Journal of virological methods最新文献_第4页

Screening of monoclonal vaccine strains based on real-time live-cell imaging technology 基于实时活细胞成像技术的单克隆疫苗株筛选

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-11-19 DOI: 10.1016/j.jviromet.2025.115305

Qiufang Huang , Tingting Zhao , Wenhao Su , Shishi Li , Jingjing Liu , Zihao Ge , Benyao Zhang , Xiuxiu Ren , Xiaohuan Zhang , Jiangbo Wei

The plaque purification is a critical step in the screening of traditional live-attenuated vaccines and recombinant viral vaccines, aiming to acquire vaccine clones with homogeneous characteristics and desirable immunogenicity to address outbreaks of emerging diseases such as monkeypox, chikungunya fever, and dengue fever. The traditional plaque purification process to screen out a vaccine strain with genetically consistent stability from a mixed pool of viral clones generally requires laborsome work. We utilized live-cell imaging technique enabling us to isolate monoclonal vaccine strains to simplify and improve the efficiency of this process. Here, we genetically engineered the vaccinia virus TianTan (VTT) using CRISPR/Cas9 system to generate recombinant VTT viruses (VTT-WS01-EGFP) that expressed enhanced green fluorescent protein (EGFP). Initially, we performed 9 rounds of plaque purification using traditional plaque assay, yielding 50 candidate clones. The Incucyte Live-Cell Imaging and Analysis system was subsequently performed to conduct a rigorous, high-resolution screening of these candidates in a more automated, sensitive and high-throughput way. Through this screening process, we ultimately obtained 31 pure viral clones that were free of parental strain contamination, followed by the analysis of plaque formation, fluorescent plaque size, and plaque morphology, and 11 candidate clones were selected for immunological evaluation. Furthermore, we found that clone 49 induced a relatively high titer of anti-VTT neutralizing antibodies and elicited the production of cross-reactive IgG against monkeypox virus antigens, thereby validating its potential as a candidate strain as a monkeypox virus vaccine. Taken together, our data demonstrates that live-cell imaging technique significantly accelerates the screening process for the isolation of monoclonal viral clones as recombinant viral vaccines, and holds considerable potential in attenuated strain selection as well as investigations into biological characteristics of viruses, including viral replication.

空斑纯化是筛选传统减毒活疫苗和重组病毒疫苗的关键步骤，旨在获得具有同质特性和理想免疫原性的疫苗克隆，以应对猴痘、基孔肯雅热和登革热等新发疾病的暴发。传统的空斑纯化过程从混合的病毒克隆池中筛选出具有遗传一致性稳定性的疫苗菌株，通常需要费力的工作。我们利用活细胞成像技术分离单克隆疫苗株，以简化和提高这一过程的效率。本研究利用CRISPR/Cas9系统对牛痘天坛病毒（VTT）进行基因工程改造，生成表达增强型绿色荧光蛋白（EGFP）的重组VTT病毒（VTT- ws01 -EGFP）。最初，我们使用传统的空斑法进行了9轮空斑纯化，产生了50个候选克隆。随后使用Incucyte活细胞成像和分析系统，以更加自动化、敏感和高通量的方式对候选细胞进行严格、高分辨率的筛选。通过这一筛选过程，我们最终获得了31个没有亲本株污染的纯病毒克隆，随后对斑块形成、荧光斑块大小和斑块形态进行了分析，并选择了11个候选克隆进行免疫学评价。此外，我们发现克隆49诱导了相对高滴度的抗vtt中和抗体，并引发了针对猴痘病毒抗原的交叉反应性IgG的产生，从而验证了其作为猴痘病毒疫苗候选株的潜力。综上所述，我们的数据表明，活细胞成像技术显著加快了单克隆病毒克隆作为重组病毒疫苗的筛选过程，并在减毒株选择以及病毒生物学特性研究（包括病毒复制）方面具有相当大的潜力。

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引用次数: 0

Metagenomic surveillance of emerging viruses in mosquito populations from high-risk regions of Iran 伊朗高危地区蚊群中新发病毒的宏基因组监测

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-11-17 DOI: 10.1016/j.jviromet.2025.115301

Ebrahim Abbasi

Background

Mosquito-borne arboviruses pose a growing threat to public health, particularly in ecologically vulnerable and climatically dynamic regions. This study aimed to investigate the diversity of emerging arboviruses in mosquito populations from high-risk provinces in southern and southeastern Iran using a metagenomic surveillance approach.

