The global mpox outbreak has highlighted critical gaps in diagnostic capabilities, particularly the need for methods that can distinguish between the high-fatality Clade I and more transmissible Clade II of the mpox virus (MPXV). Current PCR-based approaches remain reliant on laboratory infrastructure, limiting their use in resource-limited settings. There is an urgent need for a versatile diagnostic platform that can provide both accurate clade discrimination and flexible deployment across diverse healthcare environments. We developed a dual-mode detection platform by integrating recombinase-aided amplification with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a technology, creating two distinct assays: a universal assay targeting OPG034, and a Clade I-discriminatory assay targeting OPG033. The platform achieved detection sensitivities of 1 copy/reaction for both fluorescence and visual readouts with OPG034, and 10 copies (fluorescence) and 1 copy/reaction (visual) with OPG033. Both assays demonstrated high specificity, successfully distinguishing MPXV from related orthopoxviruses and common viruses. Clinical validation using 23 Clade II samples and 10 healthy controls showed that, relative to quantitative PCR (qPCR) (cycle threshold ≤37), the OPG034 fluorescence assay detected all 13 qPCR-positive samples and 2 additional positives (100% sensitivity, 80.0% specificity), while the visual assay detected 12 of 13 positives (92.3% sensitivity, 100% specificity). For Clade I-specific detection, both OPG033 fluorescence and visual assays showed 100% specificity in Clade II samples (23/23). While the clade-discriminatory capability was established through sequence-specific design, further evaluation with authentic Clade I clinical specimens is warranted to confirm typing performance. The dual-mode design provides flexibility for both laboratory and field use, advancing mpox surveillance and outbreak response.IMPORTANCEMpox is a significant zoonosis. Accurate discrimination between its highly lethal Clade I and more transmissible Clade II is critical for clinical management and outbreak control, yet current methods primarily enable only general detection. To address this, we identified novel genetic markers (OPG034 for universal detection and OPG033 for Clade I specificity) and developed a dual-mode detection platform integrating Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a with recombinase-aided amplification. Its key advantage is providing two result readouts: a sensitive fluorescence mode for laboratories and an instrument-free visual colorimetric mode for field use. The demonstrated excellent performance on clinical samples confirms that this platform meets the precision requirements of clinical laboratories while remaining suitable for resource-limited settings like field clinics. Thus, it offers a flexible and practical tool for enhancing mpox surveillance and control globally, particularly in regions with constrained medical resources.

Toxoplasma gondii is a widely spread opportunistic pathogen that can infect nearly all warm-blooded vertebrates and cause serious toxoplasmosis in immunosuppressed animals and patients. However, the relationship between the host’s innate immune system and effector proteins is poorly understood, particularly with regard to how effectors antagonize cGAS-STING signaling during T. gondii infection. In this study, the ROP5 from the PRU strain of T. gondii was found to promote cGAS-STING-mediated immune responses. Mechanistically, ROP5 interacted with STING through predicted domain 2 and modulated cGAS-STING signaling in a predicted domain 3-dependent manner. Additionally, ROP5 strengthened cGAS-STING signaling by enhancing the K63-linked ubiquitination of STING. Consistently, ROP5 deficient PRU (PRUΔROP5) induced fewer type I IFN-related immune responses and replicated faster than the parental strain in RAW264.7 cells. Taken together, this study provides new insights into the mechanism by which ROP5 regulates T. gondii infection and provides new clues for strategies to prevent and control toxoplasmosis.

