Journal of virological methods最新文献_第3页

Evaluation of dried blood spot testing for serological monitoring of epizootic and zoonotic pathogens in domestic pigs 家猪兽疫和人畜共患病原体血清学监测中干血斑点试验的评价。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-24 DOI: 10.1016/j.jviromet.2025.115336

Ranja Steinhauer , Eric Kübler , Stefan Gaugler , Cornel Fraefel , Julia Lechmann

Dried blood spots (DBS) constitute a stable, cost-efficient sampling matrix that can be collected in a minimally invasive manner. Although widely adopted in human medicine, their use in veterinary diagnostics remains limited. This study aimed to establish and validate DBS elution protocols for use in commercial ELISAs to detect antibodies against Hepatitis E virus (HEV), African swine fever virus (ASFV) and Aujeszky’s disease virus (ADV) in domestic pigs. DBS were prepared from EDTA blood, dried serum spots (DSS) from serum. Additional DBS samples were prepared after spiking blood from healthy pigs with antibodies. Various parameters were evaluated to establish the final elution protocols, i.e. number of disks, type and volume of elution buffer, incubation time of the disks in elution buffer, and volume of eluate used for detection. Once the final protocols were in place, for each pathogen 52 DBS were tested in three independent runs. The diagnostic performance was evaluated by comparing the ELISA results of DBS eluates with the corresponding serum or plasma samples. For HEV, only one out of 52 DBS samples qualitatively did not match the plasma result in any of the three runs. For ASFV, all 52 DBS samples matched the qualitative results of the corresponding liquid samples. For ADV, two samples yielded false negative results in all three runs. The results suggest that DBS represent a practical and reliable alternative to liquid blood samples for antibody detection in pigs. Further validation with field samples and large-scale testing is needed.

干血斑（DBS）构成了一种稳定、成本效益高的采样基质，可以以微创的方式采集。虽然在人类医学中被广泛采用，但它们在兽医诊断中的应用仍然有限。本研究旨在建立和验证DBS洗脱方案，用于商用elisa检测家猪戊型肝炎病毒（HEV）、非洲猪瘟病毒（ASFV）和奥杰斯基病病毒（ADV）的抗体。EDTA血制备DBS，血清干斑（DSS）制备DBS。在健康猪的血液中加入抗体后，制备了额外的DBS样本。评估各种参数以确定最终洗脱方案，包括：圆盘数、洗脱缓冲液的类型和体积、圆盘在洗脱缓冲液中的孵育时间、用于检测的洗脱液体积。一旦最终方案到位，每种病原体52个DBS在三个独立的运行中进行测试。通过比较DBS洗脱液的ELISA结果与相应的血清或血浆样品来评估诊断性能。对于HEV， 52个DBS样本中只有一个在三次运行中与血浆结果定性不匹配。对于ASFV， 52份DBS样品均与相应液体样品的定性结果相匹配。对于ADV，两个样本在所有三次运行中都产生了假阴性结果。结果表明，DBS是一种实用可靠的猪抗体检测方法，可替代液体血液样品。需要用现场样品和大规模试验进一步验证。

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引用次数: 0

Corrigendum to “An alternative model for HEV infection in the HepaRG cell line” [J. Virol. Methods 340 (2026) 115307] “HepaRG细胞系中HEV感染的另一种模型”的更正[J]。性研究。方法340(2026)115307。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-24 DOI: 10.1016/j.jviromet.2025.115334

Stacy Gellenoncourt , Marie Pellerin , Aïlona Marcadet-Hauss , Roxanne Fouillé , Michel Rivoire , Guillaume Passot , Julie Lucifora , David Durantel , Nicole Pavio , Virginie Doceul

引用次数: 0

Development of sensitive and specific indirect ELISA tests for the detection of antibodies against bovine leukemia virus in serum and milk samples 血清和牛奶中抗牛白血病病毒抗体的间接ELISA检测方法的建立。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-23 DOI: 10.1016/j.jviromet.2025.115335

Andrés Addiego , Federico Carrión , Natalia Olivero-Deibe , Martín Fló , Florencia Rammauro , Natalia Ibañez , Caroline da Silva Silveira , Franklin Riet-Correa , Lorena Tomé-Poderti , Otto Pritsch , Sergio Bianchi

