Journal of virological methods最新文献_第4页

Experimental arboviral infection of mosquito larvae: A novel approach for vector competence studies 蚊子幼虫的虫媒病毒实验感染：病媒能力研究的新方法。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-07 DOI: 10.1016/j.jviromet.2024.115061

Christin Körsten, Mandy Schäfer

Vector competence studies in mosquitoes present valuable opportunities to explore arboviral transmission and virus-vector interactions. However, oral infection studies in mosquitoes can be challenging. An alternative approach is to infect mosquitoes during their aquatic larval stage, resulting in the emergence of infected adults. To investigate the potential of this method, Culex pipiens biotype molestus larvae were infected with Usutu virus (USUV, Orthoflavivirus usutuense). For this purpose, larvae were exposed to USUV-infected mammalian and mosquito cell cultures for 24 h before being reared to adults. Subsequent analysis via RT-qPCR revealed that the Culex larvae successfully acquired USUV from the infected cells and exhibited high susceptibility to infection. Immediately after emergence, 32.10 % (26/81) of male and 41.03 % (16/39) of female mosquitoes tested positive for USUV RNA. Notably, females that were incubated for 15 days post-emergence demonstrated even higher infection rates, reaching 100.00 % (23/23). In addition, viral RNA and infectious particles were detected in some saliva samples, indicating the potential for transmission. This experimental infection of mosquito larvae thus offers the opportunity to produce infected adult mosquitoes for studies enhancing our understanding of virus-vector interactions, co-infections, and transmission routes. Such research contributes to better public health strategies addressing arboviral diseases.

蚊子的病媒能力研究为探索虫媒病毒传播和病毒与病媒的相互作用提供了宝贵的机会。然而，蚊子口腔感染研究具有挑战性。另一种方法是在蚊子的水生幼虫阶段对其进行感染，从而产生受感染的成虫。为了研究这种方法的潜力，我们用乌苏图病毒（USUV，Orthoflavivirus usutuense）感染了库蚊（Culex pipiens biotype molestus）幼虫。为此，在将幼虫饲养成成虫之前，先将其暴露于受 USUV 感染的哺乳动物和蚊子细胞培养物中 24 小时。随后的 RT-qPCR 分析表明，库蚊幼虫成功地从受感染的细胞中获得了 USUV，并表现出对感染的高度敏感性。刚出生时，32.10%（26/81）的雄蚊和 41.03%（16/39）的雌蚊的 USUV RNA 检测呈阳性。值得注意的是，出壳后孵化 15 天的雌蚊感染率更高，达到 100.00 %（23/23）。此外，在一些唾液样本中检测到了病毒 RNA 和传染性颗粒，这表明病毒有传播的可能性。因此，对蚊子幼虫的这种实验性感染为产生受感染的成蚊提供了机会，以进行研究，加深我们对病毒-媒介相互作用、合并感染和传播途径的了解。这些研究有助于制定更好的公共卫生策略来应对虫媒病毒疾病。

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引用次数: 0

Development of a concentration method for simple and sensitive detection of SARS-CoV-2 in saliva using a magnetic nanoparticle kit 利用磁性纳米粒子试剂盒开发唾液中 SARS-CoV-2 简单灵敏检测浓度法。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-01 DOI: 10.1016/j.jviromet.2024.115059

Yasuko Yamazaki , Riku Tanaka , Gladys Castillo , Adrian Miki C. Macalanda , Melbourne R. Talactac , Wataru Yamazaki

Background

When diagnosing viral infections in humans and animals, the presence of virus in a sample in trace amounts that are below the analytical sensitivity of the detection system may cause false negative results and inaccurate diagnosis. We previously reported the development of a simple virion concentration technique using 12 ml large-volume samples that can dramatically improve diagnostic sensitivity by increasing analytical sensitivity by 100-fold over conventional methods. The present study was conducted to further improve the simplicity and versatility of this method. We constructed a simple and highly sensitive method for the detection of SARS-CoV-2 in human saliva after concentration using a magnetic nanoparticle conjugated with polyethylene glycol (PEG).

Results

Performance of the method was evaluated by comparing a combination of automated nucleic acid extraction and RT-qPCR or triplex RT-LAM detection in a spiked sample of 20 ml saliva collected from healthy humans. The method theoretically achieved 300-fold concentration of spiked SARS-CoV-2 in saliva, enabling 10- to 1000-fold higher analytical sensitivity for detection compared to conventional RNA extraction methods.

