显示,重组质粒转化至大肠杆菌(Escherichia coli)BL21(DE3),经诱导表达后,目标蛋白均正确表达,SDS-PAGE分析显示,其相对分子质量分别为34和38 kD.Western blot结果显示,rSVP2-B和rSVP2-BT与SVA阳性血清均具有良好的反应原性.纯化后rSVP2-B和rSVP2-BT免疫小鼠产生的体液免疫水平无显著差异,均诱导产生了较强的特异性IgG抗体及IgG1抗体,且主要诱导偏向于Th2型的体液免疫应答.病毒中和实验结果显示,2种重组蛋白的中和抗体效价介于1:64～1:128.同时,rSVP2-B和rSVP2-BT免疫小鼠也产生了较强的细胞免疫,其中rSVP2-BT免疫组的脾淋巴细胞增殖水平、细胞因子白细胞介素-2(interleukin-2,IL-2)和干扰素-γ(interferon-γ,IFN-γ)水平显著高于rSVP2-B.以上结果表明,成功制备了具有良好免疫原性的重组串联多表位蛋白rSVP2-B和rSVP2-BT,rSVP2-BT的免疫原性优于rSVP2-B.本研究为SVA多表位疫苗研制提供理论依据.

为提高口蹄疫(FMD)抗原和疫苗的稳定性,采用正交试验设计L18(37),以溶液pH值和氯化钠、氯化镁、磷酸二氢钠、葡萄糖、精氨酸和氯化钙6种组分的质量浓度作为考察因素,以口蹄疫抗原或疫苗在37℃条件下146S抗原降解为0时保存的时间作为评价指标,以PBS(pH值8.0)作为对照,筛选对FMD 146S抗原具有热稳定性作用的化学试剂.结果显示:在4℃条件下,用研制的缓冲液重悬的抗原和配制的疫苗可长期保存;在37℃条件下,与PBS比较,FMDV/OM抗原可保存63 d,FMDV/AF-72抗原可保存84 d,疫苗分别可以保存56和96 d,说明该缓冲液对FMDV抗原和疫苗具有很好的热稳定性.另外,该缓冲液配制方法简单,化学试剂易溶解,可用于大规模生产.研制的缓冲液能提高FMD抗原和疫苗在运输和保存过程中的稳定性,为保证FMD灭活疫苗质量提供了技术支撑.

为了进一步提高口蹄疫疫苗的稳定性并打破国外佐剂垄断现象,研究近年来市面上不断出现的新型佐剂的性能,给口蹄疫疫苗生产厂家筛选国产新型疫苗佐剂提供数据支持,试验以南京威尔药业集团股份有限公司的Well佐剂和SEPPIC公司的ISA 201佐剂为研究对象,以目前口蹄疫疫苗生产厂家常规使用的ISA 206佐剂为对照,配制口蹄疫灭活疫苗后检测其物理性状、免疫效果及稳定性,从而评价佐剂效果.结果表明,Well佐剂和ISA 201佐剂的疫苗稳定性和免疫效果均低于ISA 206佐剂.

疫苗是预防和控制动物疾病感染的有效措施,疫苗制备中最重要的步骤之一就是选择合适的佐剂.本文对佐剂在动物疫苗中的应用进展进行了概述,重点归纳总结了目前几种商业化佐剂的主要成分、作用机制及对畜牧动物的适宜性,并提出了目前兽医疫苗佐剂在研究中面临的挑战.

Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease in cloven-hoofed animals, and neutralizing antibodies play critical roles in the defense against viral infections. Here, we isolated a bovine antibody (R55) using the single B cell antibody isolation technique.

近年来,为了解决新型疫苗免疫原性差的问题,研究者们投入大量的精力研制与之匹配的新型佐剂.佐剂种类较多,现从功能和机制的不同出发,将新型疫苗佐剂分为免疫调节分子类佐剂、抗原递送类佐剂、复合类佐剂三大类,分别对其研究现状和进展进行综述,指出现阶段已存在佐剂的不足及安全性问题,并指出新型免疫佐剂研发的方向.

FMDV is the causative agent of foot-and-mouth disease (FMD), which is one of the most contagious and economically devastating diseases of domestic animals. The antigenic structure of FMDV serotype O is rather complicated, especially for those sites that can elicit a cross-protective neutralizing antibody response.

