Journal of virological methods最新文献

Exploring the potential therapeutic effects of ultradiluted Crotalus horridus on antibody-dependent enhancement in dengue virus pathogenesis 探讨超稀释牛角蒿对登革热病毒发病机制中抗体依赖性增强的潜在治疗作用

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-06-01 Epub Date: 2026-02-11 DOI: 10.1016/j.jviromet.2026.115365

Tanusree Ghorai , Avipsha Sarkar , Anirban Roy , Debadatta Nayak , Satadal Das

DENV remains a global health concern, with ADE playing a major role in severe disease progression. ADE leads to excessive cytokine release, nitric oxide overproduction, and elevated levels of non-structural-1 antigen, contributing to higher vascular dysfunction, thrombocytopenia, and hemorrhagic manifestations. The present study investigated the therapeutic effect of ultradiluted Crotalus horridus on ADE-mediated DENV infection using in vitro system. MTT, colony formation, cytopathic effect observation, ELISA, and qRT-PCR, were performed to assess cytotoxicity, viral pathogenesis, and immunomodulatory effects. Ultradiluted Crotalus horridus showed no cytotoxicity at lower concentrations and preserved cell viability. Therapeutic treatment altered the expression of pro-inflammatory and anti-inflammatory mediators in HepG2 cells infected with DENV under ADE conditions. Furthermore, nitric oxide levels and non-structural 1 antigen secretion were significantly decreased, indicating a reduction in viral replication and associated pathogenic immunomodulation. Our study also revealed that the expression of TLRs were significantly downregulated during ADE of DENV infection. Following treatment with ultradiluted Crotalus horridus, TLR expression was enhanced, which might have aggravated the immune response during ADE in DENV infection. In conclusion, these findings demonstrate that ultradiluted Crotalus horridus may act as a potential therapeutic drug to alleviate the severity associated with ADE-mediated infection.

DENV仍然是一个全球性的健康问题，ADE在严重疾病进展中起着重要作用。ADE导致细胞因子过度释放、一氧化氮过量产生和非结构-1抗原水平升高，导致血管功能障碍加重、血小板减少和出血表现。本研究利用体外系统研究了超稀释虎蹄草对ade介导的DENV感染的治疗作用。采用MTT、菌落形成、细胞病变效应观察、ELISA和qRT-PCR方法评估细胞毒性、病毒发病机制和免疫调节作用。超稀释后的红花在较低浓度下无细胞毒性，且保留了细胞活力。在ADE条件下，治疗改变了DENV感染的HepG2细胞中促炎和抗炎介质的表达。此外，一氧化氮水平和非结构1抗原分泌显著降低，表明病毒复制和相关的致病性免疫调节减少。我们的研究还发现，在DENV感染ADE期间，tlr的表达显著下调。经超稀释后的虎蹄草处理后，TLR表达增强，这可能加重了DENV感染ADE期间的免疫反应。总之，这些研究结果表明，超稀释的角足草可能作为一种潜在的治疗药物，以减轻与ade介导的感染相关的严重程度。

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引用次数: 0

Distribution of Lily symptomless virus in tissues of Lanzhou lily (Lilium davidii var. unicolor) by fluorescence in situ hybridisation 利用荧光原位杂交技术研究兰州百合组织中百合无症状病毒的分布。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-06-01 Epub Date: 2026-02-15 DOI: 10.1016/j.jviromet.2026.115367

Panpan Yang , Xuefei Xu , Jingfang Wei , Wenliang Zhang , Guangya Bian , Leifeng Xu , Guodong Zhou , Jun Ming

Lily symptomless virus (LSV) is one of the main viruses that endanger Lilium davidii var. unicolor, causing billions of losses to the lily planting industry every year. The distribution of viruses in plants is uneven and identifying the virus-free tissue regions in lily is crucial for obtaining virus-free resources through in vitro culture. In this study, Lilium davidii var. unicolor carrying LSV was used as the experimental material. RNA fluorescent probes were designed and prepared based on the genomic sequence of LSV. RNA in situ hybridization experiments were conducted on the meristematic tissues (root tips and stem tips), mature tissues (leaves, stem segments, pedicel), and flower organs (ovaries) to observe the distribution of LSV in different tissues. It was found that no fluorescence signal was detected in the apical meristematic tissue (0.65–0.8 mm × 0.2–0.25 mm) and pith cells of stem. Strong LSV signal expression was observed in the root tips, epidermis, cortex and vascular bundles of stem and pedicel, epidermis, palisade tissue, spongy tissue and vein vascular bundles of leaves, ovary and ovule. Due to the difficulty in the regeneration and differentiation of pith cells in stem segments, the apical meristem of Lilium davidii var. unicolor can be used as the starting material for obtaining virus-free resources through in vitro culture.

