Porcine epidemic diarrhea virus (PEDV) causes substantial economic losses in the swine industry globally. Host factors regulating the intracellular replication of PEDV, particularly early RNA synthesis and structural protein production, are not well understood, limiting antiviral strategies. We report that glycoprotein non‑metastatic melanoma protein B (GPNMB) is a key host factor promoting PEDV infection. Initially identified as a PEDV S1-binding partner, GPNMB was confirmed to enhance infection via loss-and gain-of-function experiments in Vero and IPEC-J2 cells. Genetic knockout of GPNMB inhibited PEDV replication without affecting viral attachment or internalization. We show that GPNMB is necessary for the accumulation of double-membrane vesicles (DMVs) and promotes early viral RNA synthesis. Notably, GPNMB directly interacts with the PEDV spike (S) and nucleocapsid (N) proteins, increases their abundance, and facilitates their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, implicating it in structural protein maturation. Our work reveals a pivotal role for GPNMB in PEDV replication and nominates it as a target for host-directed antiviral intervention.

Pseudorabies virus (PRV) inflicts substantial economic losses on the global swine industry. Accurate differentiation between vaccine-derived and circulating wild-type strains is critical for effective disease control and eradication programs. However, conventional diagnostic methods often rely on costly instrumentation and entail lengthy turnaround times, thereby limiting their utility for rapid, field-deployable differential diagnosis—particularly in resource-constrained settings. In this study, we developed a rapid detection system integrating recombinase polymerase amplification (RPA) with lateral flow diagnostic strips (LFD) for the simultaneous identification of PRV wild-type and vaccine strains. The assay demonstrated high analytical specificity, reliably differentiating between PRV vaccine and wild-type strains, exhibiting no cross-reactivity with other prevalent swine pathogens. It also exhibited high sensitivity, yielding clear visual results within 20 min and achieving a limit of detection of 80 copies/µL for the PRV gE gene. Moreover, the method is operationally straightforward, requires minimal training, delivers unambiguous visual readouts, and is highly cost-effective. Collectively, the RPA-LFD platform represents a robust, field-adaptable tool for on-site surveillance of PRV field strains, offering significant potential to support large-scale screening and advance pseudorabies eradication initiatives.

Efficient separation of Staphylococcus aureus (S. aureus) from complex samples is crucial for sensitive detection. Herein, we fabricated a novel cefradine-modified magnetic nanoparticle (CMN) for selective capture of S. aureus. The biomolecule-free CMN could specifically capture target S. aureus due to the specific recognition between cefradine and penicillin binding proteins on the surface of S. aureus, and had the advantages of good stability, fast magnetic responsiveness, simple preparation, and low cost. Additionally, a new vancomycin-modified time-resolved fluorescent microsphere (VTFM) was prepared for electively labeling S. aureus captured by CMN. The antibody-free VTFM could selectively label S. aureus by the specific interaction between vancomycin and peptidoglycan on the surface of S. aureus, and could generate stable, sensitive, and easily quantifiable fluorescence signals. For proving the practicability, CMN linked VTFM (CMN-VTFM) strategy was proposed for the determination of S. aureus. Briefly, the CMN first selectively captured S. aureus from complex samples, and then the VTFM specifically labeled S. aureus captured by CMN; subsequently, CMN-bacteria-VTFM was separated and eluted to release the VTFM; finally, after removing CMN-bacteria, the VTFM was quantitatively analyzed. The biomolecule-free CMN-VTFM strategy with limit of detection of 92 CFU mL−1 for milk and 73 CFU mL−1 for spinach, was successfully used for detecting S. aureus in spiked real samples with recovery between 93.7 % and 100.1 % and relative standard deviation between 3.1 % and 6.1 %, respectively. Therefore, we believe that the CMN-VTFM strategy based on the antibiotic-based recognition has great potential in the field of ultrasensitive determination of pathogens.