Goatpoxvirus (GTPV), sheeppoxvius (SPPV), and the Lumpy skin disease virus (LSDV) is a Capripoxvirus belonging to the family poxviridae. They can cause significant economic losses in countries where this disease are endemic. However, effective and convenient diagnostic tools against sera antibody are not readily available until now. Toward this goal, a polyclonal antibody competitive enzyme-linked immunosorbent assay (c-ELISA) of detecting serogroup-specific antibody is established based on major LSDV antigen A33. Serum samples (n = 605) were collected to optimize the c-ELISA from different areas. The cut-off value for the c-ELISA was estimate using percent inhibition (PI) values. The diagnostic performance of test including sensitivity (sn) and specificity (sp) were obtained by receiver operator characteristic (ROC) analysis. Among these analysis, > 57.61% PI value was accepted as cut-off of the c-ELISA, the diagnostic sn an diagnostic sp were reached to 96.4% and 98.5%, at > 95% confidence interval. These results show that the developed competitive ELISA is sensitive, specific, and reliable, which make it appropriate for serological investigation.

正痘病毒属病毒的A33和H3L蛋白是其成员共有的中和抗体的两个主要靶标.为检测鼠痘病毒(ectromelia virus,ECTV)A33和H3L蛋白同源物的免疫原性及抗体中和活性,以ECTV-Moscow株基因组DNA为模板,PCR扩增A3 3和H3L同源基因EVM135、EVM085目的序列,分别构建pET30a-EVM135、pET30-EVM085原核表达载体,转化Rosetta感受态细胞,IPTG诱导表达,利用镍层析柱纯化并进行逐步透析复性,免疫兔制备多克隆抗体.建立间接ELISA方法测定其抗体效价分别达1∶120 000和1∶360 000,Western blot和IFA证实该多克隆抗体具有良好的特异性和反应性;ECTV病毒中和试验表明抗EVM135多克隆抗体中和效价为1∶16,而抗EVM085多克隆抗体效价为1∶128.本研究通过高效表达EVM135和EVM085蛋白,并分析其免疫原性及抗体中和活性,为深入研究痘病毒致病和免疫机理,进而快速建立其诊断方法,为猴痘防控技术措施的研究奠定基础.

本研究从疑似爆发伪狂犬病(pseudorabies,PR)猪场死亡猪中分离到1株病毒,经PCR检测、主要毒力基因gB、gC、gD和gE测序及遗传关系分析,发现它与国内流行的PRV变异毒株具有相似的突变特征,氨基酸序列与HN-ZZ/Swine(MH321405)变异株亲缘关系最近,将其鉴定为伪狂犬病变异病毒(pseudorabies virus,PRV),并命名为PRVHN-2017株,发现该毒株在Vero细胞上具有较强的适应性.为进一步探讨PRV HN-2017株的致病性,将该变异株按不同剂量经滴鼻途径接种于45日龄的SPF猪,均先后出现了发热、抽搐、呼吸困难等猪伪狂犬病典型症状,其中高剂量感染组在7 d内全部死亡;RT-PCR检测证实该病毒毒株在体内分布较广,在扁桃体中病毒载量最高,其次是脾脏、肺脏、淋巴结、脑组织、肾脏和肝脏.这表明成功分离到1株PRV变异毒株,并证实该毒株对仔猪具有很强的致病性,能导致多种重要组织器官的不同程度的损害.

细胞程序性死亡是一种受基因调控的细胞死亡方式,包括细胞凋亡、焦亡、坏死性凋亡、铁死亡、自噬等,这些多元化的细胞程序性死亡方式在病毒的复制和传播中发挥重要作用.其中,痘病毒通过编码大量的病毒蛋白(例如E3L、F1L等)参与介导痘病毒免疫逃逸,抵抗宿主细胞死亡,以此维持自身复制.本文综述了痘病毒E3L蛋白在调控细胞凋亡、焦亡、坏死性凋亡中的作用,深入剖析了 PKR、F1L和Zα结构域介导的E3L相关细胞程序性死亡分子机制,以期为痘病毒免疫逃逸机制探索、抗病毒药物开发等提供新思路.