百合无症状病毒（Lily symptomless virus， LSV）是危害百合的主要病毒之一，每年给百合种植业造成数十亿美元的损失。病毒在植物体内的分布是不均匀的，鉴定百合的无病毒组织区域是通过离体培养获得无病毒资源的关键。本研究以携带LSV的百合为实验材料。基于LSV基因组序列设计制备了RNA荧光探针。对分生组织（根尖和茎尖）、成熟组织（叶、茎节、花梗）和花器官（子房）进行RNA原位杂交实验，观察LSV在不同组织中的分布。在茎的顶端分生组织（0.65 ~ 0.8mm × 0.2 ~ 0.25mm）和髓细胞中未检测到荧光信号。在茎和花梗的根尖、表皮、皮层和维管束、叶的表皮、栅栏组织、海绵组织和脉维管束、子房和胚珠中均有强烈的LSV信号表达。由于百合茎段髓细胞难以再生分化，单色百合的顶分生组织可作为离体培养获得脱毒资源的起始材料。

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引用次数: 0

Cytokine-based rapid process control of live virus production: MCP-1 as a cross-family biomarker in Vero cells 基于细胞因子的活病毒生产快速过程控制：MCP-1在Vero细胞中作为跨家族生物标志物

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-06-01 Epub Date: 2026-02-24 DOI: 10.1016/j.jviromet.2026.115380

Johanna Bacher , Jana Barbero Calzado , Mario Nebenführ , Robert Schlegl , Alois Jungbauer

Rapid determination of infectious virus titers is essential for efficient vaccine process development, yet conventional assays such as TCID50 and plaque assays require several days and limit throughput. Cytokine secretion represents a potential surrogate marker, enabling rapid detection of productive infection. Here, we evaluated cytokine secretion in Vero cells infected with live vaccine candidates from the Flaviviridae (Japanese encephalitis virus [JEV], yellow fever virus [YFV], Zika virus [ZIKV]) and Togaviridae (Chikungunya virus [CHIKV]) families. Cytokine profiling revealed consistent induction of MCP-1 across flavivirus infections, with RANTES detectable at lower intensity, while CHIKV infection suppressed cytokine release. MCP-1 secretion showed a strong quantitative correlation with infectious titers for JEV (R² = 0.86) and YFV (R² = 0.88), best described by logarithmic models at 18 and 24 h post infection. Additionally, MCP-1 dynamics reflected infection conditions, distinguishing medium and MOI effects in roller bottle production. These results establish MCP-1 as a rapid, cross-family biomarker of infectious virus replication and highlight its potential as a Process Analytical Technology (PAT) tool for automatable in-process monitoring and accelerated vaccine manufacturing.

快速测定传染性病毒滴度对于有效的疫苗工艺开发至关重要，然而传统的检测方法，如TCID50和空斑检测需要数天时间，并且限制了通量。细胞因子分泌是一种潜在的替代标志物，可以快速检测生产性感染。本研究评估了黄病毒科（日本脑炎病毒[JEV]、黄热病病毒[YFV]、寨卡病毒[ZIKV]）和托加病毒科（基孔肯雅病毒[CHIKV]）候选活疫苗感染Vero细胞后细胞因子的分泌情况。细胞因子分析显示，MCP-1在黄病毒感染中具有一致的诱导作用，在较低强度下可检测到RANTES，而CHIKV感染抑制细胞因子释放。MCP-1的分泌与乙脑病毒（R²= 0.86）和YFV （R²= 0.88）的感染滴度有很强的定量相关性，在感染后18和24小时用对数模型最能描述。此外，MCP-1动态反映了感染条件，区分介质和MOI在滚瓶生产中的影响。这些结果确立了MCP-1作为传染性病毒复制的快速、跨家族生物标志物，并突出了其作为过程分析技术（PAT）工具用于自动化过程监测和加速疫苗生产的潜力。

