Journal of virological methods最新文献_第10页

AI-driven strategies for enhancing Mpox surveillance and response in Africa 在非洲加强麻疹监测和应对的人工智能驱动战略

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-01 Epub Date: 2025-09-24 DOI: 10.1016/j.jviromet.2025.115270

David B. Olawade , Chiamaka Norah Ezeagu , Chibuike S. Alisi , Aanuoluwapo Clement David-Olawade , Deborah Motilayo Eniola , Temitope Akingbala , Ojima Z. Wada

Mpox, a zoonotic viral disease endemic to several African countries, has re-emerged as a significant public health concern, particularly in regions with limited healthcare resources. Current public health strategies in Africa fall short due to fragmented surveillance systems, delayed diagnostic capabilities, and inadequate resource distribution networks that cannot effectively respond to rapidly evolving outbreaks in remote and underserved areas. This narrative review explores the potential of Artificial Intelligence (AI) to enhance the management and control of Mpox in Africa. AI technologies, including machine learning and predictive analytics, can significantly improve early detection, surveillance, contact tracing, case management, public health communication, and resource allocation. AI-driven tools can analyze large datasets to identify outbreak patterns, automate contact tracing through mobile data, optimize treatment plans, and tailor public health messages to specific communities. However, the successful implementation of AI faces challenges, including limited digital infrastructure, data quality issues, ethical concerns, and the need for capacity building. Furthermore, ongoing research is essential to refine AI algorithms and develop culturally sensitive applications. This review emphasizes the need for investment in infrastructure, training, and ethical frameworks to fully integrate AI into public health systems in Africa. By addressing these challenges, AI can play a pivotal role in mitigating the impact of Mpox and enhancing the resilience of healthcare systems against future infectious disease outbreaks. This represents a novel comprehensive synthesis of AI applications specifically for African Mpox control, providing a critical framework for evidence-based implementation strategies in resource-limited settings.

麻疹是一种在若干非洲国家流行的人畜共患病毒性疾病，已重新成为一个重大的公共卫生问题，特别是在卫生保健资源有限的区域。由于监测系统支离破碎、诊断能力滞后以及资源分配网络不足，无法有效应对偏远和服务不足地区迅速演变的疫情，非洲目前的公共卫生战略存在不足。本综述探讨了人工智能（AI）在加强非洲麻疹管理和控制方面的潜力。包括机器学习和预测分析在内的人工智能技术可以显著改善早期发现、监测、接触者追踪、病例管理、公共卫生沟通和资源分配。人工智能驱动的工具可以分析大型数据集以确定疫情模式，通过移动数据自动追踪接触者，优化治疗计划，并针对特定社区定制公共卫生信息。然而，人工智能的成功实施面临着挑战，包括有限的数字基础设施、数据质量问题、道德问题以及能力建设的需求。此外，正在进行的研究对于完善人工智能算法和开发具有文化敏感性的应用程序至关重要。本综述强调需要在基础设施、培训和道德框架方面进行投资，以便将人工智能充分纳入非洲的公共卫生系统。通过应对这些挑战，人工智能可以在减轻麻疹的影响和增强卫生保健系统对未来传染病暴发的抵御能力方面发挥关键作用。这代表了专门针对非洲麻疹控制的人工智能应用的一种新颖的全面综合，为在资源有限的情况下基于证据的实施战略提供了一个关键框架。

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引用次数: 0

Evaluation of two IgG-scFv bispecific antibodies for neutralizing Omicron variants of SARS-CoV-2 两种IgG-scFv双特异性抗体中和SARS-CoV-2 Omicron变体的评价

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-01 Epub Date: 2025-09-05 DOI: 10.1016/j.jviromet.2025.115258

Diana Hinojosa-Trujillo , Freddy Dehesa-Canseco , Melissa García-Vega , Verónica Mata-Haro , Mario Solís-Hernández , Mónica Reséndiz-Sandoval , Fanglei Zuo , Harold Marcotte , Jesús Hernández