Conclusions

This newly developed method allows for easy and reliable concentration of the virion in less than 60 min, improving the analytical sensitivity of the SARS-CoV-2 test. Further, the method allows for easy and reliable enrichment of the virus in less than 60 min, improving the analytical sensitivity of the SARS-CoV-2 test. This method is easily used for highly sensitive virus detection from a variety of human oral fluid samples and may also be applied to rapid and labor-saving screening tests by pooling a large number of samples.

背景：在诊断人类和动物的病毒感染时，如果样本中存在低于检测系统分析灵敏度的痕量病毒，可能会导致假阴性结果和不准确的诊断。我们以前曾报道过利用 12 毫升大容量样本开发出一种简单的病毒浓缩技术，该技术可显著提高诊断灵敏度，与传统方法相比，其分析灵敏度提高了 100 倍。本研究旨在进一步提高该方法的简便性和通用性。我们利用与聚乙二醇（PEG）共轭的磁性纳米粒子构建了一种简便、高灵敏度的方法，用于浓缩后检测人体唾液中的 SARS-CoV-2 ：通过比较自动核酸提取与 RT-qPCR 或三重 RT-LAM 检测相结合的方法，对该方法的性能进行了评估。与传统的 RNA 提取方法相比，该方法理论上可使唾液中的 SARS-CoV-2 加标浓度提高 300 倍，使检测分析灵敏度提高 10 到 1000 倍：结论：这种新开发的方法可在 60 分钟内简便可靠地浓缩病毒，提高了 SARS-CoV-2 检测的分析灵敏度。此外，该方法可在 60 分钟内简便可靠地富集病毒，提高了 SARS-CoV-2 检测的分析灵敏度。该方法易于从各种人体口腔液样本中进行高灵敏度的病毒检测，也可通过汇集大量样本用于快速、省力的筛查试验。

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引用次数: 0

Indirect ELISA for analysis of malignant catarrhal fever virus-specific antibodies in a range of species 用于分析各种物种中恶性卡他热病毒特异性抗体的间接酶联免疫吸附试验。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-10-31 DOI: 10.1016/j.jviromet.2024.115060

George C. Russell, Ann Percival, Dawn M. Grant

The culture-attenuated alcelaphine herpesvirus 1 (AlHV-1) C500 strain can be grown to high titre and has been used successfully as a candidate vaccine for wildebeest-associated malignant catarrhal fever (MCF). This vaccine virus was also used to develop an indirect ELISA to allow monitoring of virus-specific antibodies in vaccinated cattle. However the extraction method was expensive and time-consuming, and the resulting test was not suitable for use in sheep. Here we describe an improved antigen extraction method that also broadens the application of the assay, allowing its application to sheep samples. The updated assay was tested using control samples from cattle and sheep, and showed a high level of accuracy in both species. This novel assay should prove to be a useful tool in MCF diagnosis and in evaluation of MCF vaccine responses.

经培养减毒的阿尔卡拉疱疹病毒 1 (AlHV-1) C500 株可生长至高滴度，并已成功用作野蜂相关恶性卡他热 (MCF) 的候选疫苗。这种疫苗病毒还被用于开发一种间接 ELISA 方法，以监测接种疫苗的牛体内的病毒特异性抗体。然而，这种提取方法既昂贵又费时，所得到的检测结果也不适合用于绵羊。在此，我们介绍了一种改进的抗原提取方法，该方法还拓宽了检测方法的应用范围，可用于绵羊样本。我们使用牛和羊的对照样本对更新后的检测方法进行了测试，结果表明该方法在这两个物种中的准确性都很高。这种新型检测方法将被证明是诊断 MCF 和评估 MCF 疫苗反应的有用工具。

引用次数: 0

Effects of chemokine-binding protein in visceral ovine aphthae on immune regulation response 内脏鹅口疮中的趋化因子结合蛋白对免疫调节反应的影响

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-10-30 DOI: 10.1016/j.jviromet.2024.115058