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引用次数: 0

Comparison of whole-viral genome sequencing and PCR-based assay for anal HPV detection to inform clinical implementation 全病毒基因组测序与pcr检测肛门HPV的比较，为临床实施提供依据。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-06-01 Epub Date: 2026-02-13 DOI: 10.1016/j.jviromet.2026.115366

Omar Harfouch , Derek MacMath , Poorani Subramanian , Rahwa Eyasu , Habib O. Ramadhani , Peiying Ye , Ashley Davis , Meredith Zoltick , Emade Ebah , Amay Shah , Amelia Cover , Phyllis Bijole , Rachel Silk , David Sternberg , Tina Liu , Grace Garrett , Miriam Jones , Randy Kier , Henry Masur , Shyamasundaran Kottilil , Andrea Lisco

To optimize anal cancer screening strategies, we compared two molecular platforms for anal high-risk Human Papillomavirus (HR-HPV) detection: whole-viral genome sequencing (WGS) and Cobas HPV-test-PCR (RC). Anal swabs from participants at high-risk for HPV acquisition were evaluated for cytological abnormalities and HR-HPV prevalence. Compared to WGS, RC had 51 % sensitivity and 98 % specificity for HPV16, 29 % and 92 % for HPV18, and 74 % and 93 % for non-16/18 HR-HPV. In a population with high prevalence of anal HR-HPV, RC had lower sensitivity in the detection of anal HR-HPV but was more robustly associated with abnormal anal cytology compared to WGS. In addition, WGS-based identification of HPV16 sub-lineages demonstrated that the composite A1, A2, A4, and C1 sub-lineages were associated with a higher risk of abnormal cytology compared to other HPV16 sub-lineages. WGS can further delineate the natural history of anal HR-HPV and their sub-lineages in populations at high-risk for HPV acquisition.

为了优化肛门癌筛查策略，我们比较了肛门高危人乳头瘤病毒（HR-HPV）检测的两种分子平台：全病毒基因组测序（WGS）和Cobas hpv -测试- pcr （RC）。对HPV感染高风险参与者的肛门拭子进行细胞学异常和HR-HPV患病率评估。与WGS相比，RC对HPV16的敏感性为51%，特异性为98%，对HPV18的敏感性为29%，特异性为92%，对非16/18 HR-HPV的敏感性为74%，特异性为93%。在肛门HR-HPV患病率高的人群中，RC对肛门HR-HPV检测的敏感性较低，但与WGS相比，RC与肛门细胞学异常的相关性更强。此外，基于wgs的HPV16亚系鉴定表明，与其他HPV16亚系相比，复合的A1、A2、A4和C1亚系与更高的细胞学异常风险相关。WGS可以进一步描述肛门HR-HPV的自然历史及其在HPV感染高危人群中的亚系。

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引用次数: 0

Detection of cell impurities and NF-κB activation using SEAP reporter cell lines in vaccines 用SEAP报告细胞系检测疫苗细胞杂质和NF-κB活化。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-06-01 Epub Date: 2026-02-18 DOI: 10.1016/j.jviromet.2026.115375

Sarah K. Mercier , Georgia Gilmayer , Jackson Williams , Jodie Latta , Pearl Bamford , Laura McMahon , Vidyani Manatunga , Anita Carscadden , Lisa Kerr

Introduction

During the 2010 influenza season, the Therapeutic Goods Administration (TGA) reviewed adverse event (AE) signals of febrile convulsions in children following the administration of the seasonal Fluvax® trivalent vaccine. These AEs were attributed to nuclear factor kappa-beta (NF-κB) activation. In response, the TGA developed a method to detect NF-κB activation by vaccine samples.

Methods

Complementary Toll-Like Receptor (TLR)-expressing reporter cell lines THP1-Blue and Ramos-Blue enabled detection of TLR1–9 activation using the QUANTI-Blue™ system following incubation with a panel of TLR agonists, tumour necrosis factor-alpha (TNF-α), or vaccine sample.