Bispecific antibodies (bsAbs) offer an alternative to monoclonal antibody (mAb) cocktails for addressing the loss of efficacy due to the rapid emergence of SARS-CoV-2 mutants. The structure and specificity of the parental antibodies influence the development of a highly neutralizing bsAb. To design an effective bsAb, the recognition of 44 single-chain fragment variable (scFv) antibodies against variants of SARS-CoV-2 was evaluated, along with an assessment of their ability to competitively bind to the receptor-binding domain (RBD) compared to the most potent neutralizing mAbs. From this analysis, three antibodies − 19n01, 01n21, and 01n27 − were identified for their broad recognition and non-competitive binding behavior. These antibodies were selected as the parental antibodies for the design of two bsAb. The bsAb bis L and bis H were engineered as IgG-scFv constructs, each with the secondary domain oriented differently to introduce new specificities. Both bsAbs retained the neutralizing capabilities of their parental antibodies in live-virus assays, neutralizing the Omicron variants BQ.1.1 and XBB.1.

双特异性抗体（bsAbs）是单克隆抗体（mAb）鸡尾酒的替代方案，可解决由于SARS-CoV-2突变体迅速出现而导致的疗效丧失问题。亲本抗体的结构和特异性影响高度中和性bsAb的发展。为了设计一种有效的bsAb，我们评估了44种针对SARS-CoV-2变体的单链片段变量（scFv）抗体的识别能力，并与最有效的中和单克隆抗体相比，评估了它们与受体结合结构域（RBD）的竞争性结合能力。从这个分析中，鉴定出三种抗体- 19n01, 01n21和01n27，它们具有广泛的识别和非竞争性结合行为。选择这些抗体作为亲本抗体设计两种bsAb。bsAb his L和his H被设计成IgG-scFv结构，每个结构域定向不同，以引入新的特异性。在活病毒试验中，两种bsab都保留了亲本抗体的中和能力，中和了Omicron变体BQ.1.1和XBB.1。

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引用次数: 0

Evaluating the effectiveness of a novel environmental decontamination system utilizing low-energy hyper-charged photoelectrons against coronavirus 评估一种利用低能量超电荷光电子对抗冠状病毒的新型环境净化系统的有效性。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-01 Epub Date: 2025-09-20 DOI: 10.1016/j.jviromet.2025.115269

Madeeha Afzal , Mark D.P. Willcox , Stephan Praet , Murray Mcdonald , Muhammad Yasir

The COVID-19 pandemic had profound economic and social effects across the globe. The present study evaluated the virus attenuation efficacy of an environmental decontamination system named photon-mediated electron emitter (PMEE) on aerosolized and surface-associated coronavirus. The intensity of hyper-charged photoelectrons emitted by the PMEE were measured over distances of 1–5 m using a photon-detection mapping device. The antiviral efficacy of the PMEE was tested against mouse hepatitis virus (MHV-1) ATCC/VR261. For aerosolised studies, the MHV-1 was aerosolized using an electronic diffuser in an enclosed booth. Virus particles were exposed to PMEE for 10 and 15 min. For surface studies, viruses were dried on steel and laminate surfaces and then exposed to the PMEE from distances of 1 and 5-meters. The antiviral potential of the PMEE was evaluated by culturing MHV-1 in A9 ATCC/CCL 1.4 cells using a plaque assay. PMEE emission strength ranged from 1.44 to 1.86 V inside the booth and 0.83–1.86 V outside. The average size of the generated aerosol particles was 3.0 ± 0.3 µm. After 10 min exposure, the virucidal effects against particles of 2.1 µm, 1.1 µm, and 0.65 µm pore sizes were 74.5 ± 11.1 %, 79 ± 4.9 %, and 96 ± 1.4 % respectively. On surfaces, a 1-minute exposure at 1 m resulted in a 60 ± 0.5 % reduction on steel and 43 ± 2.7 % on laminate. The PMEE-based system effectively reduced the infectivity of MHV-1 both in aerosols and on surfaces, demonstrating strong potential for environmental decontamination applications.