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a disease characterized by lymphoproliferation and B-cell tumors. There is currently no available treatment or vaccine, making it a global animal health challenge and resulting in severe economic losses. The World Organization for Animal Health (WOAH) advocates for serological diagnostic tools and disease control programs to eradicate BLV. One major diagnostic technique recognized by the WOAH is the enzyme-linked immunosorbent assay (ELISA). We developed and optimized three indirect ELISAs for detecting anti-BLV antibodies in serum and milk. These assays utilize viral Env ectodomain and p24 recombinant proteins expressed in Drosophila melanogaster S2 cells and Escherichia coli systems, respectively. We compared their performance with a widely used commercial ELISA kit for completeness, which detects antibodies against BLV gp51 protein. Our immunoassay using recombinant p24 protein (anti-p24-S ELISA) showed comparable strength to the reference method (kappa index: 84.4 %, in 952 samples evaluated). Results for the anti-ectoEnv-S ELISA in serum samples closely matched the reference kit, with higher concordance than the anti-p24-S ELISA (kappa index: 94.9 %, in 1781 samples evaluated). Similarly, our in-house anti-ectoEnv-M ELISA in milk samples exhibited high concordance with both the commercial reference kit and the anti-ectoEnv-S (kappa index between the three tests: 95.6 %, in 479 blood and milk paired samples). Thus, we have developed, optimized, and validated three indirect in-house ELISAs for detecting anti-BLV antibodies in cattle, demonstrating good sensitivity and specificity values, using two different recombinant viral proteins.

牛白血病病毒（BLV）是地方性牛白血病（EBL）的病原体，EBL是一种以淋巴细胞增殖和b细胞肿瘤为特征的疾病。目前没有可用的治疗方法或疫苗，使其成为全球动物卫生挑战，并造成严重的经济损失。世界动物卫生组织（WOAH）提倡使用血清学诊断工具和疾病控制计划来根除BLV。世界卫生组织认可的一种主要诊断技术是酶联免疫吸附试验（ELISA）。我们开发并优化了3种间接elisa检测血清和乳汁中的抗blv抗体。这些实验分别利用在果蝇S2细胞和大肠杆菌系统中表达的病毒Env外结构域和p24重组蛋白。我们将其性能与广泛使用的商用ELISA试剂盒进行了比较，该试剂盒检测BLV gp51蛋白的抗体。我们使用重组p24蛋白（抗p24- s ELISA）进行免疫分析，其强度与参考方法相当（在评估的952份样品中kappa指数为84.4%）。血清样品中抗ectoenv - s酶联免疫吸附试验结果与参考试剂盒的一致性较好，且高于抗p24- s酶联免疫吸附试验（kappa指数为94.9%，共1781份）。同样，我们在牛奶样品中的抗ectoenv - m ELISA与商业参考试剂盒和抗ectoenv - s均表现出高度的一致性（在479份血液和牛奶配对样品中，三种检测的kappa指数为95.6%）。因此，我们利用两种不同的重组病毒蛋白，开发、优化并验证了三种用于检测牛体内抗blv抗体的间接elisa，显示出良好的灵敏度和特异性。

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引用次数: 0

Rapid establishment and validation of a PMAxx-RT-qPCR method for infectious titer detection of freeze-dried live attenuated hepatitis A vaccine (H2 Strain) 冻干甲型肝炎减毒活疫苗H2株感染效价的pmax - rt - qpcr快速检测方法的建立与验证

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-23 DOI: 10.1016/j.jviromet.2025.115333

Yongrong Wang , You Gao , Wei Wang , Qin Liu , Yadong Li , Haiyuan Zeng , Lifei Chen , Chen Cheng , Fan Wu

The infectious titer of hepatitis A virus (HAV) is a critical quality parameter for evaluating potency of freeze-dried live attenuated hepatitis A (HepA-L-fd) vaccines. In this study, we developed a rapid PMAxx-RT-qPCR for detecting the infectious titer of HepA-L-fd vaccines. This method enables virus titer detection to be completed within a day. Systematic optimization of experimental parameters yielded a validated methodology demonstrating excellent precision (coefficient of variation, CV < 1 %), accuracy (recovery rate 107.6 ± 3.7 %), and a linear range of 6.23–7.73 log₁₀CCID₅₀/mL. Comparing with the measurements from the traditional method, results showed no significant differences among the six batches of vaccine finished products (P = 0.7452). This optimized PMAxx-RT-qPCR assay provides a rapid and reliable analytical means for the infectious titer assay in HepA-L-fd vaccines.