Methods

Adult mosquitoes were collected from 36 sites across Hormozgan, Sistan and Baluchestan, and Khuzestan provinces. Specimens were pooled by species and location, followed by RNA extraction and high-throughput sequencing. Bioinformatics analysis was performed to identify viral taxa and assess phylogenetic relationships.

Results

A total of 4275 mosquitoes representing six species were analyzed. Virome analysis revealed 43 viral taxa, including medically important arboviruses such as dengue virus serotype 2 (DENV-2), chikungunya virus (CHIKV), and West Nile virus (WNV). Multiple novel viral sequences were also detected, including putative members of Phenuiviridae and Orthomyxoviridae. Viral diversity was highest in Hormozgan province and positively correlated with ambient temperature.

Conclusion

This study provides the first comprehensive metagenomic insight into mosquito viromes in Iran, revealing both endemic and potentially novel arboviruses. These findings underscore the need for integrated genomic surveillance and regional vector-borne disease preparedness.

背景：蚊媒虫媒病毒对公众健康构成日益严重的威胁，特别是在生态脆弱和气候动态地区。本研究旨在利用宏基因组监测方法调查伊朗南部和东南部高风险省份蚊子种群中新发虫媒病毒的多样性。方法：在霍尔木兹甘省、锡斯坦省、俾路支斯坦省和胡齐斯坦省36个地点采集成蚊。标本按物种和地点汇总，然后进行RNA提取和高通量测序。进行生物信息学分析以确定病毒分类群并评估系统发育关系。结果：共捕获蚊虫6种4275只。病毒组分析揭示了43个病毒分类群，包括医学上重要的虫媒病毒，如2型登革热病毒（DENV-2）、基孔肯雅病毒（CHIKV）和西尼罗河病毒（WNV）。此外，还检测到多个新的病毒序列，包括疑似的苯病毒科和正粘病毒科成员。病毒多样性在霍尔木兹甘省最高，且与环境温度呈正相关。结论：本研究首次对伊朗蚊子病毒群进行了全面的宏基因组研究，揭示了地方性和潜在的新型虫媒病毒。这些发现强调了综合基因组监测和区域媒介传播疾病防范的必要性。

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引用次数: 0

Establishment of a nucleic acid detection method for foot-and-mouth disease virus serotype O utilizing RPA-CRISPR/Cas12a technology 利用RPA-CRISPR/Cas12a技术建立O型口蹄疫病毒核酸检测方法

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-11-17 DOI: 10.1016/j.jviromet.2025.115304

Zhen Zhong , Guoyan Li , Guanglin Liang , Tongwei Ren , Cuijing Teng , Jingpin Xiong , Guangqiang Ji , Min Zheng , Yan Pan , Yifeng Qin , Kang Ouyang , Yeshi Yin , Ying Chen , Weijian Huang , Zuzhang Wei

This study aimed to develop a rapid and visually interpretable nucleic acid detection assay for Foot-and-Mouth Disease Virus serotype O (FMDV-O) by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Specific RPA primers and CRISPR RNA (crRNA) sequences were designed and optimized based on the conserved 3D gene region of FMDV-O. An assay combining RPA pre-amplification with Cas12a-mediated cleavage was subsequently established. The sensitivity and specificity of the RPA-CRISPR/Cas12a method were systematically evaluated, and its diagnostic utility was further assessed using clinical samples. The results demonstrated that the primer set RPA-F1/R1 paired with crRNA1 constituted the optimal combination, with an ideal reaction system comprising 50 nM Cas12a protein and 200 nM crRNA. This system exhibited a detection limit of 2.60 × 10² copies/μL for target plasmid DNA following a 20-minute incubation at 37°C. Specificity analysis confirmed positive detection exclusively for FMDV-O plasmids, with no cross-reactivity observed with other tested pathogens. When applied to clinical samples, the proposed method demonstrated a superior detection rate relative to conventional PCR. In conclusion, a novel diagnostic platform for FMDV-O was successfully developed based on RPA-CRISPR/Cas12a. This method is characterized by its rapidity, operational simplicity, high sensitivity, and excellent specificity, holding significant promise for application in clinical diagnostics, epidemiological surveillance, and field-based testing.