The monkeypox epidemic has attracted global attention to poxviruses. The cytoplasmic replication of poxviruses requires extensive protein synthesis, challenging the capacity of the endoplasmic reticulum (ER). However, the role of the ER in the life cycle of poxviruses is unclear. In this study, we demonstrate that infection with the lumpy skin disease virus (LSDV), a member of the poxvirus family, causes ER stress in vivo and in vitro, further facilitating the activation of the unfolded protein response (UPR). Although UPR activation aids in the restoration of the cellular environment, its significance in the LSDV life cycle remains unclear. Furthermore, the significance of ER imbalance for viral replication is also unknown. We show that LSDV replication is hampered by an unbalanced ER environment. In addition, we verify that the LSDV replication depends on the activation of PERK‐eIF2α and IRE1‐XBP1 signaling cascades rather than ATF6, implying that global translation and reduced XBP1 cleavage are deleterious to LSDV replication. Taken together, these findings indicate that LSDV is involved in the repression of global translational signaling, ER chaperone transcription, and ATF6 cleavage from the Golgi into the nucleus, thereby maintaining cell homeostasis; moreover, PERK and IRE1 activation contribute to LSDV replication. Our findings suggest that targeting UPR elements may be applied in response to infection from LSDV or even other poxviruses, such as monkeypox.

Guanylate-binding proteins (GBPs) are highly expressed interferon-stimulated genes (ISGs) that play significant roles in protecting against invading pathogens. Although their functions in response to RNA viruses have been extensively investigated, there is limited information available regarding their role in DNA viruses, particularly poxviruses. Ectromelia virus (ECTV), a member of the orthopoxvirus genus, is a large double-stranded DNA virus closely related to the monkeypox virus and variola virus. It has been intensively studied as a highly effective model virus. According to the study, GBP2 overexpression suppresses ECTV replication in a dose-dependent manner, while GBP2 knockdown promotes ECTV infection. Additionally, it was discovered that GBP2 primarily functions through its N-terminal GTPase activity, and the inhibitory effect of GBP2 was disrupted in the GTP-binding-impaired mutant GBP2K51A. This study is the first to demonstrate the inhibitory effect of GBP2 on ECTV, and it offers insights into innovative antiviral strategies.

目的 克隆羊口疮病毒020基因,构建其重组真核表达质粒并转染HeLa细胞,分析020基因编码E3L蛋白的亚细胞定位,分析该蛋白生物信息学特征.方法 提取羊口疮病毒基因组,PCR扩增020基因并构建重组真核表达质粒,转染HeLa细胞,采用Western blot检测其在该细胞中的表达,免疫荧光法检测该蛋白的亚细胞定位.利用DNAstar软件分析不同毒株ORFV E3L氨基酸序列特点及其遗传演化关系;利用SOMPA软件分析其二级结构;利用SignalP4.0软件预测信号肽;利用TMHMM2.0软件预测跨膜结构;利用NetPhos 3.1预测磷酸化位点;利用NetNGlyc-1.0预测糖基化位点;应用IDEB和SYFPEITHI预测ORFV E3L蛋白的抗原表位.结果 成功克隆得到ORFV 020基因,全长549 bp,编码183个氨基酸;亚细胞定位显示该蛋白定位于细胞核和细胞质.生物信息学分析ORFV E3L蛋白α螺旋约占40.44％、β折叠约占5.46％、延伸链约占16.94％、无规则卷曲约占37.16％;该蛋白无信号肽和跨膜结构,可能存在15个磷酸化位点,7个N-糖基化位点,5个B细胞线性表位,2个CTL表位,3个Th细胞表位,3个B细胞和CTL联合表位,3个B细胞和Th细胞联合表位.结论 ORFV E3L蛋白定位于细胞核和细胞质,含有多个抗原表位,可能具有免疫原性.该蛋白高度保守,具有成为优势保护性抗原的潜力.为进一步揭示ORFV E3L蛋白的功能奠定基础,为ORFV诊断方法的建立及疫苗的研制提供了理论依据.