Results

Using TNF-α as a positive control and specific Toll-like receptor (TLR) agonists, specificity was confirmed for each cell line based on extracellular (THP1-Blue) or endosomal (Ramos-Blue) TLR expression. Agonists for each cell line demonstrated linearity within the working range, met precision criteria for repeatability and precision. The assay tested viral protein, messenger RNA (mRNA), and bacterial protein vaccines, met accuracy parameter of 80–120 % recovery of spiked controls.

Discussion

We established and validated a robust, sensitive and reliable analytical approach for detecting possible NF-κB activation by bioactive components in vaccines.

Interpretation

This assay enables manufacturers and regulators to meet the growing demand for reliable impurity screening assays that support the characterization of vaccines activating the NF-κB pathway. Future studies must further characterise NF-κB activation by individual TLRs across a broader range of vaccines.

在2010年流感季节，美国药品管理局（TGA）审查了接种季节性Fluvax®三价疫苗后儿童发热性惊厥的不良事件（AE）信号。这些ae归因于核因子κ b （NF-κB）活化。为此，TGA开发了一种检测疫苗样品中NF-κB活化的方法。方法：使用一组TLR激动剂、肿瘤坏死因子-α (TNF-α)或疫苗样品孵育后，使用QUANTI-Blue™系统检测TLR1-9的激活。结果：使用TNF-α作为阳性对照和特异性toll样受体（TLR）激动剂，基于细胞外（THP1-Blue）或内体（Ramos-Blue） TLR表达证实了每种细胞系的特异性。每种细胞系的激动剂在工作范围内呈线性，满足重复性和精密度的精度标准。该方法检测病毒蛋白、信使RNA （mRNA）和细菌蛋白疫苗，符合加标对照回收率80-120%的准确性参数。讨论：我们建立并验证了一种强大、灵敏和可靠的分析方法，用于检测疫苗中生物活性成分可能激活NF-κB。解释：该检测使制造商和监管机构能够满足日益增长的对可靠杂质筛选检测的需求，这些检测支持对活化NF-κB通路的疫苗进行表征。未来的研究必须在更广泛的疫苗范围内进一步表征单个tlr对NF-κB的激活。

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引用次数: 0

Development and application of truncated cap protein-based indirect ELISA for screening antibodies against porcine circovirus 3 基于截断帽蛋白间接ELISA法筛选猪圆环病毒3型抗体的建立与应用

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-06-01 Epub Date: 2026-02-18 DOI: 10.1016/j.jviromet.2026.115376

D. Dinesh , Sonalika Mahajan , P. Madhankumar , Monalisa Sahoo , Karikalan Mathesh , Gaurav Kumar Sharma , Amit Kumar , Triveni Dutt

Porcine circovirus type 3 (PCV3) is an emerging pathogen linked with reproductive failure, respiratory disease, dermatitis, nephropathy, and multisystemic inflammation, posing growing threats to the swine industry worldwide. Although molecular evidence of PCV3 circulation has been reported in India, there is no published information on its seroprevalence, leaving the extent of exposure and population immunity largely unknown. Serological tools are critical for epidemiological surveillance and monitoring vaccine responses; however, validated assays for PCV3 remain scarce. In the present study, we developed and evaluated an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of PCV3-specific antibodies, based on a recombinant truncated Cap protein expressed in Escherichia coli. The antigenic fragment of the Cap gene, excluding the N-terminal nuclear localization signal, was used as coating antigen and assay parameters like optimal antigen concentration, serum dilution, and conjugate conditions, were standardized. The developed ELISA exhibited high specificity, with no cross-reactivity to antisera against other common porcine viruses, and the relative diagnostic sensitivity and specificity were estimated to be 97.0 % and 94.5 %, respectively. The assay also demonstrated strong repeatability and reproducibility and was validated at four different laboratories with κ- values indicating perfect agreement Application of the assay to field sera revealed widespread seropositivity (64.25 %), underscoring its utility for epidemiological surveillance and sero-monitoring of PCV3. This study provides the first indigenous ELISA with potential application in India, and is the first report indicating widespread seropositivity of PCV3 in India.