本研究旨在将重组酶聚合酶扩增（RPA）技术与CRISPR/Cas12a技术相结合，建立一种快速、视觉可解释的口蹄疫病毒O型（FMDV-O）核酸检测方法。基于FMDV-O保守的3D基因区域，设计并优化特异性RPA引物和CRISPR RNA （crRNA）序列。随后建立了RPA预扩增与cas12a介导的裂解相结合的实验。系统评估RPA-CRISPR/Cas12a方法的敏感性和特异性，并通过临床样本进一步评估其诊断实用性。结果表明，引物组RPA-F1/R1与crRNA1配对是最优组合，理想反应体系为50nM Cas12a蛋白与200nM crRNA。该系统在37℃条件下培养20分钟后，靶质粒DNA的检出限为2.60 × 10²copies/μL。特异性分析证实仅对FMDV-O质粒呈阳性检测，未观察到与其他检测病原体的交叉反应。当应用于临床样品时，该方法相对于传统PCR具有更高的检出率。综上所述，基于RPA-CRISPR/Cas12a成功构建了新型FMDV-O诊断平台。该方法具有快速、操作简便、灵敏度高、特异性好等特点，在临床诊断、流行病学监测和现场检测等方面具有重要的应用前景。

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引用次数: 0

Development of a LAMP-based diagnostic method for HTLV-1 using Iranian clinical samples 利用伊朗临床样本建立基于lamp的HTLV-1诊断方法

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-11-16 DOI: 10.1016/j.jviromet.2025.115303

Yousef Douzandegan , Ali Kargar Kheirabad , Sayed‑Hamidreza Mozhgani , Gholamreza Tavoosidana , Abbas Rahimi Foroushani , Mehdi Norouzi

Background

Human T-lymphotropic virus type 1 (HTLV-1) is a delta-retrovirus responsible for severe diseases such as adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Accurate and accessible diagnostic tools are required, especially in endemic regions such as Iran, to manage reasonably and prevent HTLV-1 infections.

Objective

This study aimed to develop and validate an HTLV-1 detection protocol using loop-mediated isothermal amplification (LAMP) method on clinical samples from Iranian patients.

Methods

A LAMP assay was designed using optimized primers targeting the HTLV-1 genome. It was validated with clinical samples, and its sensitivity and specificity were evaluated compared to the polymerase chain reaction (PCR) method. Reaction conditions were optimized for fast and specific amplification, and results were visually analyzed utilizing a Luminator device and a real-time thermocycler.

Results

The LAMP assay revealed a sensitivity of 100 % and specificity of 94.7 % for HTLV-1 detection. Within 15–30 min, the method produced results in isothermal conditions with good performance. The fluorescence-based detection approach is easy to use. It does not need a sophisticated laboratory facility and has a limit of detection of 2.5 copies/µL.

Conclusion

This study demonstrates the feasibility of a LAMP-based method for HTLV-1 detection that is rapid, cost-effective, and accessible compared to PCR. For this reason, this alternative could enhance HTLV-1 diagnosis in both qualitative or quantitative form in endemic and low-resource scenarios. Future research should explore further validation in more diverse populations and integration with new technologies for digital detection.