新冠肺炎疫情在全球范围内产生了深刻的经济社会影响。本研究评估了一种名为光子介导电子发射器（PMEE）的环境去污系统对雾化和表面相关冠状病毒的病毒衰减效果。利用光子探测测绘装置测量了PMEE发射的超电荷光电子的强度，距离为1至5m。研究了PMEE对小鼠肝炎病毒（MHV-1） ATCC/VR261的抗病毒作用。对于雾化研究，MHV-1在封闭的隔间中使用电子扩散器雾化。将病毒颗粒暴露在PMEE中10分钟和15分钟。在表面研究中，病毒在钢铁和层压板表面干燥，然后在距离1米和5米的地方暴露在PMEE中。通过斑块实验，在A9 ATCC/CCL 1.4细胞中培养MHV-1，评估PMEE的抗病毒潜力。PMEE发射强度在展台内为1.44 ~ 1.86mV，展台外为0.83 ~ 1.86mV。产生的气溶胶颗粒平均粒径为3.0±0.3µm。暴露10min后，对孔径为2.1µm、1.1µm和0.65µm的颗粒的毒力分别为74.5±11.1%、79±4.9%和96±1.4%。在表面上，在1米处暴露1分钟，钢的磨损减少60±0.5%，层压板的磨损减少43±2.7%。基于pme的系统有效地降低了MHV-1在气溶胶和表面上的传染性，显示出在环境净化应用中的强大潜力。

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引用次数: 0

Cryopreservation of chicken and duck tracheal rings and precision-cut lung slices: A promising tool for the rapid characterization of avian influenza viruses 鸡鸭气管环和精确切割肺片的低温保存：一种有前途的禽流感病毒快速表征工具

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-01 Epub Date: 2025-09-02 DOI: 10.1016/j.jviromet.2025.115257

Alessandra Napolitan , Elisa Mazzacan , Niccolò Fonti , Sofia Tomasoni , Erica Melchiotti , Claudia Zanardello , Lucrezia Vianello , Sami Ramzi , Valentina Panzarin , Marika Crimaudo , Ranieri Verin , Francesco Bonfante , Eva Mazzetto

Since its emergence in 1996, highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/96 lineage have diversified into multiple clades, culminating in the 2020–2021 global panzootic caused by H5N1 viruses of the clade 2.3.4.4b. Further reassortment events have significantly diversified the phenotypes of these viruses, underscoring the need for continuous monitoring and strain characterization to better adjust control measures and mitigate the impact of the disease in wild birds and poultry. Standardized, ready-to-use ex vivo tissue platforms for rapid phenotyping of avian influenza viruses (AIVs) offer a valid alternative to in vivo models that are financially, ethically and logistically demanding. We optimized explant production and cryopreservation protocols for chicken and duck tracheal organ cultures (cTOCs and dTOCs) and precision-cut lung slices (cPCLS and dPCLS), assessing post-thaw viability, histological integrity, and susceptibility to AIV infection. Trehalose supplementation of cryopreservation solutions based on dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) significantly improved tissue viability. Although cryopreserved tissues were less viable than the fresh explants, viral replication was similar and only a modest reduction in susceptibility to infection was observed. Finally, we used duck and chicken TOCs to assess the ability of cryopreserved explants to discriminate viruses based on their divergent fitness and host preference. These findings underscore the potential of cryopreserved TOCs and PCLS as additional tools for the phenotypic characterisation of emerging AIVs.

自1996年出现以来，A/Goose/Guangdong/1/96谱系的高致病性禽流感（HPAI）已分化成多个分支，最终在2020-2021年由2.3.4.4b分支的H5N1病毒引起的全球大流行中达到高潮。进一步的重配事件使这些病毒的表型显著多样化，强调需要持续监测和品系表征，以更好地调整控制措施并减轻该疾病对野生鸟类和家禽的影响。标准化的、即用型的离体组织平台可用于禽流感病毒（AIVs）的快速表型分析，为在体模型提供了一种有效的替代方案，而体内模型在经济、伦理和后勤方面都要求很高。我们优化了鸡和鸭气管器官培养（cTOCs和dTOCs）和精确切割肺切片（cPCLS和dPCLS）的外植体生产和冷冻保存方案，评估了解冻后的活力、组织学完整性和对AIV感染的易感性。以二甲亚砜（DMSO）和胎牛血清（FBS）为基础的冷冻保存液中添加海藻糖可显著提高组织活力。虽然冷冻保存的组织比新鲜外植体的存活率低，但病毒复制相似，对感染的易感性仅略有降低。最后，我们用鸭和鸡的TOCs来评估冷冻保存的外植体根据病毒的不同适应度和宿主偏好来区分病毒的能力。这些发现强调了冷冻保存的TOCs和PCLS作为新兴aiv表型表征的额外工具的潜力。