Objectives: Stability is vital for potency of food-and-mouth disease virus vaccine preparation. However, the assembly of inactivated foot-and-mouth disease virus is poor stable and prone to dissociate into 12s under mild acidic or heating conditions, especially emulsified with oil-adjuvant. Thus, it is crucial to explore a suitable medium and condition to improve the stability and efficiency of inactivated FMDV vaccine. Results: In this study, the basic solution buffer and a serious of potential stabilizers, such as carbohydrate, amino acid, antioxidant, salt and antioxidant were screened for evaluating stable effect on FMDV antigen with aid of high performance size exclusion chromatography (HPSEC). On this basis, orthogonal experiment was performed to optimize and finally confirm the formulation. Anti-aging test were carried out to asses the efficiency of formulation on vaccine stability and the results showed that the vaccine was more stabler either stored at 37℃or 4℃. Moreover, physicochemical monitoring revealed that formulation had no influence on the properties of vaccine. The combined results suggested that the novel solution buffer would lower degradation and prolong shelf life of vaccine. In a word, the novel buffer is beneficial to make FMD vaccine more stable and effective, reducing the dependence on cold delivery and storage. This study also provides insight into the processes of optimization and inactivated vaccine development.

The development of a universal vaccine against foot-and-mouth disease virus (FMDV) is hindered by cross-serotype antigenic diversity and by a lack of knowledge regarding neutralization of the virus in natural hosts. In this study, we isolated serotype O-specific neutralizing antibodies (NAbs) (F145 and B77) from recovered natural bovine hosts by using the single B cell antibody isolation technique. We also identified a serotype O/A cross-reacting NAb (R50) and determined virus-NAb complex structures by cryo-electron microscopy at near-atomic resolution. F145 and B77 were shown to engage the capsid of FMDV-O near the icosahedral threefold axis, binding to the BC/HI-loop of VP2. In contrast, R50 engages the capsids of both FMDV-O and FMDV-A between the 2- and 5-fold axes and binds to the BC/EF/GH-loop of VP1 and to the GH-loop of VP3 from two adjacent protomers, revealing a previously unknown antigenic site. The cross-serotype neutralizing epitope recognized by R50 is highly conserved among serotype O/A. These findings help to elucidate FMDV neutralization by natural hosts and provide epitope information for the development of a universal vaccine for cross-serotype protection against FMDV.

为建立一种简单快速、敏感特异、高通量的牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)抗体检测方法,采用原核表达方法对BVDV E2基因中免疫原性强的1段序列进行截短表达,获得了具有良好反应活性的重组E2蛋白,以重组E2蛋白为包被抗原建立了检测BVDV抗体的间接ELISA方法.结果 显示:该方法检测牛副流感病毒3型(BPIV-3)、牛轮状病毒(BRV)、牛冠状病毒(BCV)、牛传染性鼻气管炎病毒(IBRV)阳性血清均为阴性,检测BVDV抗体的灵敏度可达1∶12 800,批内和批间重复性试验的变异系数分别小于5％和10％,与中和试验的符合率为94.44％.应用该方法检测国内外5个生产厂家的53批次细胞培养用牛血清样品,阳性污染率达39.62％;检测589份临床牛血清样品,阳性感染率为34.80％.研究表明,建立的BVDV重组E2蛋白间接ELISA抗体检测方法具有良好的特异性、敏感性、重复性和适用性,为BVDV感染的监测提供了重要工具.

为了建立一种敏感、特异的猪圆环病毒3型(PCV3)抗体检测方法,根据GenBank公布的PCV3毒株基因组序列,选择ORF2基因中免疫原性较强的一段序列进行截短表达,以获得的重组Cap蛋白为包被抗原,通过间接ELISA反应条件的优化,建立PCV1重组Cap蛋白间接ELISA抗体检测方法,该方法检测PCV2、CSFV、PRRSV、PRV、PPV、PEDV和TGEV阳性血清均为阴性,对PCV3抗体的最低检出效价可达1:25600,批内和批间变异系数分别小于5％和10％,应用该方法检测采集于甘肃及周边地区的857份临床猪血清样品,阳性感染率达21.12％.建立的PCV3重组Cap蛋白间接ELISA抗体检测方法具有良好的特异性、敏感性、可重复性和临床适用性,将为临床中PCV3感染的诊断和流行病学调查等提供一种敏感、特异、简便、快速、高通量的血清学检测技术.

疫苗作为预防多种疾病的主要手段,具有接种方便、安全有效、副作用小等优点.随着人们防疫意识的提高,疫苗需求量的日益增长,疫苗产业亟需增强目前的生产能力、降低成本价格.与此同时,国内外药品监管机构也对疫苗产品的质量提出更高的要求.该文分别从哺乳动物细胞培养和疫苗质量控制的角度,回顾了国内外工业化疫苗生产技术,分析了动物细胞培养的现状和疫苗生产中质量的控制策略,并提出人用灭活疫苗生产技术的发展方向.