背景：人类嗜t淋巴病毒1型（HTLV-1）是一种delta逆转录病毒，可导致成人t细胞白血病/淋巴瘤（ATL）和HTLV-1相关脊髓病/热带痉挛性截瘫（HAM/TSP）等严重疾病。需要准确和可获得的诊断工具，特别是在伊朗等流行地区，以便合理管理和预防HTLV-1感染。目的：本研究旨在建立并验证环介导等温扩增（LAMP）方法对伊朗患者临床样品的HTLV-1检测方案。方法：采用优化后的引物，设计针对HTLV-1基因组的LAMP检测方法。用临床样本进行验证，并与聚合酶链反应（PCR）法比较其敏感性和特异性。优化反应条件以实现快速特异扩增，并利用Luminator装置和实时热循环仪对扩增结果进行可视化分析。结果：LAMP法检测HTLV-1的灵敏度为100%，特异性为94.7%。该方法在等温条件下15-30min内生成结果，性能良好。基于荧光的检测方法易于使用。它不需要复杂的实验室设施，检测限为2.5拷贝/µL。结论：本研究证明了基于lamp的HTLV-1检测方法的可行性，与PCR相比，该方法快速，经济，易于获取。因此，在流行和资源匮乏的情况下，这种替代方法可以提高HTLV-1的定性或定量诊断。未来的研究应该探索在更多样化的人群中进一步验证，并与数字检测的新技术相结合。

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引用次数: 0

Design and development of a diagnostic method for HPV-16 virus with nanoball DNA based on vertical flow assay 基于垂直流式试验的纳米球DNA诊断HPV-16病毒方法的设计与开发。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-11-13 DOI: 10.1016/j.jviromet.2025.115300

Mansoreh Abdolhosseini , Mohammad H. Ghahremani , Moloud Absalan , Somayeh Jalilvand , Farshid Zandsalimi , Mohammad Panji , Mona Arianejad , Gholamreza Tavoosidana

The Human papillomavirus is one of the deadliest causes of cancer in women. This study aimed to design and develop a diagnostic method based on Rolling Circle Amplification (RCA) and vertical Flow Assay (VFA) for detection of HPV16. DNA nanoball synthesis was optimized using Phi29 DNA polymerase and probes. Nitrocellulose membranes were saturated with saline-sodium citrate buffer (SSC), and different probe concentrations were tested for optimal color production and intensity measurement. The results were checked with Image J software. Membrane blocking and washing buffer formulations were optimized to minimize background noise and increase test accuracy. Real-time PCR was used as a gold standard, confirming 39 HPV16-positive clinical samples. The assay was validated with positive HPV16 and HSV samples, and the sensitivity and specificity of the VFA-RCA method were determined. The limit of detection (LOD) for HPV16 was 0.5 ng, equivalent to 472 DNA copies/ml, with a sensitivity of 94 % and a specificity of 88 %. The RCA-VFA method offers a reliable, sensitive, and specific diagnostic approach for the early detection of HPV16, demonstrating potential to identify cancer at an early stage.

人类乳头瘤病毒是女性癌症最致命的原因之一。本研究旨在设计和建立一种基于滚动循环扩增（RCA）和垂直流动试验（VFA）的HPV16检测方法。利用Phi29 DNA聚合酶和探针优化了DNA纳米球的合成。用柠檬酸盐缓冲液（SSC）使硝化纤维素膜饱和，并对不同浓度的探针进行了最佳显色和强度测量。用Image J软件对结果进行核对。优化了膜阻断和洗涤缓冲配方，以最大限度地减少背景噪音，提高测试精度。以Real-time PCR为金标准，确认39例hpv16阳性临床样本。用HPV16和HSV阳性样品验证了该方法，并确定了VFA-RCA方法的敏感性和特异性。HPV16的检出限（LOD）为0.5ng，相当于472个DNA拷贝/ml，灵敏度为94%，特异性为88%。RCA-VFA方法为HPV16的早期检测提供了一种可靠、敏感和特异性的诊断方法，显示了在早期发现癌症的潜力。

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引用次数: 0

Development of monoclonal antibody based SPCE logistic regression model to predict the protective status of animals vaccinated against FMD virus type A 建立基于单克隆抗体的SPCE logistic回归模型预测动物接种口蹄疫A型病毒的保护状况。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-11-07 DOI: 10.1016/j.jviromet.2025.115299

Rajamanickam Hema Sayee , Madhusudan Hosamani , Narayanan Krishnaswamy , Subramaniyan Shanmuganathan , Manchikanti Sri Sai Charan , Dasu Ramadoss Vignesh , Veerakyathappa Bhanuprakash