猪圆环病毒3型（PCV3）是一种与生殖衰竭、呼吸系统疾病、皮炎、肾病和多系统炎症相关的新兴病原体，对全球养猪业构成越来越大的威胁。尽管在印度报告了PCV3病毒循环的分子证据，但没有关于其血清流行率的公开信息，因此暴露程度和人群免疫力在很大程度上未知。血清学工具对于流行病学监测和监测疫苗反应至关重要；然而，有效的PCV3检测方法仍然很少。在本研究中，我们开发并评估了一种间接酶联免疫吸附试验（ELISA），用于检测pcv3特异性抗体，该试验基于大肠杆菌中表达的重组截断Cap蛋白。将除去n端核定位信号的Cap基因抗原片段作为包被抗原，并对最佳抗原浓度、血清稀释度、偶联条件等检测参数进行标准化。所建立的ELISA具有较高的特异性，与其他常见猪病毒的抗血清无交叉反应，相对诊断敏感性和特异性分别为97.0%和94.5%。该方法还具有很强的重复性和再现性，并在四个不同的实验室进行了验证，κ-值表明完全一致。该方法在现场血清中显示广泛的血清阳性（64.25%），强调了其在流行病学监测和PCV3血清监测中的实用性。该研究为印度提供了首个具有潜在应用价值的本土ELISA，也是首个表明PCV3在印度广泛存在血清阳性的报告。

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引用次数: 0

Presentation of multiple copies of a non-dominant surface epitope by circular RNA effectively induce an immune response against SARS-CoV-2 环状RNA呈递非显性表面抗原表位的多个拷贝，可有效诱导针对SARS-CoV-2的免疫应答。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-06-01 Epub Date: 2026-02-20 DOI: 10.1016/j.jviromet.2026.115378

Mehrasa Asghari , Masoud Golalipour , Ali Memarian , Touraj Farazmandfar

Background

There is a pressing need for vaccine development platforms that are rapid, effective, and adaptable to emerging pathogens such as SARS-CoV-2. Circular RNA (circRNA) offers advantages in stability and non-immunogenicity over traditional mRNA approaches. We aimed to investigate the effect of increasing the amplification of a non-dominant surface epitope in the circRNA structure on the induction of an immune response to that epitope in a mouse model.

Methods

Two circRNA constructs containing one and ten copies of an epitope were designed using immunoinformatic methods, and their impact on immune responses was investigated. Immunogenicity in mice was assessed by antibody assays, virus neutralization assays, and cytokine analysis.

Results

The titer of anti-SARS-CoV-2 RBD-specific IgG antibody in mice treated with circRNA containing ten copies of the epitope was significantly higher than that in mice treated with circRNA containing a single copy of the epitope. The level of neutralizing antibodies in the circRNA containing ten copies of the epitope was also significantly higher than that in the single-copy group. In addition, the levels of cytokines IFN-γ, TNF-α, and IL-2 were significantly higher in the circRNA containing ten copies of the epitope than in the single copy group.

Conclusion

Combining copy number amplification of a candidate epitope with a circRNA platform could lead to a powerful construct for enhancing immune responses. This approach may offer significant potential for controlling emerging infectious diseases in the future.

背景：迫切需要针对SARS-CoV-2等新发病原体快速、有效、适应性强的疫苗开发平台。与传统的mRNA方法相比，环状RNA （circRNA）在稳定性和非免疫原性方面具有优势。我们的目的是在小鼠模型中研究增加circRNA结构中非显性表面表位的扩增对诱导对该表位的免疫反应的影响。方法：采用免疫信息学方法设计含有1个和10个表位拷贝的环状rna构建体，并研究其对免疫应答的影响。通过抗体试验、病毒中和试验和细胞因子分析来评估小鼠的免疫原性。结果：含有10个表位拷贝的circRNA处理小鼠的抗sars - cov -2 rbd特异性IgG抗体滴度显著高于含有单个表位拷贝的circRNA处理小鼠的滴度。在含有10个表位拷贝的circRNA中，中和抗体的水平也显著高于单拷贝组。此外，在含有10个表位拷贝的circRNA中，细胞因子IFN-γ、TNF-α和IL-2的水平显著高于单拷贝组。结论：将候选表位的拷贝数扩增与circRNA平台结合，可以构建一种增强免疫应答的强大结构。这种方法在未来可能为控制新出现的传染病提供巨大的潜力。