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引用次数: 0

Potential of recombinant avian adeno-associated virus as a viral vector for CRISPR/Cas9 delivery to avian cells 重组禽腺相关病毒作为CRISPR/Cas9载体的潜力

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-01 Epub Date: 2025-09-09 DOI: 10.1016/j.jviromet.2025.115263

Takumi Terada , Sodai Fujii , Nanako Yamanishi , Ryota Kajihara , Tenkai Watanabe , Ryo Ezaki , Hiroyuki Horiuchi , Mei Matsuzaki

While genome editing has been established in chickens, where cultured primordial germ cell (PGC) systems are available, the implementation of genome editing remains a major challenge in many other birds due to the lack of robust PGC culture methods. Therefore, the development of reliable and efficient tools can significantly accelerate precision genome modification in avian species. Here, we evaluated the applicability of recombinant avian adeno-associated virus (rA3V) as a delivery vector for a CRISPR/Cas9 construct in avian cells using Staphylococcus aureus-derived Cas9 (SaCas9) and single-guide RNA (sgRNA). Infection with rA3V particles carrying an EGFP expression cassette (rA3V-EGFP) successfully induced EGFP expression in chicken fibroblasts (DF-1) cells, with approximately 80 % EGFP-positive cells at the maximum multiplicity of infection (MOI = 10,000). In plasmid-based transfection experiments, sgRNAs targeting the chicken tyrosinase locus and SaCas9 exhibited DNA cleavage activity in DF-1 cells. Furthermore, infection with rA3V particles encoding these CRISPR components successfully introduced indel mutations into the tyrosinase gene in DF-1 cells, with a calculated indel frequency of approximately 5.4 % at MOI = 40,000 without drug selection. Although EGFP expression was observed in quail fibrosarcoma cells, the percentage of EGFP-positive cells was much lower than that in DF-1 cells. In addition, in vivo infection with rA3V-EGFP of the chicken blastoderm failed to induce EGFP expression in germline cells, even at the highest applicable viral dose. In summary, rA3V can be used as a genome-editing vector in birds, although further investigation of its infectivity and tropism is necessary to expand its applicability to diverse avian species.

虽然基因组编辑已经在鸡中建立，其中培养的原始生殖细胞（PGC）系统是可用的，但由于缺乏强大的PGC培养方法，在许多其他鸟类中实施基因组编辑仍然是一个主要挑战。因此，开发可靠、高效的工具可以显著加快鸟类基因组的精确修饰。在这里，我们利用金黄色葡萄球菌衍生的Cas9 （SaCas9）和单导RNA （sgRNA）评估了重组禽腺相关病毒（rA3V）作为CRISPR/Cas9构建物在禽细胞中的传递载体的适用性。携带EGFP表达盒的rA3V颗粒（rA3V-EGFP）感染鸡成纤维细胞（DF-1）成功诱导EGFP表达，在最大感染倍数（MOI = 10,000）时，EGFP阳性细胞约占80%。在质粒转染实验中，靶向鸡酪氨酸酶位点和SaCas9的sgRNAs在DF-1细胞中表现出DNA切割活性。此外，编码这些CRISPR成分的rA3V颗粒感染成功地将indel突变引入DF-1细胞的酪氨酸酶基因中，在MOI为40000时计算出的indel频率约为5.4%，没有药物选择。虽然在鹌鹑纤维肉瘤细胞中观察到EGFP的表达，但EGFP阳性的细胞比例远低于DF-1细胞。此外，即使在最高适用病毒剂量下，rA3V-EGFP在鸡胚皮体内感染也不能诱导种系细胞表达EGFP。综上所述，rA3V可以作为鸟类基因组编辑载体，但需要进一步研究其感染性和嗜性，以扩大其在不同鸟类物种中的适用性。

