Journal of virological methods最新文献_第4页

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 : 2026-04-01 Epub 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

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 : 2026-04-01 Epub 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

Development of end-point and quantitative polymerase chain reaction assays to detect and discriminate between blueberry scorch virus and blueberry virus S 建立检测和区分蓝莓焦毒和蓝莓病毒S的终点和定量聚合酶链反应方法。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-04-01 Epub Date: 2025-12-16 DOI: 10.1016/j.jviromet.2025.115324

D.E.V. Villamor, D. Stainton, I.E. Tzanetakis

Blueberry scorch virus (BlScV) and blueberry virus S (BVS) are closely related carlaviruses with the latter being only recently characterized. Most available PCR assays for BlScV fail to detect some isolates of the virus, as well as BVS. Here, we describe PCR assays designed based on all available virus sequences in public databases that can discriminate BlScV from BVS. Initially, a triplex endpoint PCR assay was developed that yields different amplicon sizes for each virus and incorporates an internal control which assesses the quality of the nucleic acids and the efficiency of the enzymatic reactions. Subsequently, a duplex quantitative PCR was designed employing primers targeting both viruses and discriminatory probes specific to each. Comparison between end-point and qPCR assays revealed near parallel sensitivity. The availability of the two PCR formats reliably improves the detection assays of the viruses in diagnostic facilities, quarantine, and clean plant programs.

蓝莓焦病毒（BlScV）和蓝莓病毒S （BVS）是密切相关的卡拉病毒，后者是最近才发现的。大多数可用的BlScV PCR检测方法都不能检测到病毒的某些分离株以及BVS。在这里，我们描述了基于公共数据库中所有可用的病毒序列设计的PCR检测，可以区分BlScV和BVS。最初，开发了一种三重终点PCR试验，为每种病毒产生不同的扩增子大小，并纳入内部控制，以评估核酸的质量和酶促反应的效率。随后，设计了一种双工定量PCR，采用引物靶向两种病毒和针对每种病毒的歧视性探针。终点和qPCR检测的比较显示接近平行敏感性。两种PCR格式的可用性可靠地改善了诊断设施、检疫和清洁工厂计划中病毒的检测分析。

引用次数: 0

Evaluation of ∑-MM™ molecular medium for the SARS-CoV-2 RNA stability over 90 days under different temperature conditions 不同温度条件下∑-MM™分子培养基对SARS-CoV-2 RNA 90天稳定性的评价

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-04-01 Epub Date: 2025-12-05 DOI: 10.1016/j.jviromet.2025.115322

Giasemi C. Eptaminitaki, Alexandros Zafiropoulos, George Sourvinos

The collection and storage of clinical specimens are critical determinants of reliable and accurate diagnostic outcomes. Swabs are commonly utilized for specimen collection aimed at the detection and identification of microorganisms. In this study, the ∑-MM™ Molecular Medium (Medical Wire and Equipment), which rapidly inactivates microorganisms, including bacteria, mycobacteria, and viruses, was evaluated for its capacity to preserve the stability of SARS-CoV-2 for up to 90 days under various temperature conditions. A total of 42 newly collected SARS-CoV-2 positive samples were selected to evaluate the performance of the ∑-MM™. Aliquots were stored under different conditions: at 4°C for 7 days, at room temperature for 7 days, at −80°C for one month, at room temperature for one month, at −80°C for three months, and at room temperature for three months. Samples were subsequently tested for SARS-CoV-2 RNA by Real-Time PCR. No statistically significant differences were observed across all storage conditions, when compared to the baseline measurements. The ∑-MM™ effectively preserves SARS-CoV-2 for up to 90 days at ambient temperatures, without statistically significant changes in CT values compared to baseline. These results validate ∑-MM™ as a reliable transport medium that enables specimen storage and transport under ambient conditions without compromising diagnostic accuracy. Given the demonstrated stability, future studies will explore the performance of ∑-MM™ over extended storage durations.