Glucocorticoids play a key role in a variety of physiological processes, but their extensive use in the environment has brought potential health hazards. Herein, it is of great necessity to develop a rapid and efficient method for the detection of glucocorticoids. In this work, a hydrophilic core-shell structured magnetic covalent organic framework (HMCOF) was fabricated via a post-modification strategy for the efficient magnetic solid-phase extraction (MSPE) of five glucocorticoids from tap water and milk samples. The HMCOF featured a Fe₃O₄@SiO₂ magnetic core encapsulated by a porous COF shell modified with polyethylene glycol, endowing it with a hydrophilic outer layer, porous structure and sufficient paramagnetism. The adsorption studies showed that HMCOF exhibited high adsorption capacities (50.77-80.25 mg g-1) for glucocorticoids. Notably, HMCOF retained 80 % of its adsorption capacity after 5 cycles, confirming its reusability. Under the optimal conditions of MSPE, the HMCOF-based MSPE-UPLC method was developed to test five glucocorticoids, which demonstrated good linearity (5-150 ng mL-1, R2 ≥ 0.9991), low detection limits (0.2-1.4 ng mL-1) and satisfactory spiked recovery rates (89.5-114.2 %) with intra-day variability below 4.3 % and inter-day precision within 6.3 %. The method underscores the potential of HMCOF serving as an adsorbent for MSPE, providing a promising approach for the analysis of glucocorticoids within food products.

BACKGROUND:Pseudorabies (PR) is a highly contagious disease, and it causes significant economic losses to the global swine industry. Vaccination plays an important role in the prevention and control of pseudorabies virus (PRV). To evaluate vaccine efficacy, there is a need for a quick and straight forward method to monitor PRV-induced antibody levels in practice. RESULTS:A time-resolved fluorescence immunochromatographic (TRFIC) strip was developed for the serological detection of PRV gB antibodies in swine. Following systematic analysis and evaluation, the assay demonstrated a high degree of correlation with established reference method. The positive and negative coincidence rates between the TRFIC strip and ELISA were 96.8% and 94.2%, respectively. Furthermore, comprehensive analytical and comparative assessments revealed that the TRFIC strip exhibited no cross-reactivity with antibodies against other porcine pathogens. CONCLUSION:Given its high specificity, sensitivity, and convenience, the TRFIC strip is suitable for on-site detection of PRV gB antibodies and can serve as a valuable tool for monitoring PRV immune status in animal populations.

Porcine parvovirus (PPV), a non-envelope single-stranded DNA virus, causes severe reproductive disorders in swine worldwide, characterized by fetal mortality, mummification, and reduced boar fertility. As a highly prevalent pathogen in Chinese swine herds, PPV imposes substantial economic burdens on intensive pig production systems. This review systematically synthesizes recent advances in PPV virology, focusing on genomic evolution of emerging strains (PPV1–PPV8), epidemiological dynamics of emerging strains, molecular pathogenesis, and novel diagnostic tools. Furthermore, we critically evaluate current vaccine strategies, highlighting their limitations in cross-protective efficacy and viral shedding control. By integrating multi-omics insights with immunological profiling, this work delineates actionable pathways for next-generation vaccine design and proposes a roadmap for rational antigen selection. This review consolidates foundational knowledge and establishes a translational bridge between basic virology and prevention and control of porcine parvovirus, addressing critical gaps in porcine reproductive disease management.

RESEARCH HIGHLIGHTS:A sandwich ELISA was developed to detect EDSV using the mAbs 5G4 and HRP-6G6.The sandwich ELISA maintained high specificity and sensitivity.The sandwich ELISA had equivalent consistency with real-time PCR assay.

Summary Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone‐dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 μg provided effective protection in pigs without interference from pre‐existing antibodies. Crystal structure and small‐angle X‐ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.

[目的]建立一种基于量子点(quantum dots,QDs)技术的猪伪狂犬病病毒(Pseudorabies virus,PRV)gB抗体免疫层析试纸,为PRV疫苗免疫效果评估提供一种快速、简便的检测技术.[方法]选用昆虫细胞表达系统表达的gB蛋白,采用羧基修饰的水溶性QDs,在偶联剂1-乙基-3[3-二甲基氨基丙基]碳二亚胺盐酸盐(EDC)的作用下制备gB-QDs荧光标记物.将金黄色葡萄球菌蛋白A(Staphylococcus aureus protein A,SPA)和抗gB蛋白的单克隆抗体分别固定在硝酸纤维素膜上作为检测线和质控线,将样品垫、标记垫、吸水垫、支撑底板按生产工艺组装成基于QDs的荧光免疫层析试纸.检测该试纸的特异性、敏感性及与商品化ELISA试剂盒的符合率.[结果]敏感性试验结果显示,该试纸的敏感性为1∶6 400.特异性试验结果显示,该试纸与猪瘟病毒(CSFV)、口蹄疫病毒(FMDV)、猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)阳性血清均无交叉反应,特异性为100％.通过检测田间猪血清样品,对比商品化ELISA检测试剂盒,结果显示,113份田间猪血清样品中,两种方法检测结果不一致的血清有11份,结果一致的血清有102份,该试纸与商品化ELISA试剂盒的符合率为90.3％.[结论]本研究制备的试纸检测线紫外灯下显色清晰可见,辨识度高,具有较高的特异性和敏感性,可用于PRV gB抗体的检测.