甲型肝炎病毒（HAV）的感染效价是评价冻干甲型肝炎减毒活疫苗效价的重要质量参数。在这项研究中，我们建立了一种快速检测HepA-L-fd疫苗感染滴度的pmax - rt - qpcr。此方法可在一天内完成病毒滴度检测。对实验参数进行系统优化，得到精密度高（变异系数CV < 1%）、准确度高（回收率107.6±3.7%），线性范围为6.23 ~ 7.73 log10CCID50/mL。与传统方法比较，6批疫苗成品的测定结果无显著差异（P = 0.7452）。优化后的pmax - rt - qpcr检测方法为检测HepA-L-fd疫苗的感染滴度提供了一种快速可靠的分析方法。

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引用次数: 0

Application of digital PCR and CRISPR/Cas13a-based fluorescent assay for accurate and on-site detection of cotton leafroll dwarf virus 数字PCR和基于CRISPR/ cas13的荧光技术在棉花卷叶矮缩病毒准确和现场检测中的应用

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-19 DOI: 10.1016/j.jviromet.2025.115332

Vijay Sheri , Pankaj K. Verma , SaiKrishna Lekkala, Madhusudhana R. Janga

Cotton leafroll dwarf virus (CLRDV) is an emerging viral pathogen posing a significant threat to cotton production in the United States. Early and accurate detection is critical for effective disease surveillance and management. Although traditional reverse transcription PCR (RT-PCR) is commonly employed for CLRDV diagnosis, it suffers from limitations in sensitivity, quantification accuracy, and involves labor-intensive workflows. In this study, we evaluated two advanced molecular diagnostic approaches for detecting CLRDV, digital PCR (dPCR) and CRISPR/Cas13a-based fluorescent assay. Symptomatic cotton leaf samples from Lubbock and Brownfield, Texas, were screened and confirmed positive by RT-PCR. Digital PCR analysis enabled absolute quantification of viral load, revealing significantly higher titers in Brownfield (F2) samples and offered improved sensitivity over RT-PCR, particularly in samples with low viral loads. However, dPCR is resource-intensive and requires specialized instrumentation. To address the need for rapid, field-deployable diagnostics, we developed a CRISPR/Cas13a-based assay targeting the conserved ORF3, ORF2, and ORF3a regions of the CLRDV genome. Adapted from the SHERLOCK platform, this fluorescence-based assay uses collateral cleavage activity of Cas13a to enable highly specific visual detection. While the assay successfully enabled direct detection from crude leaf extracts without RNA purification, the sensitivity analysis was conducted using purified, in vitro transcribed RNA. Fluorescence signals were reliably observed with as few as 50 RNA copies, defining the assay’s practical limit of detection. While dPCR is optimal for quantitative laboratory analysis, the CRISPR/Cas13a-based assay offers a rapid, sensitive, and cost-effective tool for field-level detection. Together, these complementary tools enhance CLRDV surveillance and management in cotton.

棉花卷叶矮病毒（CLRDV）是一种新兴的病毒性病原体，对美国棉花生产构成重大威胁。早期和准确的检测对于有效的疾病监测和管理至关重要。虽然传统的逆转录PCR （RT-PCR）通常用于CLRDV的诊断，但它在灵敏度、定量准确性方面存在局限性，并且涉及劳动密集型的工作流程。在这项研究中，我们评估了检测CLRDV的两种先进的分子诊断方法，数字PCR （dPCR）和基于CRISPR/ cas13的荧光检测。对来自德克萨斯州Lubbock和Brownfield的有症状棉花叶片样本进行筛选，并通过RT-PCR确认为阳性。数字PCR分析实现了病毒载量的绝对定量，在Brownfield （F2）样品中显示出明显更高的滴度，并提供了比RT-PCR更高的灵敏度，特别是在低病毒载量的样品中。然而，dPCR是资源密集型的，需要专门的仪器。为了满足快速、可现场部署诊断的需求，我们开发了一种基于CRISPR/ cas13的检测方法，针对CLRDV基因组的保守ORF3、ORF2和ORF3a区域。这种基于荧光的检测方法改编自SHERLOCK平台，利用Cas13a的侧切活性来实现高度特异性的视觉检测。虽然该试验成功地从粗叶提取物中直接检测而无需纯化RNA，但使用纯化的体外转录RNA进行敏感性分析。荧光信号被可靠地观察到，只有50个RNA拷贝，确定了该分析的实际检测极限。虽然dPCR是定量实验室分析的最佳选择，但基于CRISPR/ cas13的分析为现场检测提供了快速、敏感和经济高效的工具。这些互补工具共同加强了棉花中CLRDV的监测和管理。