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引用次数: 0

A lyophilized stabilizer formulation enhances thermostability and preserves the immunogenicity of Senecavirus A post thermal challenge 一种冻干稳定剂配方提高了赛尼卡病毒A热激后的热稳定性和免疫原性。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-06-01 Epub Date: 2026-02-24 DOI: 10.1016/j.jviromet.2026.115379

Jiankun Huang, Huiying Zhou, Zhenru Hu, Fengshi Xie, Xiaobo Wen, Xuhua Ran

Senecavirus A (SVA) is a pathogenic virus that causes vesicular lesions, locomotor disorders, and neonatal mortality in pigs. Its increasing global prevalence poses a persistent threat to the swine industry. Vaccination remains the primary control strategy; however, SVA exhibits pronounced thermal instability, and exposure to unsuitable temperatures during storage or transport can impair vaccine immunogenicity. We developed a heat-stable lyoprotectant formulation for live SVA vaccines to overcome this limitation. The formulation, composed of 10% trehalose, 2% inulin, 2% dextran 40, 2% mannitol, 2% polyethylene glycol, 5% tryptone, 1% glycine, and 1% gelatin, demonstrated superior preservation during lyophilization and significantly greater thermal stability than conventional skim milk protectants. It exhibited no cytotoxicity at a 10% concentration and induced strong neutralizing antibody responses in mice, even after heat exposure. Collectively, this study establishes an effective lyoprotectant formulation that enhances the thermal stability and immunogenic integrity of live SVA, offering a practical advancement for improving vaccine quality and field efficacy.

塞内卡病毒A （SVA）是一种致病性病毒，可引起猪的水疱性病变、运动障碍和新生儿死亡。其日益增长的全球流行对养猪业构成了持续的威胁。疫苗接种仍然是主要的控制策略；然而，SVA表现出明显的热不稳定性，在储存或运输期间暴露在不合适的温度下会损害疫苗的免疫原性。为了克服这一限制，我们开发了一种热稳定的SVA活疫苗冻干保护剂配方。该配方由10%海藻糖、2%菊粉、2%葡聚糖40、2%甘露醇、2%聚乙二醇、5%色氨酸、1%甘氨酸和1%明胶组成，在冻干过程中表现出优越的保存性，并且比传统的脱脂牛奶保护剂具有更大的热稳定性。在10%的浓度下，它没有表现出细胞毒性，并在小鼠中诱导强烈的中和抗体反应，即使在热暴露后也是如此。总之，本研究建立了一种有效的冻干保护剂配方，提高了活SVA的热稳定性和免疫原性完整性，为提高疫苗质量和田间药效提供了实际的进展。

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引用次数: 0

Molecular evolutionary insights into the host-virus relationship between Marburg Virus and its bat reservoir 马尔堡病毒及其蝙蝠宿主-病毒关系的分子进化研究。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-06-01 Epub Date: 2026-02-19 DOI: 10.1016/j.jviromet.2026.115377

Nikita Kar , Srinjay Kumar Bharadaj , Stella Bharadaj , Supriyo Chakraborty

Bats, often silent carriers of lethal pathogens, serve as natural reservoirs for numerous viruses capable of crossing species barriers with devastating effects. Among them, Rousettus aegyptiacus (R. aegyptiacus) has emerged as a pivotal species in understanding viral spillovers. This fruit bat hosts the Marburg virus (MARV), supporting its replication and occasional transmission to humans, where it causes fatal hemorrhagic fever. However, a striking paradox remains while MARV infection is deadly in humans, R. aegyptiacus remains asymptomatic. Driven by this contrast, our study explores the codon usage patterns to reveal evolutionary mechanisms underlying viral adaptation and host tolerance. Far from being a passive carrier, R. aegyptiacus represents an insightful model for antiviral research. Our analysis revealed distinct codon usage patterns in MARV, indicating fine-tuned evolutionary alignment with the host’s codon preferences. MARV exhibited low codon bias, suggesting broad compatibility with the host’s translational machinery. Further, COA and PR2 analyses suggested that ecological factors shape codon selection in both virus and host. RNA modification analysis revealed frequent cytosine-to-thymine transitions, implying conserved mutational pressures or host-driven editing. Variations in RCDI values indicated deoptimized codon usage.