临床标本的收集和储存是可靠和准确诊断结果的关键决定因素。拭子通常用于标本采集，目的是检测和鉴定微生物。在本研究中，研究人员评估了在各种温度条件下保持SARS-CoV-2稳定性长达90天的∑-MM™分子培养基（医用线材和设备）的能力，该培养基可以快速灭活微生物，包括细菌、分枝杆菌和病毒。选取新采集的42份SARS-CoV-2阳性样本，评价∑-MM™的性能。等分在不同条件下保存：4℃保存7天，室温保存7天，-80℃保存1个月，室温保存1个月，-80℃保存3个月，室温保存3个月。随后用Real-Time PCR检测样品的SARS-CoV-2 RNA。与基线测量值相比，在所有存储条件下均未观察到统计学上的显著差异。∑-MM™在环境温度下可有效保存SARS-CoV-2长达90天，与基线相比，CT值没有统计学上的显著变化。这些结果验证了∑-MM™是一种可靠的运输介质，可以在环境条件下存储和运输标本，而不会影响诊断的准确性。鉴于已证明的稳定性，未来的研究将探索∑-MM™在延长储存时间内的性能。

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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 : 2026-04-01 Epub 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

Evaluation of Tasso T20 to collect dried blood for the quantification of HIV-1 RNA Tasso T20采集干血用于HIV-1 RNA定量的评价。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-04-01 Epub Date: 2025-12-11 DOI: 10.1016/j.jviromet.2025.115326

Sara W.F. Geffert , Soya S. Sam , Karen Chandran , Curt G. Beckwith , Rami Kantor , Angela M. Caliendo

Tasso T20 (T20) is a finger stick-free, self-sampling device capable of at-home blood sample collection. We optimized HIV-1 RNA elution from T20, evaluated its analytical performance, compared its performance to Whatman dried blood spots (DBS), and assessed HIV-1 RNA stability. We used mock viral samples to optimize HIV-1 RNA elution using viral concentrations 10,000 and 3000 copies/mL. Limit of detection (LOD) and limit of quantification (LOQ) were determined, and linearity and reproducibility were verified. Analytical agreement between T20 and DBS was assessed using eight concentrations, from 100,000 to 1000 copies/mL. T20 HIV-1 RNA stability was assessed over time. All samples were tested using the m2000 RealTime HIV-1 DBS assay. The optimized elution conditions selected were 30 min of sonication followed by 30 min of incubation at 55 °C. The 95 % LOD and LOQ were determined to be 3.56 and 3.70 log₁₀ copies/mL, respectively. Good linearity was observed with a strong correlation (R²=0.98) and excellent reproducibility. Analytical agreement analysis demonstrated a mean difference of −0.36 log₁₀ copies/mL (SD ± 0.21 log₁₀ copies/mL). ANOVA indicated all samples remained stable (p value=0.33, F=1.22). The Tasso T20 is capable of quantifying HIV-1 viral loads, with results comparable to DBS.

Tasso T20 （T20）是一种无需手指粘连的自采样装置，能够在家中采集血液样本。我们优化了T20中HIV-1 RNA的洗脱，评估了其分析性能，将其与Whatman干血点（DBS）进行了比较，并评估了HIV-1 RNA的稳定性。我们使用模拟病毒样本优化HIV-1 RNA洗脱，病毒浓度分别为10000和3000拷贝/mL。测定了检测限（LOD）和定量限（LOQ），并进行了线性和重复性验证。T20和DBS之间的分析一致性评估使用8个浓度，从100,000到1000拷贝/mL。随着时间的推移评估T20 HIV-1 RNA的稳定性。所有样本均使用m2000 RealTime HIV-1 DBS检测。选择的最佳洗脱条件为：超声30min， 55℃孵育30min。95% LOD和LOQ分别为3.56和3.70 log10 copies/mL。线性良好，相关性强（R2=0.98），重现性好。分析一致性分析显示，平均差异为-0.36 log10 copies/mL （SD±0.21 log10 copies/mL）。方差分析显示所有样本保持稳定（p值=0.33，F=1.22）。Tasso T20能够定量HIV-1病毒载量，其结果与DBS相当。