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引用次数: 0

Development and analytical validation of a quantitative PCR assay for the detection of Magellanic penguin herpesvirus 1 麦哲伦企鹅疱疹病毒1型定量PCR检测方法的建立及分析验证

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-01 Epub Date: 2025-08-28 DOI: 10.1016/j.jviromet.2025.115255

Maris J. Daleo , Laura A. Adamovicz , Karisa N. Tang , Matthew C. Allender

Herpesviruses are associated with disease in several aquatic bird species, including penguins. Magellanic penguin herpesvirus 1 (MagHV1) was initially detected in 58.3 % of oiled Magellanic penguins (Spheniscus magellanicus) in South America presenting with respiratory distress characterized by a combination of necrohemorrhagic tracheitis, fibrinous air sacculitis, pneumonia, and death. Additional exploration is needed to understand how herpesviruses affect penguin health; however, there is currently a lack of rapid, sensitive, and specific methods for detecting and quantifying herpesvirus infections in this taxon. To address this problem, we developed a real-time quantitative PCR (qPCR) assay for the detection of MagHV1 in penguins. Using a commercial program, TaqMan-MGB primer-probes targeting the DNA polymerase gene were designed in silico. Inter- and intra-assay variability, dynamic range, limit of detection, and analytical specificity were assessed to validate the assay per MIQE guidelines. The resulting assay was highly specific for MagHV1, failing to amplify fifteen closely related avian herpesviruses. It performed with high efficiency (slope =-3.336, R² = 0.999, efficiency 99.40 %) and low inter- and intra-assay variability (coefficient of variation < 1.67 % at all dilutions). Reaction efficiency was not impacted by the presence of penguin DNA from known-negative tracheal swabs. This qPCR assay has a linear range of detection from 10⁷ to 10¹ viral copies per reaction and provides a valuable tool in the surveillance and characterization of MagHV1 epidemiology in penguins. This assay can further be used to detect asymptomatic birds and as an effective tool to monitor infectious individuals.

疱疹病毒与包括企鹅在内的几种水禽的疾病有关。麦哲伦企鹅疱疹病毒1 （MagHV1）最初在南美洲58.3% %的油麦哲伦企鹅（Spheniscus magellanicus）中检测到，表现为呼吸窘迫，其特征是坏死性出血性气管炎、纤维性空气囊炎、肺炎和死亡。需要进一步的探索来了解疱疹病毒如何影响企鹅的健康；然而，目前缺乏快速、敏感和特异性的方法来检测和定量该分类群中的疱疹病毒感染。为了解决这一问题，我们建立了一种实时定量PCR （qPCR）方法来检测企鹅体内的MagHV1。利用商业程序，设计了针对DNA聚合酶基因的TaqMan-MGB引物探针。评估测定间和测定内的变异性、动态范围、检测限和分析特异性，以根据MIQE指南验证测定。结果对MagHV1具有高度特异性，不能扩增15种密切相关的禽疱疹病毒。该方法效率高（斜率=-3.336,R2 = 0.999，效率99.40 %），测定间和测定内变异性低（所有稀释度的变异系数<； 1.67 %）。反应效率不受已知阴性气管拭子中企鹅DNA存在的影响。该方法具有107 ~ 101个线性检测范围，为企鹅MagHV1流行病学监测和鉴定提供了有价值的工具。该试验可进一步用于检测无症状禽类，并作为监测感染个体的有效工具。

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引用次数: 0

Evaluation of the Applied Biosystems™ TaqPath™ Seq HIV-1 Genotyping Kit for HIV-1 drug resistance testing from dried blood spot specimens 应用Biosystems™TaqPath™Seq HIV-1基因分型试剂盒对干血斑点标本进行HIV-1耐药检测的评价