为进一步了解猪圆环病毒2型(PCV2)分子进化特点,以PCV2主要蛋白编码序列ORF1和ORF2为研究对象,对74株不同亚型(2a-2e)PCV2进行核酸组成、相对同义密码子使用频率(RSCU)、有效密码子数目(ENC)、中性绘图和密码自适应指数等生物信息学分析,以了解PCV2密码子使用模式及其影响因素.结果显示,相比AU含量,PCV2 GC含量较低(低于50％),但更倾向于使用以C和U结尾的密码子;RSCU值和对应分析显示在密码子使用上,各亚型PCV2间差异较小,但PCV2各亚型与寄主Sus.s间则存在较大的差异;ENC分析和中性分析显示,相比突变压力,各亚型PCV2密码子使用模式受到较大程度的自然选择的影响;CAI值分析结果进一步确定,相比PCV2 c和PCV2 e,PCV2 a、2 b和2 d在密码子使用上更适应其寄主.论文揭示了自然选择是影响PCV2密码子使用模式的重要因素,并分析了影响各PCV2亚型流行的因素,该结果有助于PCV2分子进化的研究和理解该病毒在其寄主内翻译的机制.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a coronavirus which can cause severe human respiratory diseases with a fatality rate of almost 36%. In this study, we report the generation, characterization and epitope mapping of several monoclonal antibodies against the spike receptor-binding domain (RBD) of MERS-CoV. Two monoclonal antibodies (4C7 and 6E8) that can react with linearized RBD have been selected for subsequent identification of RBD mAb-binding epitopes. Two distinct novel linear epitopes, 423FTCSQIS429 and 546SPLEGGGWL554,were precisely located at the outermost surface of RBD by dot-blot hybridization and ELISAs. Multiple sequence alignment analysis showed that these two peptides were highly conserved. Alanine (A)-scanning mutagenesis demonstrated that residues 423F, 428I, and 429S are the crucial residues for the linear epitope 423FTCSQIS429 while residues 548L, 550G, 553W, 554L for epitope 546SPLEGGGWL554. These findings may be helpful for further understanding of the function of RBD protein and the development of subsequent diagnosis and detection methods.

[目的]建立口蹄疫病毒(FMDV)感染与免疫鉴别诊断及免疫评价二联试纸稳定的生产工艺,促进其产业化生产及临床应用.[方法]本研究以胶体金免疫探针结合猪IgG,FMDV结构蛋白(SP)及非结构蛋白(NSP)的表位多肽偶联载体蛋白制备人工抗原,设置两条检测线精准拦截抗体的检测模式,以试纸条特异性及敏感性为评价指标,对胶体金免疫探针用蛋白、试纸拦截用多肽抗原、检测线位置及喷膜缓冲液、金标蛋白保存液、样品垫缓冲液以及样品稀释液等参数进行优化.采用优化后的试纸检测266份田间猪血清样品,并与口蹄疫0型抗体液相阻断ELISA检测试剂盒(LPB ELISA)和3ABC阻断ELISA抗体检测试剂盒(3ABC ELISA)检测结果进行对比,计算该试纸与商品ELISA试剂盒的符合率.[结果]经优化后,口蹄疫感染与免疫鉴别诊断及免疫评价二联试纸生产工艺参数如下:以胶体金标记金黄色葡萄球菌A蛋白(SPA)为免疫探针;选择混合多肽的形式设置拦截线,以非结构蛋白多肽(BSA-NSPs)喷涂T1线,结构蛋白多肽(BSA-SPs)喷涂T2线,以0.1 mol/L Tris-HCl为喷膜缓冲液;以ddH20 含 15 mg/mL BSA、13.25 mg/mL Na2 HPO4·12H2O、15.9 mg/mL NaH2PO4·2H2O、10％Trition X-100 和0.3 mg/mL NaN3 为金标蛋白保存液;以 0.02 mol/L Na2B4O7·10H2O 含 10 mg/mL 酪蛋白、5％Trition X-100、0.3 mg/mL NaN3为样品垫缓冲液;以生理盐水含1.0％Tween-20为样品稀释溶液.制备的FMDV感染与免疫鉴别诊断及免疫评价二联试纸与3ABC阻断ELISA抗体检测试剂盒的符合率为96.20％,与口蹄疫O 型抗体液相阻断ELISA检测试剂盒的符合率为94.36％.[结论]通过对试纸生产工艺的优化,建立了稳定的生产工艺,制备的二联试纸检测线显色清晰可见,肉眼识别度高,试纸的检测特异性和敏感性良好.本研究为该试纸的批量生产和产业化应用奠定了基础,为基层口蹄疫的检测提供了稳定、特异、敏感、准确的检测方法.