In enzootic regions like India, foot-and-mouth disease (FMD) is managed through vaccination and strict biosecurity. Traditionally, FMD vaccine potency is assessed via in vivo challenge, which raises animal welfare concerns. As an alternative, this study evaluated a serology-based indirect potency test using 87 serum samples derived from calves 28 days post-vaccination. Samples were analyzed via virus neutralization test (VNT) and SPCEs employing polyclonal (pAb) and monoclonal antibodies (mAbs 2C4G11 and 6E8D11). Log₁₀ serum titres (ti) were correlated with protection outcomes from live virus challenge (FMDV serotype A/40/2000) using logistic regression. Four logit models were developed, and ROC analysis determined ti cut-offs for 50 %, 75 %, and 95 % protection probabilities (pi). VNT and mAb SPCEs showed sigmoid logit curves, indicating strong ti–pi correlation. Based on AIC and Somers’ D, mAb 2C4G11 SPCE was the best-fit model. At 75 % pi, ti thresholds for VNT, pAb, mAb 2C4G11, and mAb 6E8D11 were > 1.204, > 1.041, > 1.204, and > 1.204, respectively, with mAb 6E8D11 showing highest accuracy. Thus, mAbs 2C4G11 and 6E8D11 SPCEs are promising tools for predicting homologous protection. Further validation with larger sample sizes is recommended.

在印度等动物地方病流行地区，通过接种疫苗和严格的生物安全措施来控制口蹄疫。传统上，口蹄疫疫苗效力是通过体内挑战来评估的，这引起了对动物福利的关注。作为替代方案，本研究评估了基于血清学的间接效力测试，使用了接种后28天小牛的87份血清样本。采用多克隆抗体（pAb）和单克隆抗体（mAbs 2C4G11和6E8D11）对样品进行病毒中和试验（VNT）和spce分析。log₁0血清滴度（ti）与活病毒攻击（FMDV血清型A/40/2000）的保护结果相关。建立了四个logit模型，ROC分析确定了50%、75%和95%保护概率（pi）的截止值。VNT和mAb spce呈s形logit曲线，具有较强的ti-pi相关性。基于AIC和Somers' D， mAb 2C4G11 SPCE是最适合的模型。在75%的pi值下，VNT、pAb、mAb 2C4G11和mAb 6E8D11的阈值分别为>.204、>1.041、>1.204和>1.204，其中mAb 6E8D11的准确度最高。因此，单抗2C4G11和6E8D11 spce是预测同源保护的有效工具。建议使用更大的样本量进行进一步验证。

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引用次数: 0

Development of multiplex reverse-transcription digital PCR assay for co-detection of bovine leukemia virus and bovine viral diarrhea virus in cattle 牛白血病病毒和牛病毒性腹泻病毒多重反转录数字PCR联合检测方法的建立。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-11-06 DOI: 10.1016/j.jviromet.2025.115298

Shakir Ullah , Kosuke Notsu , Akatsuki Saito , Tamaki Okabayashi , Hirohisa Mekata , Mai Shiokawa , Hiroshi Aoki , Satoshi Sekiguchi

Bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) are important transboundary pathogens that cause substantial economic losses in the cattle industry globally. Their early detection and control are critical for preventing disease transmission and minimizing their impact on livestock health and productivity. Therefore, establishing a sensitive and robust diagnostic method capable of simultaneously detecting BLV and BVDV is vital for implementing timely control measures. Here, we developed a multiplex RT-dPCR assay to detect BLV and BVDV in a single-tube reaction using nucleic acid extracted from whole blood of infected cattle. The multiplex RT-dPCR assay successfully detected both BLV and BVDV with high specificity, exhibiting no cross-reactivity with other bovine viruses including Akabane virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, bovine herpesvirus 1, and bovine immunodeficiency virus. The assay demonstrated the ability to detect BVDV-1 and BVDV-2 at minimum titers of 10² and 10 ³ TCID₅₀/mL, respectively. For BLV, the multiplex RT-dPCR assay exhibited a detection limit as low as 18.7 viral copies. Our findings represent a significant advance in the detection of BLV and BVDV from extracted RNA and cDNA, highlighting the potential of assays for achieving improved diagnostic accuracy and early disease intervention.