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-01 Epub Date: 2025-10-08 DOI: 10.1016/j.jviromet.2025.115277

Obiageli Okafor , Jane Cameron , Andrea Garcia , Charmaine Hinahon , Carmen Salvador-Palomeque , Mitchell Starr , Philip H. Cunningham

Objectives

Monitoring HIV drug resistance is crucial for HIV treatment success. Logistical challenges restrict the use of liquid venous blood specimens for HIV drug resistance (HIVDR) testing, but dried blood spots (DBS) offer a more accessible alternative. This study evaluates the performance of TaqPath™ Seq HIV-1 Genotyping Kit (TaqPath kit) for detecting resistance-associated mutations in the protease (PR), reverse transcriptase (RT), and integrase (INI) regions of HIV-1 pol gene using DBS specimens.

Methods

A total of 79 DBS samples collected from ART-naive and ART-experienced individuals with viral loads ranging from 2830 to 9,095,161 copies/mL and HIV-1 subtypes A, B, C, F, and CRF01_AE were genotyped with TaqPath kit. Performance was assessed using the WHO HIVDR assay validation criteria.

Results

The assay met the amplification sensitivity criterion, achieving 95.7 % for PR/RT and 92.7 % for INI in high viral load samples. However, lower viral load samples between 2000 and 5000 copies/mL demonstrated reduced sensitivity of 50 % and 30 % for the PR/RT and INI regions respectively. Intra- and inter-assay agreement exceeded 99 % and mutation detection showed strong correlation with the reference assay, identifying over 95 % of reference mutations.

Conclusions

The TaqPath kit met the WHO acceptance criteria for HIVDR assay validation, except in the INI region at low viral loads, where amplification sensitivity was below the required threshold. Dried blood spot specimens are compatible with TaqPath kit for baseline and treatment-failure HIVDR genotyping, offering a reliable alternative to plasma in resource-constrained and remote settings.

目的监测HIV耐药性对HIV治疗成功至关重要。后勤方面的挑战限制了液体静脉血标本用于艾滋病毒耐药性（HIVDR）检测的使用，但干血点（DBS）提供了一种更容易获得的替代方法。本研究评估了TaqPath™Seq HIV-1基因分型试剂盒（TaqPath Kit）检测HIV-1 pol基因蛋白酶（PR）、逆转录酶（RT）和整合酶（INI）区域耐药相关突变的性能。方法采用TaqPath试剂盒对病毒载量为2830 ~ 9,095,161拷贝/mL、HIV-1亚型A、B、C、F和CRF01_AE的初接受art治疗和已接受art治疗的79例DBS患者进行基因分型。使用世卫组织HIVDR测定验证标准评估其性能。结果在高病毒载量样品中，PR/RT和INI的扩增灵敏度分别达到95.7% %和92.7 %，符合扩增灵敏度标准。然而，在2000和5000拷贝/mL之间的病毒载量较低的样本中，PR/RT和INI区域的敏感性分别降低了50% %和30% %。测定内和测定间的一致性超过99% %，突变检测与参考测定具有很强的相关性，鉴定出95% %以上的参考突变。结论TaqPath试剂盒符合WHO对HIVDR检测验证的接受标准，但在INI区域低病毒载量时，扩增敏感性低于要求的阈值。干血斑标本可与TaqPath试剂盒兼容，用于基线和治疗失败的hiv - dr基因分型，在资源受限和偏远地区提供可靠的血浆替代方案。

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引用次数: 0

A dual-target real-time PCR for proactive detection of Mpox variants 主动检测m痘变异的双目标实时PCR。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-01 Epub Date: 2025-09-05 DOI: 10.1016/j.jviromet.2025.115260

Tracy D. Lee , Alan O’Dwyer , Michael Chan , Branco Cheung , Frankie Tsang , John R. Tyson , Kathleen L. Kolehmainen , Natalie A. Prystajecky , Agatha N. Jassem