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引用次数: 0

Development of a rapid, real-time RT-RAA assay for sensitive detection of bovine enterovirus in fecal samples 建立一种快速、实时RT-RAA检测方法，用于灵敏检测粪便样品中的牛肠病毒。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-19 DOI: 10.1016/j.jviromet.2025.115331

Cuilan Wu , Xixian Liu , Lan Jia , Zhijie Mo , Yeheng Gao , Shuhong Zhong , Shiwen Feng , Zhongwei Chen , Xian Li , Xindong Wang , Xiongbiao Xuan , Huili He , Shuai Hu , Changting Li , Zuzhang Wei , Yongping Xie , Hao Peng , Yingyi Wei , Jun Li

Bovine enterovirus (BEV) is a significant pathogen affecting bovine intestinal health, posing challenges to cattle production and public health. Existing detection methods, such as RT-PCR, RT-qPCR, and LAMP, are time-consuming and dependent on specialized laboratory facilities, highlighting the need for rapid and field-deployable diagnostic tools. In this study, we developed a real-time reverse transcription recombinase-aided amplification (RT-RAA) assay designed for BEV detection, capable of delivering results within 20 min at 39℃. The real-time RT-RAA assay showed no cross-reactivity to other eight pathogens, including BCoV, BVDV, BKV, BRV, BAstV, STEC, FMDV, and PEV. The sensitivity assay revealed that the real-time RT-RAA method could reliably detect a minimum plasmid concentration of 5.50 × 10 ¹ copies/reaction. Clinical validation conducted with 780 field samples revealed performance comparable to those of conventional RT-PCR and RT-qPCR methods, achieving a positivity rate of 23.59 %. The RT-RAA method operates at low temperatures, requires minimal instrumentation, and reduces technical demands, making it ideal for resource-limited settings. Its portability, cost-effectiveness, and robustness position it as a transformative tool for BEV surveillance, enhancing outbreak control, livestock health management, and food safety.

牛肠病毒（BEV）是影响牛肠道健康的重要病原体，对牛生产和公共卫生构成挑战。现有的检测方法，如RT-PCR、RT-qPCR和LAMP，耗时且依赖于专门的实验室设施，突出了对快速和可现场部署的诊断工具的需求。在本研究中，我们开发了一种用于BEV检测的实时逆转录重组酶辅助扩增（RT-RAA）方法，能够在39℃下20分钟内得出结果。实时RT-RAA检测结果显示，与BCoV、BVDV、BKV、BRV、BAstV、STEC、FMDV和PEV等其他8种病原体无交叉反应。灵敏度分析结果表明，实时RT-RAA法可可靠地检测到5.50 × 10¹拷贝/反应的最小质粒浓度。780份现场样品的临床验证结果表明，该方法的检测结果与传统RT-PCR和RT-qPCR方法相当，阳性率为23.59%。RT-RAA方法在低温下工作，需要最少的仪器，降低了技术要求，使其成为资源有限环境的理想选择。它的便携性、成本效益和稳健性使其成为BEV监测、加强疫情控制、牲畜健康管理和食品安全的变革性工具。

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引用次数: 0

Search and prove of efficient inhibitors against papain-like protease from SARS-CoV-2 SARS-CoV-2抗木瓜蛋白酶有效抑制剂的寻找和证明。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-19 DOI: 10.1016/j.jviromet.2025.115329