牛白血病病毒（BLV）和牛病毒性腹泻病毒（BVDV）是重要的跨界病原体，对全球养牛业造成重大经济损失。它们的早期发现和控制对于预防疾病传播和尽量减少它们对牲畜健康和生产力的影响至关重要。因此，建立一种能够同时检测BLV和BVDV的灵敏且鲁棒的诊断方法对于及时实施控制措施至关重要。在这里，我们建立了一种多重RT-dPCR方法，利用从感染牛的全血中提取的核酸，在单管反应中检测BLV和BVDV。多重RT-dPCR试验成功地检测出BLV和BVDV具有高特异性，与其他牛病毒（包括Akabane病毒、牛冠状病毒、牛副流感病毒3、牛呼吸道合胞病毒、牛疱疹病毒1和牛免疫缺陷病毒）无交叉反应。该分析证明能够分别以最小滴度10²和10³TCID₅₀/mL检测BVDV-1和BVDV-2。对于BLV，多重RT-dPCR检测的检测限低至18.7个病毒拷贝。我们的研究结果代表了从提取的RNA和cDNA中检测BLV和BVDV的重大进展，突出了检测方法在提高诊断准确性和早期疾病干预方面的潜力。

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引用次数: 0

Development and evaluation of a molecular assay for the detection and quantification of SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus from municipal wastewater in British Columbia 不列颠哥伦比亚省城市污水中SARS-CoV-2、甲型流感、乙型流感和呼吸道合胞病毒分子检测方法的建立和评价

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-11-05 DOI: 10.1016/j.jviromet.2025.115297

Jennifer Kopetzky , Sarah C. Mansour , Rachel Floyd , Liam Byrne , Christine Tchao , Natalie Prystajecky

The ability to monitor the prevalence of respiratory viruses in a community depends on testing infrastructure, surveillance programs, and community engagement. Wastewater-based surveillance has recently evolved as a tool to monitor disease at a population level with reduced costs, diverse surveillance coverage, and inherently passive population participation. This study designed a molecular assay for the detection and quantification of multiple respiratory viruses—SARS-CoV-2, influenza A, influenza B, and Respiratory Syncytial Virus (RSV)—from wastewater samples. The presence of inhibitory substances in the wastewater matrix and multiple molecular targets did not prove to be limiting factors for the performance of the assay. Evaluation of the assay using municipal wastewater treatment plant samples showed a strong concordance with publicly available surveillance and clinical case data in British Columbia, Canada. Existing and future community testing programs for respiratory viruses should integrate wastewater-based surveillance to capture asymptomatic/non-testing populations, expand surveillance coverage, and provide greater insight into the community dynamics of respiratory viruses.

监测社区中呼吸道病毒流行的能力取决于检测基础设施、监测规划和社区参与。基于废水的监测最近已发展成为一种在人口层面监测疾病的工具，其成本较低，监测覆盖范围多样化，而且人口参与本身就是被动的。本研究设计了一种用于从废水样品中检测和定量多种呼吸道病毒（sars - cov -2、甲型流感、乙型流感和呼吸道合胞病毒（RSV））的分子分析方法。废水基质中存在的抑制物质和多个分子靶标并未被证明是测定性能的限制因素。使用城市污水处理厂样本进行的分析评估显示，与加拿大不列颠哥伦比亚省公开获得的监测和临床病例数据高度一致。现有和未来的社区呼吸道病毒检测规划应整合基于废水的监测，以捕获无症状/未检测人群，扩大监测覆盖范围，并更深入地了解呼吸道病毒的社区动态。

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引用次数: 0

Saliva samples in SARS-Cov-2 virus detection compared to the nasopharyngeal RT-PCR findings in individuals with suspected COVID-19 infection 唾液样本中SARS-Cov-2病毒检测结果与疑似COVID-19感染个体鼻咽RT-PCR结果的比较

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-11-03 DOI: 10.1016/j.jviromet.2025.115295

Anu E. Jääskeläinen , Anne Pitkäranta , Anu Haaramo , Johanna Nokso-Koivisto , Enni Sanmark