In 2022, cases of Monkeypox virus (MPXV) in California contained a mutation in the TNF receptor gene (GR2G) that rendered the virus undetectable using a widely adopted public health diagnostic qPCR assay. This underscored the need for a dual-target PCR approach and prompted validation of a second target by the BCCDC Public Health Laboratory. In addition to the GR2G target validated in the original qPCR assay (and duplexed with the endogenous target human β-globin (HBG)), GP113 (OPG128) was identified and validated using both clinical samples and MPXV DNA controls. Mutations in GR2G and GP113 (found in the emerging clade Ib) were also addressed along with the updated target. The new triplex assay (GR2G/GP113/HBG) had 100 % inclusivity and 100 % accuracy with all clinical samples tested and did not cross-react with herpes simplex virus-1 or −2, varicella zoster virus, or enterovirus. It showed < 5 % coefficient of variance between replicates and had a limit-of-detection of 10 copies/μL for GR2G and GP113. The use of two targets presents redundancy against further mutations in MPXV and is recommended for use with all viral qPCR assays moving forward.

2022年，加利福尼亚州的猴痘病毒（MPXV）病例包含TNF受体基因（GR2G）突变，这使得使用广泛采用的公共卫生诊断qPCR检测无法检测到该病毒。这强调了双靶点PCR方法的必要性，并促使BCCDC公共卫生实验室对第二个靶点进行验证。除了在最初的qPCR实验中验证的GR2G靶点（并与内源性靶点人β-珠蛋白（HBG）双活）外，GP113 （OPG128）在临床样本和MPXV DNA对照中被鉴定和验证。GR2G和GP113的突变（在新兴分支Ib中发现）也与更新的靶标一起得到了解决。新的三重检测（GR2G/GP113/HBG）对所有临床样本检测具有100%的包容性和100%的准确性，并且不会与单纯疱疹病毒-1或-2、水痘带状疱疹病毒或肠病毒发生交叉反应。它显示

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引用次数: 0

Comparative effects of three SARS-CoV-2 inactivation methods on cytokine detection using LEGENDplex™ bead-based immunoassays 三种SARS-CoV-2灭活方法对LEGENDplex™免疫检测细胞因子的比较效果

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-01 Epub Date: 2025-08-24 DOI: 10.1016/j.jviromet.2025.115244

Yifan Chen , Ting Zhang , Jie Zhang , Xixuan Dong , Lixiang Xue , Zhongnan Yin

In virology-related studies, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is routine to inactivate body fluid samples carrying the virus to reduce the spread of the virus and guarantee the safety of biobankers and researchers. However, inactivation treatments may affect the molecular structure of proteins in biological samples, and it is necessary to select an inactivation method that has the least impact on the target molecule associated with protein detection techniques. Cytometric Bead Array (CBA), a novel and powerful technology, allows the simultaneous quantification of up to 10–30 different soluble proteins from one sample, with a particular focus on various cytokines and chemokines in human body fluids. But only a few studies have investigated the effect of inactivation methods on relevant assays. Therefore, this study aims to investigate various viral inactivation methods and evaluate their impact on LEGENDplex™ bead-based immunoassays. By detecting eight plasma samples and eight ascites samples, we assessed the impacts of heat denaturation, γ-irradiation, and paraformaldehyde (PFA) inactivation methods on certain protein components in plasma and ascites by LEGENDplex™ bead-based immunoassays. The results showed that heat treatment and γ-irradiation treatment had little effect on LEGENDplex™ bead-based immunoassays in both blood and ascites, while PFA treatment resulted in changes in multiple cytokines and chemokines. Among twenty-six cytokines and chemokines, perforin, I-TAC (CXCL11), Eotaxin (CCL11), and MIP-3α (CCL20) were more vulnerable to heat denaturation, γ-irradiation, and PFA treatments. To sum up, our findings provide evidence to inform the selection of optimal inactivation methods for reliable cytokine profiling in SARS-CoV-2-infected samples.