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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 : 2026-04-01 Epub 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 : 2026-04-01 Epub 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

Alternatives to central nervous system samples for enhanced rabies surveillance: Exploring natural infections of Lyssavirus rabies in striped skunks (Mephitis mephitis) in Northern Colorado 加强狂犬病监测的中枢神经系统样本替代方案：探索科罗拉多州北部条纹臭鼬（Mephitis Mephitis）中狂犬病溶血病毒的自然感染。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-04-01 Epub Date: 2025-12-09 DOI: 10.1016/j.jviromet.2025.115325

Clara C.P. Mankowski , Matthew W. Hopken , Terry R. Spraker , Amy T. Gilbert

Enhanced rabies surveillance (ERS) of wildlife populations complements passive public health surveillance through active field-based sampling. The combined data augment rabies management decision-making through tracking virus distribution and movement, rapidly identifying potential outbreaks, and informing containment and elimination strategies at regional and landscape scales. Laboratory diagnostic methods are critical to ERS and several gold standard methods are recommended for detection of Lyssavirus rabies (RV); each validated using central nervous system (CNS) tissue which requires necropsy and cold storage, posing challenges and risks in remote field settings. Alternative sample types to CNS tissue may improve ERS efficiency and field personnel safety, provided they offer robust diagnostic specificity and sensitivity. We evaluated multiple sample types opportunistically collected postmortem from naturally infected striped skunks (Mephitis mephitis) and used real-time reverse transcriptase PCR (rtRT-PCR) to evaluate diagnostic sensitivity and specificity of each sample type compared to CNS tissue. The detection rate of RV RNA was 97 % (95 % CI 81–100) for oral swabs and 96 % (95 % CI 80–100) for eye swabs; when swab types were combined, the detection rate was 100 % (95 % CI 85–100) and comparable to detection from CNS. Other sample types demonstrated compromised diagnostic sensitivities (33–76 %). All sample types were 100 % specific for RV diagnosis. The combination of eye and oral swabs as ERS samples may expand sampling opportunities that improve efficiency, mitigate sample collection and storage challenges, and decrease RV exposure risk among field staff while enhancing the information provided to estimate impacts of RV management on target wildlife populations.

加强野生动物种群的狂犬病监测（ERS）通过主动实地抽样补充了被动的公共卫生监测。这些综合数据通过跟踪病毒分布和移动、快速识别潜在疫情以及在区域和景观尺度上通报遏制和消除战略，增强了狂犬病管理决策。实验室诊断方法对ERS至关重要，推荐几种金标准方法用于检测狂犬溶血病毒（RV）；每种方法都使用中枢神经系统（CNS）组织进行验证，这需要尸检和冷藏，这在偏远地区带来了挑战和风险。替代中枢神经系统组织的样本类型可以提高ERS的效率和现场人员的安全，前提是它们具有强大的诊断特异性和敏感性。我们评估了从自然感染的条纹臭鼬（Mephitis Mephitis）死后收集的多种样本类型，并使用实时逆转录酶PCR （rtRT-PCR）来评估每种样本类型与中枢神经系统组织相比的诊断敏感性和特异性。口腔拭子RV RNA检出率为97% (95% CI 81 ~ 100)，眼拭子检出率为96% (95% CI 80 ~ 100)；当结合拭子类型时，检出率为100% (95% CI 85-100)，与中枢神经系统的检出率相当。其他样本类型表现出较差的诊断敏感性（76%至33%）。所有样本类型对RV诊断的特异性均为100%。将眼拭子和口腔拭子结合作为ERS样本可以增加采样机会，从而提高效率，减轻样本收集和储存的挑战，降低现场工作人员暴露于RV的风险，同时增强提供的信息，以估计RV管理对目标野生动物种群的影响。

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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 : 2026-04-01 Epub 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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