Accurate and cost-effective testing for SARS-CoV-2 is an ongoing need in public health. Nasopharyngeal swab (NPS) samples have been the benchmark as testing material but there are challenges connected to sample collection. We examined the usability of saliva as sample material for high-throughput laboratory molecular testing for SARS-CoV-2. Saliva samples from 108 individuals with suspected acute SARS-CoV-2 infection were collected and tested with cobas® SARS-CoV-2 and cobas® SARS-CoV-2 Duo tests (both Roche Molecular Diagnostics) to detect SARS-CoV-2 nucleic acids and compared to findings from NPS samples. Both cobas® tests performed well for saliva samples with 96 % positive percent agreement for both tests, 98 % negative percent agreement for cobas® SARS-CoV-2 test, and 100 % negative percent agreement for cobas® SARS-CoV-2 Duo test when compared to NPS samples. Saliva is a potential sample material for detecting SARS-CoV-2 infection in adult outpatients and can be considered as sample material to conserve health care resources.

对SARS-CoV-2进行准确和具有成本效益的检测是公共卫生领域的持续需求。鼻咽拭子（NPS）样本一直是测试材料的基准，但在样本收集方面存在挑战。我们研究了唾液作为高通量实验室SARS-CoV-2分子检测样本材料的可用性。收集108例疑似急性SARS-CoV-2感染患者的唾液样本，采用cobas®SARS-CoV-2和cobas®SARS-CoV-2双重检测（均为罗氏分子诊断公司）检测SARS-CoV-2核酸，并与NPS样本的结果进行比较。与NPS样本相比，两种cobas®测试在唾液样本中表现良好，两种测试的阳性率均为96%，cobas®SARS-CoV-2测试的阴性率为98%，cobas®SARS-CoV-2 Duo测试的阴性率为100%。唾液是检测成人门诊患者SARS-CoV-2感染的潜在样本材料，可作为节约医疗资源的样本材料。

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引用次数: 0

A split GFP approach to assay SARS-CoV-2 spike-dependent cell fusion 分裂GFP法检测SARS-CoV-2刺突依赖性细胞融合

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-11-01 DOI: 10.1016/j.jviromet.2025.115296

M. Jane Morwitzer, Ying Yi Zheng, Heather Friberg, Jeffrey R. Currier

The SARS-CoV-2 spike (S) protein plays a central role in viral entry through receptor binding and membrane fusion, making it a key target for therapeutic interventions. While existing assays for studying spike-mediated fusion can be complex and lack real-time monitoring, we present a split GFP-based cell fusion assay that provides a straightforward and adaptable platform for evaluating fusion dynamics. This assay utilizes a split GFP system in non-adherent 293-F cells to detect fusion events, allowing for reliable assessment of spike-targeting monoclonal antibodies and fusion inhibitors. Our study demonstrates the effectiveness of this approach in evaluating the inhibitory potential of therapeutics against multiple SARS-CoV-2 variants. The results highlight the assay’s ability to distinguish variant-specific fusion characteristics and inhibitor responses, particularly in the context of Omicron’s altered entry pathways. By enabling the quantitative, real-time assessment of spike-mediated fusion, this platform provides a valuable tool for therapeutic screening, supporting future efforts to develop antiviral strategies against SARS-CoV-2 and other emerging coronaviruses.

SARS-CoV-2刺突蛋白(S)在病毒通过受体结合和膜融合进入病毒中起着核心作用，使其成为治疗干预的关键靶点。虽然现有的研究尖峰介导的融合的方法可能很复杂，缺乏实时监测，但我们提出了一种基于gfp的分裂细胞融合实验，为评估融合动力学提供了一个简单且适应性强的平台。该试验利用非贴壁293-F细胞中的分裂GFP系统来检测融合事件，从而可以可靠地评估尖峰靶向单克隆抗体和融合抑制剂。我们的研究证明了这种方法在评估治疗方法对多种SARS-CoV-2变体的抑制潜力方面的有效性。结果强调了该检测方法区分变异特异性融合特征和抑制剂反应的能力，特别是在Omicron改变进入途径的背景下。通过对刺突介导的融合进行定量、实时评估，该平台为治疗筛选提供了有价值的工具，支持未来开发针对SARS-CoV-2和其他新兴冠状病毒的抗病毒策略。

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