Fu Guowen , Li Rong , Zhang Ruixue, Zeng Qin, Yuan Jiarui, Liu Yabo, Fan Yueyuan

ORFV of the family poxvirus，which produces a pustular dermatitis both in humans and animals.,Previous studies have found an fatal case caused by the infection of ORFV in the viscera. However, the mechanisms of ORFV how to infect the viscera remain unknown. Our sequencing results revealed that the CBP of the visceral infection strain lacked a 24-base pair segment at position 217 comparison to the oral infection strain. Subsequently, we successfully packaged the recombinant adenoviruses pAd-CBP-K and pAd-CBP-N in HEK-293A cells and carried it to infect lymphocytes. RT-PCR analysis showed that the CBP protein was expressed in lymphocytes, and pAd-CBP-N group exhibited a significantly higher CBP expression level compared to the pAd-CBP-K group. At 4, 8, and 12 hours post-infection, both pAd-CBP-K and pAd-CBP-N were found to downregulate the expression of MIP-1 and CCL-5 in the supernatant of lymphocytes. However, the expression of IL-2, IL-6, IL-8, IL-12, INF-γ, and TNF-α showed a significant up-regulation. Furthermore, the inflammatory factors relative expression levels of IL-2, IL-6, IL-8, IL-8, IL-12, IFN-γ and TNF-α were significantly up-regulated in the both group. Interestingly, a significant increase in the expression of IL-6, IL-8 and TNF-α were detected in the pAd-CBP-N group at both 8 and 12 hours compared to pAd-CBP-N. Taken together, these findings showed that CBP can regulate the expression of free chemokines and activate the expression of inflammatory factors, and provide a basis for follow-up research.

ORFV是痘病毒科的一种病毒，在人类和动物身上都会产生脓疱性皮炎。然而，ORFV如何感染内脏的机制仍然未知。我们的测序结果显示，与口腔感染株相比，内脏感染株的 CBP 在 217 位缺少一个 24 碱基对片段。随后，我们成功地将重组腺病毒 pAd-CBP-K 和 pAd-CBP-N 包装到 HEK-293A 细胞中，并携带它感染淋巴细胞。RT-PCR分析表明，CBP蛋白在淋巴细胞中表达，pAd-CBP-N组的CBP表达水平明显高于pAd-CBP-K组。在感染后 4、8 和 12 小时，pAd-CBP-K 和 pAd-CBP-N 均能下调淋巴细胞上清液中 MIP-1 和 CCL-5 的表达。然而，IL-2、IL-6、IL-8、IL-12、INF-γ 和 TNF-α 的表达却出现了明显的上调。此外，IL-2、IL-6、IL-8、IL-8、IL-12、IFN-γ 和 TNF-α 的炎症因子相对表达水平在两组中均显著上调。有趣的是，与 pAd-CBP-N 组相比，pAd-CBP-N 组 IL-6、IL-8 和 TNF-α 的表达在 8 小时和 12 小时都有明显增加。综上所述，这些研究结果表明 CBP 可以调节游离趋化因子的表达，激活炎症因子的表达，为后续研究提供了依据。

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引用次数: 0

Performance of an envelope glycoprotein-based multiplex immunoassay for Ebola virus antibody detection in a cohort of Ebola virus disease survivors 基于包膜糖蛋白的多重免疫测定在一组埃博拉病毒病幸存者中检测埃博拉病毒抗体的性能。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-10-24 DOI: 10.1016/j.jviromet.2024.115057

McKenna D. Roe , Grace Hood , Spencer L. Sterling , Lianying Yan , Joseph Akoi Boré , Tom Tipton , Craig Thompson , Miles W. Carroll , Eric D. Laing

Serological surveillance in animal and human hosts can be a cost-effective strategy for orthoebolavirus detection, but is challenged by accurate estimates of seroprevalence, potential pauci-symptomatic disease presentation, and antigenic cross-reactivity. Here, we describe the use of an envelope glycoprotein (GP)-based multiplex microsphere immunoassay, consisting of nine filovirus GP antigens for the detection of anti-Ebola virus (EBOV) antibodies in a well-characterized cohort of Guinean Ebola virus disease (EVD) survivors and contacts from the 2013 – 2016 West African EVD outbreak. We examined sensitivity and specificity for the detection of anti-EBOV antibodies by GP expressed as recombinant trimeric ectodomains, yielding an assay performance of 95.96 % sensitivity and 98.61 % specificity. Additionally, agreement between the multiplex test and a whole virus ELISA and virus neutralization test showed strong correlations. The results demonstrate that this filovirus multiplex test is a sensitive tool for high-throughput serosurveillance