蝙蝠通常是致命病原体的无声携带者，是许多病毒的天然宿主，能够跨越物种障碍，造成破坏性影响。其中，埃及库蚊（Rousettus aegyptiacus， R. aegyptiacus）已成为了解病毒溢出的关键物种。这种果蝠是马尔堡病毒（MARV）的宿主，支持其复制并偶尔传播给人类，导致致命的出血热。然而，一个惊人的矛盾仍然存在，尽管人类感染MARV是致命的，埃及伊蚊仍然无症状。在这种对比的驱使下，我们的研究探索了密码子的使用模式，以揭示病毒适应和宿主耐受性的进化机制。埃及伊蚊远不是一个被动的携带者，它代表了抗病毒研究的一个有见地的模型。我们的分析揭示了MARV中不同的密码子使用模式，表明与宿主密码子偏好的微调进化一致。MARV表现出低密码子偏倚，表明与宿主的翻译机制具有广泛的兼容性。此外，COA和PR2分析表明，生态因素影响了病毒和宿主的密码子选择。RNA修饰分析揭示了频繁的胞嘧啶到胸腺嘧啶的转变，这意味着保守的突变压力或宿主驱动的编辑。RCDI值的变化表明密码子使用不优化。

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引用次数: 0

Integrated validation of internally controlled one-step multiplex real-time RT-PCR for dengue virus serotyping and monoplex assay for quantification 内控一步多重实时RT-PCR检测登革病毒血清分型和单组分定量分析的综合验证

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-06-01 Epub Date: 2026-03-02 DOI: 10.1016/j.jviromet.2026.115381

Le Thi Phuong Thuy , Vi Thuy Tran , Huynh Le Anh Huy , Tran Thai Hung , Nguyet Nguyen Minh , Ho Quang Chanh , Tran Bang Huyen , Nguyen Lam Vuong , Cameron P. Simmons , Duong Thi Hue Kien , Sophie Yacoub

Dengue is a major global health challenge, yet no commercially validated molecular kits currently provide both serotyping and viral load quantification in a single integrated format. To address this gap, we developed and internally validated a one-step multiplex real-time RT-PCR assay that simultaneously detects all four dengue virus (DENV) serotypes in a single reaction (5-PLEX), together with a complementary monoplex system for quantitative viral load measurement. The 5-PLEX assay demonstrated high specificity and reliable serotyping performance during acute and early critical phases of dengue infection (day of illness ≤4). Monoplex assays for each serotype showed strong linearity, precision, and low analytical limits of detection (3–10 copies/reaction), enabling accurate quantification. Based on these findings, we propose a diagnostic framework: 5-PLEX for samples collected within the first four days of illness, the previously validated 3-PLEX assay for samples collected from day 5 of illness onward, and monoplex assays for serotype-specific viral load measurement. Internal and external process controls were incorporated to support accuracy and reproducibility within the validated framework and to facilitate future multi-laboratory evaluation. This harmonized is designed to facilitate standardized implementation across laboratories and supports improved inter-laboratory consistency in clinical diagnostics, genomic surveillance, therapeutic trials, and pathogenesis research.

登革热是一项重大的全球卫生挑战，但目前尚无经商业验证的分子试剂盒以单一的综合格式提供血清分型和病毒载量定量。为了解决这一空白，我们开发并内部验证了一种一步多重实时RT-PCR检测方法，该方法可以在单一反应（5-PLEX）中同时检测所有四种登革热病毒（DENV）血清型，以及用于定量病毒载量测量的互补单一系统。在登革热感染的急性和早期关键阶段（发病天数≤4天），5-PLEX检测显示出高特异性和可靠的血清分型性能。每种血清型的单组分分析均显示出较强的线性、精密度和较低的检测限（3-10份/反应），能够实现准确的定量。基于这些发现，我们提出了一种诊断框架：发病前四天采集的样本采用5- plex检测，发病后第5天采集的样本采用先前验证的3-PLEX检测，血清型特异性病毒载量检测采用单效检测。纳入了内部和外部过程控制，以支持在验证框架内的准确性和可重复性，并促进未来的多实验室评估。该协调文件旨在促进跨实验室的标准化实施，并支持在临床诊断、基因组监测、治疗试验和发病机制研究方面提高实验室间的一致性。

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