Infectious bursal disease (IBD) is a highly contagious immunocompromising disorder that caused great economic losses in the poultry industry. The field-level control over IBD is primarily via vaccination. The development of a highly effective IBV vaccine has drawn great attention worldwide. Chitosan/Calcium Phosphate (CS/CaP) nanoparticle was a newly developed effective biological delivery system for drug and antigen. Ginsenoside Rb1 is one of the main bioactive components of ginseng root extract, which has antioxidant, anti-inflammatory and immunological enhancement effects. Until now, the combined effect of CS/CaP and ginsenoside Rb1 on the chicken immune response had remained unknown. In this study, the GRb1 and IL-4 were encapsulated into Calcium phosphate and chitosan core structure nanoparticles microspheres (GRb1/IL-4@CS/CaP), and the effect of a newly developed delivery system on an infectious bursal disease virus (IBDV) attenuated vaccine was further evaluated. The results demonstrated that GRb1/IL-4@CS/CaP treatment could induce the activation of chicken dendritic cells (DCs), with the upregulated expression of MHCII and CD80, and the increased production of IL-1β and TNF-α. Importantly, GRb1/IL-4@CS/CaP could trigger a higher level of IBDV-specific IgG and a higher ratio of IgG2a/IgG1 than the traditional adjuvant groups, promoting the production of cytokine, including IFN-γ, TNF-α, IL-4, IL-6, IL-1α, and IL-1β, in chicken serum after 28 d and 42 d post-vaccine. Taken in all, GRb1/IL-4@CS/CaP could elicit prolonged vigorous immune responses for IBDV attenuated vaccine in chicken, which might provide an effective adjuvant system for avian vaccine development.

为了建立一种用于猪细小病毒（PPV）7型的特异、灵敏、快速、简便检测方法，采用环介导等温扩增技术（LAMP）与侧向流动层析试纸（LFD）相结合，根据GenBank发布的PPV7保守区NS1蛋白基因序列设计1套带生物素标签的特异性引物和异硫氰酸（FITC）探针，以PPV7-pET28a重组质粒为阳性模板建立了PPV7-LAMP-LFD检测方法，对LAMP-LFD的反应温度、反应时间及胶体金标记FITC单克隆抗体（FITC-mAb）比例进行优化，分析该方法的敏感性和特异性。结果：建立的PPV7-LAMP-LFD检测方法最适反应温度为64℃,最佳反应时间为60 min;标记1 mL胶体金需要FITC-mAb量为15.6μg;该检测方法的最低检测限为1 copies/μL,其灵敏度比琼脂糖凝胶电泳和荧光定量PCR高10倍；对伪狂犬病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型、乙型脑炎病毒、猪瘟病毒和PPV1～6型病毒检测为阴性，特异性良好；不同批次引物和探针检测结果一致，重复性好；对86份血样的诊断结果与荧光定量PCR进行比对，LAMP-LFD检测方法的阳性率为9.30%（8/86）,检测灵敏度为100%（8/8）,特异性为100%（78/78）,2种方法的符合率为98.83%（85/86）。本研究为PPV7感染的监测与防控提供了技术支撑。

Antigen proteins, assembled on nanoparticles, can be recognized by antigen-presenting cells effectively to enhance antigen immunogenicity. The ability to simultaneously display multiantigens on the same nanoparticle could have numerous applications but remained technical challenges. Here, we described a method for precise assembly of multiple antigens on nanoparticles with specially designed affinity peptides. First, we designed and screened affinity peptides with high affinity and specificity, which could respectively target the key amino acid residues of classical swine fever virus (CSFV) E2 protein or porcine circovirus type 2 capsid protein (PCV2 Cap) accurately. Then, we conjugated the antigen proteins to poly(lactic acid-glycolic acid) copolymer (PLGA) and Gram-positive enhancer matrix (GEM) nanoparticles through the peptides and perfectly assembled two kinds of multiantigen display nanoparticles with different particle sizes. Subsequently, the immunological properties of the assembled nanoparticles were tested. The results showed that the antigen display nanoparticles could promote the maturation, phagocytosis, and proinflammatory effects of antigen-presenting cells (APCs). Besides, compared with the antigen proteins, multiantigen display nanoparticles could induce much higher levels of antibodies and neutralizing antibodies in mice. This strategy may provide a technical support for the study of protein structure and the research and development of polyvalent vaccines.