Wenfa Zhang , Xiao Yuan Chen , Sheng-Xiang Lin

Papain-like protease (PLpro) is a key protease encoded by SARS-CoV-2, essential for viral polyprotein processing, replicase-transcriptase complex assembly, and interference with host immune responses. New COVID-19 cases continue to emerge, and WHO has identified coronaviruses with similar PLpro structures as potential pandemic threats. Using molecular modeling, docking, and protease activity assays, we identified four potent inhibitors of SARS-CoV-2 /PLpro with IC₅₀ values of 6.96–20.21 µM. Their binding affinities, determined by fluorescence titration, yielded dissociation constants (KD) of 4.18–13.06, supporting their inhibitory activity. In silico toxicity studies suggest that most inhibitors have LD₅₀ values comparable to GRL0617, a known PLpro inhibitor. Structural analysis revealed that all inhibitors interact with key residues Asp¹⁶⁴, Pro²⁴⁸, Tyr²⁶⁴, Tyr²⁶⁸, and Gln²⁶⁹ of PLpro, but differ in specific hydrogen bonding and hydrophobic interactions. Notably, compound P636–0664 exhibits the most extensive interactions, forming 2 hydrogen bonds and 13 hydrophobic contacts. These findings provide a structural basis for the optimization of inhibitors targeting the SARS-CoV-2 PLpro, contributing directly to anti-coronavirus drug discovery.

木瓜样蛋白酶（PLpro）是SARS-CoV-2编码的关键蛋白酶，在病毒多蛋白加工、复制酶-转录酶复合物组装和干扰宿主免疫反应中至关重要。新的COVID-19病例不断出现，世卫组织已将具有类似PLpro结构的冠状病毒确定为潜在的大流行威胁。通过分子建模、对接和蛋白酶活性测定，我们确定了四种有效的SARS-CoV-2 /PLpro抑制剂，IC₅₀值为6.96-20.21µM。通过荧光滴定测定它们的结合亲和力，得到的解离常数（KD）为4.18-13.06，支持它们的抑制活性。在硅毒性研究中，大多数抑制剂的LD₅0值与GRL0617相当，GRL0617是一种已知的PLpro抑制剂。结构分析表明，所有抑制剂都与PLpro的关键残基Asp164、Pro248、Tyr264、Tyr268和Gln269相互作用，但在特异性氢键和疏水相互作用方面存在差异。值得注意的是，化合物P636-0664表现出最广泛的相互作用，形成2个氢键和13个疏水接触。这些发现为优化靶向SARS-CoV-2 PLpro的抑制剂提供了结构基础，直接有助于抗冠状病毒药物的发现。

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引用次数: 0

Evaluation of two commercial multiplex RT-PCR tests for broad genotypic detection of viral gastroenteritis 两种商用多重RT-PCR检测方法对病毒性肠胃炎广泛基因型检测的评价。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-17 DOI: 10.1016/j.jviromet.2025.115328

Chiara Filizzolo , Mariangela Pizzo , Marika Munacò , Giovanni M. Giammanco , Gianvito Lanave , Vito Martella , Floriana Bonura , Simona De Grazia

Acute gastroenteritis (AGE) remains a global health concern, particularly affecting children under five. While mortality rates have declined in industrialized countries, AGE continues to pose a significant burden on healthcare systems and remains a major threat in developing regions. This study evaluates the diagnostic performance of two commercial molecular assays for enteric viruses, GastroIntestinal (GI) Viral PLUS ELITe MGB® and GI Norovirus PLUS ELITe MGB®, for the detection of a broad panel of viral genotypes associated with AGE. A total of 222 archived stool samples, previously characterized by reference biomolecular methods, were analyzed. These included 50 rotavirus (RVA)-positive, 57 norovirus (NoV)-positive, 39 adenovirus (AdV)-positive, 45 astrovirus (HAstV)-positive samples, and 10 co-infections. Both assays showed excellent agreement with reference methods (κ ≥ 0.95), with sensitivity ranging from 99.4 % to 100 % and specificity from 98.1 % to 100 %, confirming their ability to detect a broad spectrum of circulating viral genotypes. Minor discrepancies were observed in NoV GII.6 strains, misclassified as Genogroup I (GI), likely due to overlapping annealing profiles. These findings support the use of GI Viral PLUS and GI Norovirus PLUS ELITe MGB® assays as reliable tools for rapid and accurate genotypic diagnosis of viral AGE. Their implementation may enhance patient management, reduce inappropriate antimicrobial use, and support timely outbreak containment, especially in healthcare settings.