牛结节性皮肤病(lumpy skin disease,LSD)是由痘病毒科山羊痘病毒属的结节性皮肤病病毒(lumpy skin disease virus,LSDV)感染牛而引起的一种热性、接触性、嗜上皮性病毒病.2019年8月3日,我国首次在新疆维吾尔自治区伊犁州确诊暴发了牛结节性皮肤病疫情,目前LSD已扩散至我国东南、西南、中部及北部等大部分地区且呈蔓延态势.对LSD高效、准确、快速的诊断,是成功控制和根除LSD的基础.本文对LSD的临床特征、病原学、分子生物学和血清学等诊断方法进行综述,为LSD快速有效的诊断及科学防控提供理论支持.

Poxviruses have been associated with humans for centuries. From smallpox to mpox to lumpy skin disease virus (LSDV), members of the poxvirus family have continued to threaten the lives of humans and domestic animals. A complete understanding of poxvirus-mediated cellular processes will aid in the response to challenges from the viruses. In this study, we demonstrate that LSDV infection results in an abnormal ultrastructure of the endoplasmic reticulum (ER) lumen in primary bovine embryonic fibroblast (BEF) cells, and we further show that an ER imbalance occurs in LSDV-infected BEF cells. Additionally, we believe that ER stress-related apoptosis plays a role in the late apoptosis of BEF cells infected with LSDV, primarily through the activation of the CCAAT/enhancer binding protein homologous protein (CHOP)-Caspase-12 signal. In addition to cell apoptosis, a further investigation showed that LSDV could also activate autophagy in BEF cells, providing additional insight into the exact causes of LSDV-induced BEF cell death. Our findings suggest that LSDV-induced BEF cell apoptosis and autophagy may provide new avenues for laboratory diagnosis of lumpy skin disease progression and exploration of BEF cell processes.

To investigate the effect of bovine G3BP1(bG3BP1)on the enzymatic activity of DNA recog-nition receptor cGAS,the total RNA from bovine blood lymphocytes was extracted and reversely tran-scribed into cDNA by RT-PCR and the bG3BP1 gene were cloned.Subsequently,the prokaryotic expression vector pET-28a-SUMO-bG3BP1 was constructed and transformed into competent E.coli cells.The recombi-nant protein bG3BP1 was purified by nickel column affinity chromatography and purified further after removing SUMO label proteins to reach high purity.Finally,the bG3BP1 was used for the enzymatic reac-tion of bovine cGAS(bcGAS)in vitro,and the product of 2'3'-cGAMP was detected by ELISA.The results showed that soluble bG3BP1 protein promoted the enzymatic reaction of bcGAS in vitro and increased the yield of the second messenger molecule 2'3'-cGAMP,which provided technical methods and research ideas for the large-scale production and application of 2'3'-cGAMP.

The recent spread of the monkeypox virus among humans has heightened concerns regarding orthopoxvirus infections. Consequently, conducting a comprehensive study on the immunobiology of the monkeypox virus is imperative for the development of effective therapeutics. Ectromelia virus (ECTV) closely resembles the genetic and disease characteristics of monkeypox virus, making it a valuable research tool for studying orthopoxvirus–host interactions. Guanylate-binding proteins (GBPs), highly expressed interferon-stimulated genes (ISGs), have antagonistic effects against various intracellular pathogenic microorganisms. Our previous research has shown that GBP2 has a mild but statistically significant inhibitory effect on ECTV infection. The presence of a significant number of molecules in the poxvirus genome that encode the host immune response raises questions about whether it also includes proteins that counteract the antiviral activity of GBP2. Using IP/MS and co-IP technology, we discovered that the poly(A) polymerase catalytic subunit (PAPL) protein of ECTV is a viral regulatory molecule that interacts with GBP2. Further studies have shown that PAPL antagonizes the antiviral activity of GBP2 by reducing its protein levels. Knocking out the PAPL gene of ECTV with the CRISPR/Cas9 system significantly diminishes the replication ability of the virus, indicating the indispensable role of PAPL in the replication process of ECTV. In conclusion, our study presents preliminary evidence supporting the significance of PAPL as a virulence factor that can interact with GBP2.