在包括SARS-CoV-2在内的病毒学相关研究中，常规做法是对携带病毒的体液样本进行灭活，以减少病毒的传播，保证生物银行和研究人员的安全。然而，失活处理可能会影响生物样品中蛋白质的分子结构，与蛋白质检测技术相关，有必要选择对靶分子影响最小的失活方法。CBA是一种新颖而强大的技术，可以同时定量多达10-30种不同的可溶性蛋白质，特别关注人体体液中的各种细胞因子和趋化因子。但只有少数研究探讨了灭活方法对相关测定的影响。因此，本研究旨在研究各种病毒灭活方法，并评估它们对LEGENDplex™珠免疫测定的影响。通过检测8份血浆样品和8份腹水样品，我们采用LEGENDplex™珠免疫分析法，评估热变性、γ辐照和多聚甲醛（PFA）失活方法对血浆和腹水中某些蛋白质成分的影响。结果显示，热处理和γ辐照处理对血液和腹水中基于LEGENDplex™珠的免疫测定影响不大，而PFA处理导致多种细胞因子和趋化因子的变化。在26种细胞因子和趋化因子中，穿孔素、I-TAC （CXCL11）、Eotaxin （CCL11）和MIP-3α (CCL20)更容易受到热变性、γ辐照和PFA处理的影响。综上所述，我们的研究结果为选择最佳的失活方法提供了证据，以可靠地分析sars - cov -2感染样本的细胞因子。

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引用次数: 0

Duplex droplet digital PCR enables simultaneous quantification of algal giant virus DSLLAV1 and virophage DSLV8 in natural and laboratory samples 双液滴数字PCR能够同时定量天然和实验室样品中的藻类巨病毒DSLLAV1和噬菌体DSLV8。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-01 Epub Date: 2025-10-03 DOI: 10.1016/j.jviromet.2025.115273

Ting Chu , Jiabei Yu , Qinran Wang , Chen Hu , Lanming Chen , Yongxin Yu , Yongjie Wang

Cell–virus–virophage (CVv) systems involve virophages parasitizing giant viruses within eukaryotic hosts, forming unique virus–virus interactions with complex ecological implications. However, quantitative tools for studying such systems—particularly in freshwater algae—remain limited. In this study, we developed and optimized a duplex droplet digital PCR (ddPCR) assay to simultaneously detect and quantify Dishui Lake Large Algal Virus 1 (DSLLAV1), a Mimiviridae-like algal giant virus, and its associated Dishui Lake virophage 8 (DSLV8) in the Dishui Lake ecosystem. Target-specific primers and TaqMan probes were designed based on viral genomic sequences, and assay conditions were optimized for annealing temperature, primer/probe concentrations, and droplet separation. The established assay demonstrated high specificity and sensitivity, with detection limits of 0.13 and 0.16 copies/µL for DSLLAV1 and DSLV8, respectively. The method outperformed qPCR in sensitivity and maintained stability across environmental and infection-derived samples. This ddPCR method provides a robust platform for monitoring virus–virophage dynamics and offers new opportunities for investigating the ecological and evolutionary roles of CVv systems in aquatic environments.

细胞-病毒-病毒噬菌体（CVv）系统涉及病毒噬菌体寄生于真核宿主内的巨型病毒，形成具有复杂生态意义的独特病毒-病毒相互作用。然而，用于研究这类系统的定量工具——尤其是淡水藻类——仍然有限。本研究建立并优化了双液滴数字PCR （ddPCR）方法，用于同时检测和定量滴水湖生态系统中滴水湖大藻病毒1 （DSLLAV1）及其相关的滴水湖病毒噬菌体8 （DSLV8）。根据病毒基因组序列设计了特异引物和TaqMan探针，并对退火温度、引物/探针浓度和液滴分离等条件进行了优化。建立的检测方法具有较高的特异性和敏感性，DSLLAV1和DSLV8的检测限分别为0.13和0.16 copies/µL。该方法在敏感性上优于qPCR，并在环境和感染衍生样品中保持稳定性。这种ddPCR方法为监测病毒-噬菌体动力学提供了一个强大的平台，并为研究CVv系统在水生环境中的生态和进化作用提供了新的机会。

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引用次数: 0