急性胃肠炎（AGE）仍然是一个全球健康问题，尤其影响五岁以下儿童。虽然工业化国家的死亡率已经下降，但年龄增长继续对卫生保健系统构成重大负担，并仍然是发展中地区的主要威胁。本研究评估了胃肠道（GI）病毒PLUS ELITe MGB®和胃肠道诺如病毒PLUS ELITe MGB®两种商用肠道病毒分子检测方法的诊断性能，用于检测与AGE相关的广泛病毒基因型。共有222份存档的粪便样本，以前通过参考生物分子方法进行了分析。其中轮状病毒（RVA）阳性50例，诺如病毒（NoV）阳性57例，腺病毒（AdV）阳性39例，星状病毒（HAstV）阳性45例，合并感染10例。两种检测方法均与参考方法（κ≥0.95）非常吻合，灵敏度为99.4% ~ 100%，特异性为98.1% ~ 100%，证实了它们能够检测广谱的循环病毒基因型。在11月GII.6菌株中观察到轻微的差异，可能是由于重叠的退火谱而被错误分类为基因组I （GI）。这些发现支持使用胃肠道病毒PLUS和胃肠道诺如病毒PLUS ELITe MGB®检测作为快速准确的病毒性AGE基因型诊断的可靠工具。它们的实施可以加强患者管理，减少不适当的抗菌素使用，并支持及时控制疫情，特别是在医疗保健环境中。

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引用次数: 0

Targeting Yellow-Fever Virus: Development of a specific aptamer to NS1 protein 靶向黄热病病毒：NS1蛋白特异性适配体的研制

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-17 DOI: 10.1016/j.jviromet.2025.115330

Mariane Izabella Abreu de Melo, Alessandra Nunes Duarte Miranda, Antero Silva Ribeiro de Andrade

Yellow Fever Virus (YFV), a mosquito-borne flavivirus, remains a significant public health concern despite the availability of effective vaccines. Accurate differential diagnosis continues to be challenging due to the high antigenic similarity among flaviviruses such as dengue and Zika, which compromises the specificity of current serological assays. Aptamers have emerged as promising alternatives to antibodies for diagnostic applications because of their high specificity, thermal stability, and ease of synthesis. In this study, we developed a DNA aptamer (Flav5) targeting the nonstructural protein 1 (NS1) of YFV using capillary electrophoresis–based SELEX (CE-SELEX). After three selection rounds, aptamer pools were sequenced and analyzed. Specificity assays demonstrated minimal cross-reactivity of Flav5 with NS1 proteins from dengue virus (serotypes 1–4) and Zika virus. The aptamer exhibited a high binding affinity for YFV-NS1, with a dissociation constant (Kd) of 34.5 ± 8.2 nM, as determined by quantitative PCR. The three-dimensional structure of Flav5 was modeled and docked to the tetrameric YFV-NS1 using the HDOCK server, revealing a stable binding interface within the inter-subunit cavity of NS1, supported by high confidence scores (>0.95). The Flav5 aptamer demonstrates strong potential for incorporation into YFV-specific diagnostic platforms, particularly in regions where multiple clinically relevant flaviviruses co-circulate. Its combination of high affinity, specificity, and favorable molecular docking characteristics, position it as a promising candidate for point-of-care diagnostic tools.

黄热病病毒（YFV）是一种蚊媒黄病毒，尽管已有有效疫苗，但仍是一个重大的公共卫生问题。由于登革热和寨卡等黄病毒之间的抗原高度相似，因此准确的鉴别诊断仍然具有挑战性，这损害了当前血清学分析的特异性。适配体因其高特异性、热稳定性和易于合成而成为抗体诊断应用的有希望的替代品。在这项研究中，我们利用毛细管电泳的SELEX （CE-SELEX）技术开发了一个针对YFV非结构蛋白1 （NS1）的DNA适体（Flav5）。经过三轮筛选，对适体池进行测序和分析。特异性分析显示，Flav5与登革热病毒（血清型1-4）和寨卡病毒的NS1蛋白的交叉反应性极小。该适配体对YFV-NS1具有较高的结合亲和力，其解离常数（Kd）为34.5±8.22nM。利用HDOCK服务器对Flav5的三维结构进行建模并与四聚体YFV-NS1对接，揭示了NS1亚基间空腔内稳定的结合界面，具有高置信度分数（>0.95）。Flav5适体显示了整合到yfv特异性诊断平台的强大潜力，特别是在多种临床相关黄病毒共同传播的区域。它结合了高亲和力、特异性和良好的分子对接特性，使其成为一种有希望的即时诊断工具。