The Japanese encephalitis virus (JEV) is a leading cause of mosquito-borne viral encephalitis worldwide. Clinical symptoms other than encephalitis, on the other hand, are substantially more prevalent with JEV infection, demonstrating the relevance of peripheral pathophysiology. We studied the peripheral immunopathogenesis of JEV using IFNAR deficient (IFNAR–/–) mice infected with the SA14-14-2 strain under the BSL-2. The body weight and survival rate of infected-IFNAR–/–mice decreased significantly. Infected-IFNAR–/–mice’s liver and spleen demonstrated obvious tissue damage and inflammatory cell infiltration. There was also extensive viral replication in the organs. IFN-α/β protein expression was dramatically elevated in peripheral tissues and serum, although the related interferon-stimulated genes (ISGs) remained low in the spleen and liver of infected-IFNAR–/–animals. Consistently, the differentially expressed genes (DEGs) analysis using RNA-sequencing of spleens showed inflammatory cytokines upregulation, such as IL-6, TNF-α, and MCP-1, and IFN-γ associated cytokine storm. The infiltration of macrophages and neutrophils in the spleen and liver of SA14-14-2-infected IFNAR–/– mice was dramatically elevated. However, there was no significant difference in tissue damage, viral multiplication, or the production of IFNα/β and inflammatory cytokines in the brain. Infection with the JEV SA14-14-2 strain resulted in a lethal peripheral inflammatory response and organ damage without encephalitis in IFNAR–/– mice. Our findings may help shed light on the peripheral immunopathogenesis associated with clinical JEV infection and aid in developing treatment options.

Schistosoma is a genus of parasitic trematodes that undergoes complex migration in final hosts, finally developing into adult worms, which are responsible for egg production and disease dissemination. Recent studies documented the importance of extracellular vesicles (EVs) in the regulation of host-parasite interactions. Herein, we investigated the microRNA (miRNA) profiles of EVs isolated from host plasma at different stages of Schistosoma japonicum infection (lung stage: 3 days post-infection (dpi), and liver stages: 14 and 21 dpi) to identify miRNA cargo potentially involved in the pathogenesis and immune regulation of schistosomiasis. Characterization of the isolated plasma EVs revealed their diameter to be approximately 100 nm, containing typical EV markers such as Hsp70 and Tsg101. Deep sequencing analysis indicated the presence of 811 known and 15 novel miRNAs with an increasing number of differential miRNAs from the lung stage (27 miRNAs) to the liver stages (58 and 96 miRNAs at 14 and 21 dpi, respectively) in the plasma EVs of infected mice compared to EVs isolated from the uninfected control. In total, 324 plasma EV miRNAs were shown to be co-detected among different stages of infection and the validation of selected miRNAs showed trends of abundance similar to deep sequencing analysis. For example, miR-1a-3p and miR-122-5p showed higher abundance, whereas miR-150-3p and miR-126a showed lower abundance in the plasma EVs of infected mice at 3, 14, and 21 dpi as compared to those of uninfected mice. In addition, bioinformatic analysis combined with PCR validation of the miRNA targets, particularly those associated with the immune system and parasitic infectious disease, indicated a significant increase in the expression of Gbp7 and Ccr5 in contrast to the decreased expression of Fermt3, Akt1 , and IL-12a . Our results suggested that the abundance of miRNA cargo of the host plasma EVs was related to the stages of Schistosoma japonicum infection. Further studies on the roles of these miRNAs may reveal the regulatory mechanism of the host-parasite interaction. Moreover, the differentially abundant miRNA cargo in host EVs associated with S. japonicum infection may also provide valuable clues for identifying novel biomarkers for schistosomiasis diagnosis.