试验旨在探究葡萄糖调节蛋白78(glucose regulatory protein 78,GRP78)基因的理化性质和结构特点,阐述GRP78在猪流行性腹泻病毒复制中的分子伴侣作用和调控机制.通过RT-PCR方法扩增GRP 78基因,并插入到pMD20-T Simple载体进行克隆测序,利用生物信息学方法对其氨基酸序列、跨膜结构、糖基化位点、磷酸化位点、三级结构等进行预测和分析.将GRP 78基因插入pCDNA 3.1中,然后转染Vero-E6细胞.Western blotting检测Vero-E6细胞中GRP78的表达.生物信息学分析结果表明,GRP 78基因全长1965 bp,编码654个氨基酸,蛋白质分子质量为72.33 ku,理论等电点为5.07,分子式为C3189 H 5153 N865 O1019 S13.遗传进化树分析显示,猴源GRP 78基因与双峰驼、犬、猫、大猩猩、蝙蝠、狒狒、猪、马的氨基酸序列同源性为99.5％～99.8％;遗传进化分析显示,大猩猩和狒狒亲缘关系最为接近.跨膜区和信号肽预测结果显示,该蛋白存在信号肽但不存在跨膜结构.GRP78蛋白无N-糖基化修饰位点,存在6个O-糖基化位点、28个磷酸化位点,表明GRP78可能有与激酶磷酸化有关的PKC、PKA特异性蛋白激酶的结合位点,可能参与己糖代谢和单糖代谢.

Classical swine fever virus (CSFV) is a member of the genus Pestivirus, which causes serious economic losses. The re-emergence of the disease in Japan in 2018 has increased awareness of CSFV. In this study, Balb/c mice were immunized with plant-derived E2 protein, and four monoclonal antibodies (mAbs) 4B11, 7B3, 11A5 and 6F3 were generated. Two of these mAbs, 4B11 and 7B3, effectively blocked CSFV infection of PK-15 cells. Both mAbs recognized a novel linear epitope, 256CLIGNTTVKVHASDER271. The neutralizing ability of anti-CSFV serum decreased 63%, when pre-incubated with the linear peptide at 200 μg/mL. Structural analysis showed that this linear epitope is present at the border of Domain C and Domain D on the surface of the E2 protein. Alignment of amino acid sequences showed that the epitope was conserved in different subgroups of CSFV but not in other members of the Pestivirus genus. Consistently with the analysis above, this epitope distinguished antibodies against CSFV from those against bovine viral diarrhea virus (BVDV). Our study provides an ideal candidate peptide for new vaccine design and differential diagnosis of CSFV. These findings will contribute to the control and eradication of classical swine fever.

Classical swine fever (CSF), caused by CSF virus (CSFV), is one of the most devastating viral epizootic diseases of swine in many countries. To control the disease, highly efficacious and safe live attenuated vaccines have been used for decades. However, the main drawback of these conventional vaccines is the lack of differentiability of infected from vaccinated animals (DIVA concept). Advances in biotechnology and our detailed knowledge of multiple basic science disciplines have facilitated the development of effective and safer DIVA vaccines to control CSF. To date, two types of DIVA vaccines have been developed commercially, including the subunit vaccines based on CSFV envelope glycoprotein E2 and chimeric pestivirus vaccines based on infectious cDNA clones of CSFV or bovine viral diarrhea virus (BVDV). Although inoculation of these vaccines successfully induces solid immunity against CSFV, none of them could ideally meet all demands regarding to safety, efficacy, DIVA potential, and marketability. Due to the limitations of the available choices, researchers are still striving towards the development of more advanced DIVA vaccines against CSF. This review summarizes the present status of candidate CSFV vaccines that have been developed. The strategies and approaches revealed here may also be helpful for the development of new-generation vaccines against other diseases.