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引用次数: 0

Real time PCR-based evaluation of live attenuated lumpy skin disease virus vaccines for immunogenicity and efficacy 基于实时pcr的疙瘩性皮肤病病毒减毒活疫苗免疫原性和有效性评价

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-16 DOI: 10.1016/j.jviromet.2025.115327

Mohamed Samy Abousenna, Heba A. Khafagy, Amal Abd El Moneim Mohamed, Sara El Sawy Ahmed, Fady Abd El Mohsen Shasha, Nermeen Gouda Shafik

Lumpy skin disease (LSD) is a transboundary viral disease of cattle caused by lumpy skin disease virus (LSDV), resulting in substantial economic losses. Control of the disease relies primarily on live attenuated vaccines, making accurate potency assessment essential to ensure protective efficacy. Quantitative real-time PCR (qPCR) has emerged as a rapid and sensitive method for viral detection and quantification. In this study, ten commercial batches of live attenuated LSDV vaccines were evaluated using qPCR, and the molecular titers were compared with conventional tissue culture titration and in vivo protection in a calf challenge model. Tissue culture titration was performed on MDBK cells, qPCR quantified viral genome copy numbers, and in vivo challenge testing involved 56 calves. Statistical analyses, including Pearson and Spearman correlations and linear regression, were used to assess the relationships between qPCR-derived titers, tissue culture results, and protection outcomes. Eight of the ten vaccine batches met the WOAH-recommended potency range (3–4 log₁₀ TCID₅₀/mL), while two batches were suboptimal. qPCR-derived titers showed a strong correlation with tissue culture titers (Pearson’s r = −0.994; p < 0.0001) r = −0.994; p < 0.0001) and with protection outcomes following virulent challenge (r = 0.777; p = 0.008), with lower molecular titers corresponding to reduced protection. These results demonstrate that qPCR is a rapid, sensitive, and reliable surrogate for conventional potency testing, offering biosafe and time-efficient evaluation. Integrating molecular quantification into routine vaccine assessment can improve quality control, reduce reliance on in vivo challenge, facilitate harmonized vaccine deployment, and ultimately enhance field protection against LSD.

肿块性皮肤病（LSD）是由肿块性皮肤病病毒（LSDV）引起的跨界牛病毒性疾病，造成重大经济损失。该病的控制主要依靠减毒活疫苗，因此准确的效力评估对于确保保护效果至关重要。实时荧光定量PCR （qPCR）是一种快速、灵敏的病毒检测和定量方法。本研究采用qPCR技术对10批LSDV减毒活疫苗进行了评价，并与传统的组织培养滴定法和小牛攻毒模型的体内保护作用进行了分子效价比较。对MDBK细胞进行组织培养滴定，qPCR量化病毒基因组拷贝数，并对56头小牛进行体内攻毒试验。统计分析，包括Pearson和Spearman相关性和线性回归，用于评估qpcr衍生滴度、组织培养结果和保护结果之间的关系。十个疫苗批次中有八个达到了世界卫生组织推荐的效力范围（3-4log₁₀TCID₅₀/mL），而两个批次则不理想。qpcr衍生的滴度与组织培养滴度（Pearson’s r = -0.994; p < 0.0001）和毒力攻击后的保护结果（r = 0.777; p = 0.008）有很强的相关性，较低的分子滴度对应于较低的保护。这些结果表明，qPCR是一种快速、灵敏、可靠的替代传统效价检测的方法，具有生物安全性和时效性。将分子定量纳入常规疫苗评估可以改善质量控制，减少对体内挑战的依赖，促进疫苗的协调部署，并最终增强对LSD的野外保护。

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