Antibody development is the integral process of generating and characterizing an antibody. It commences by inoculating the antigen of interest into laboratory animals, allowing the immune system develops large quantities of antibodies. This was aimed at developing antibodies against the virion of Goatpox and Sheeppox virus vaccines. The ability of Goatpox and Sheeppox vaccines was assessed. Regarding this study, the antibody titers against both Goatpox and Sheeppox viruses was increased in the same manner. The amount of IgG was determined to be 2.29 μg/μl and 2.18 μg/μl against virions of Goatpox virus and Sheeppox respectively. The purified IgG was analyzed by SDS-PAGE. Different bands of the purified antibodies were clearly visualized, and the molecular weight of IgG was estimated to be 67 kDa and 25 kDa. Additionally, antigen/antibody binding was confirmed by Western blot using GTPV A27 antigen. No significant differences in antibody titers were observed between the two groups (p < 0, 05).

2019年8月我国新疆伊犁地区首次暴发牛结节性皮肤病(LSD),在不到一年内结节性皮肤病病毒从我国最西北部传到最东南部诸省区,之后LSD疫情迅速传播和扩散,2021年又波及我国多个省市区,呈大流行态势,对我国养牛业特别是奶牛业造成巨大危害和威胁.近年来,世界上特别是俄罗斯等国家又发现了LSD疫苗样疾病以及新的变异毒株,对LSD的防控提出了新挑战.为做好这一外来病的防控工作,通过LSD疫情流行现状与趋势、发现的新流行毒株与问题,探讨我国LSD防控的新策略,为有效控制、净化和根除该病提供指导.

近交系小型猪(Sus scrofa)的培育是大型动物实验动物化的重点方向和趋势,中国农业科学院兰州兽医研究所以1对合作猪为系祖,采用"近亲交配"和异地实验动物化培育,历时18年培育成功了国际上首个高原型近交系小型猪.该研究成果填补了国内外高原型小型猪的研究空白,为大型哺乳动物实验动物化培育和应用提供了理论依据和技术基础.本文介绍了实验用合作小型猪的基本特性与利用优势,培育过程与遗传控制,以及其在抗病免疫研究中的应用.

为实现对猪瘟野毒株与疫苗株之间的快速鉴别,本试验建立了 一种猪瘟野毒株与疫苗株的双重TapMan实时定量PCR的检测方法.通过对GenBank公布的猪瘟病毒野毒株及疫苗株的全基因组序列进行比对和分析,设计出2对特异性引物及2条TapMan探针,经反应体系及反应条件优化后,对其特异性、稳定性及敏感性进行了验证.结果显示,本方法能特异性地对猪瘟野毒株及疫苗株进行鉴别检测,并且不与其他常见的猪源病毒产生交叉反应,组内和组间检测变异系数均小于1％,敏感性较普通RT-PCR分别高出100倍和1 000倍以上,且野毒株与疫苗株的最低检测下限分别在1.3 copies/μL和1.58 copies/μL.应用本方法对实验室保存的甘肃某猪场猪瘟活疫苗免疫前、后的56份已知背景的样本进行检测,结果,阳性符合率高达100％.本方法特异性强,稳定性和灵敏度高,不仅能够准确定量,并且可快速地对同一个样品中猪瘟病毒野毒株、疫苗毒株进行检测,为后续猪瘟净化效果的评估提供了新的技术手段.

The three poxviruses' namely lumpy skin disease, Goatpox, and Sheeppox viruses are categorized under the genera of Capripoxvirus based on their genome similarities. The disease caused by these viruses is complex and difficult to diagnose; the most important reasons for this are the visible variability in the host range, the clinical similarities, and the inability to differentiate by serological assays. By considering their antigenic and immunogenic similarities of capripoxviruses, this study aimed at the comparison of the immunogenicity of GTPV A27R, GTPV L1R, and SPPV A33R recombinant proteins. Concerning this study, 35 kDa, 26 kDa, and 19 kDa recombinant proteins were purified as regards GTPV A27R, GTPV L1R, and SPPV A33R respectively. The best antibody titer was revealed by GTPV A27R with the test sensitivity of 90 %, 100 %, 90 %, and diagnostic accuracy of 100 %, 90 %, and 100 % for sera of Cattle, Sheep, and Goat correspondingly. Based on the available results GTPV A27R protein showed more immunogenicity while compared with the rest of the two proteins and further studies are required in the same area for the future.