对动物和人类宿主进行血清学监测是一种具有成本效益的正粘病毒检测策略，但在准确估计血清流行率、潜在的无症状疾病表现和抗原交叉反应等方面面临挑战。在此，我们介绍了一种基于包膜糖蛋白（GP）的多重微球免疫测定方法，该方法由九种丝状病毒 GP 抗原组成，用于检测几内亚埃博拉病毒病（EVD）幸存者和 2013 - 2016 年西非埃博拉病毒病爆发接触者队列中的抗埃博拉病毒（EBOV）抗体。我们检测了以重组三聚体外显子形式表达的 GP 检测抗 EBOV 抗体的灵敏度和特异性，结果显示检测灵敏度为 95.96%，特异性为 98.61%。此外，多重检测与全病毒酶联免疫吸附和病毒中和检测之间的一致性显示出很强的相关性。结果表明，这种丝状病毒多重检验是高通量血清监测的灵敏工具。

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引用次数: 0

Preliminary survey of three mosquito-borne viruses using a self-established multiplex RT-qPCR assay in Chinese blood donors 利用自建的多重 RT-qPCR 分析法对中国献血者中的三种蚊媒病毒进行初步调查。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-10-20 DOI: 10.1016/j.jviromet.2024.115055

Xipeng Yan , Xinwei Wang , Jinlian Li , Bin Li , Baoren He , Linbin Huang , Jingheng Liang , Min Xu , Limin Chen

Background

Mosquito-borne pathogens pose a significant threat to both human health and blood safety. The primary mosquito-borne viruses that present this threat are Zika virus, Chikungunya virus, and Dengue virus. At present, there are limited efficacious vaccines or therapeutic drugs for the prevention and treatment of these viral infections. Blood donors can remain asymptomatically infected and unfortunately, screening for these three viruses in Chinese blood donors are not mandatory, leaving the residual risk to transfusion recipients uncertain. Objective: To address this, we developed a single-tube multiplex RT-qPCR assay for ZCD detection and was preliminarily employed to screen a total of 10,566 blood donations in Nanning Blood Center in order to assess the prevalence risk of these pathogens in blood donors. Results: None of the blood samples was reactive for ZCD by nucleic acid test (NAT). One out of 173 donations (1/173, 0.58 %) was IgG positive for ZIKV and 14 (14/173, 8.4 %) were IgG positive for DENV. None of these 173 donations was IgG positive for Chikungunya virus. These findings suggest that the prevalence of ZCD infection in blood donors in Nanning is very low although past DENV infection (IgG positive) was relatively common. Conclusion: A single-tube multiplex RT-qPCR assay for simultaneous detection of ZCD viruses was successfully established and applied for screening in blood donors. The residual risk of ZCD infection through transfusion is currently low in Nanning, China. The NAT assay for ZCD will serve as a technical reserve in response to future epidemic or pandemic of mosquito-borne pathogens.

背景：蚊子传播的病原体对人类健康和血液安全都构成重大威胁。造成这种威胁的主要蚊媒病毒是寨卡病毒、基孔肯雅病毒和登革热病毒。目前，预防和治疗这些病毒感染的有效疫苗或治疗药物十分有限。献血者可能无症状地受到感染，遗憾的是，中国献血者对这三种病毒的筛查并非强制性的，这使得输血受体的残余风险不确定：针对这一问题，我们开发了一种用于检测 ZCD 的单管多重 RT-qPCR 检测方法，并在南宁血液中心初步使用该方法对 10,566 份献血者血液进行了筛查，以评估这些病原体在献血者中的流行风险：结果：通过核酸检测（NAT），没有一份血样对ZCD有反应。173 例献血者中有 1 例（1/173，0.58%）ZIKV IgG 阳性，14 例（14/173，8.4%）DENV IgG 阳性。这 173 份捐赠中没有一份对基孔肯雅病毒 IgG 呈阳性。这些结果表明，南宁献血者中ZCD感染率很低，但过去的DENV感染（IgG阳性）相对常见：结论：成功建立并应用单管多重 RT-qPCR 法同时检测 ZCD 病毒，对献血者进行筛查。目前，中国南宁通过输血感染 ZCD 的残余风险较低。针对 ZCD 的 NAT 检测方法将作为应对未来蚊媒病原体流行或大流行的技术储备。

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引用次数: 0

Adjusting susceptibilities of C57BL/6 mice to orthoflaviviruses for evaluation of antiviral drugs by altering the levels of interferon alpha/beta receptor function 通过改变干扰素α/β受体功能水平，调整C57BL/6小鼠对正黄病毒的敏感性，以评估抗病毒药物。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-10-18 DOI: 10.1016/j.jviromet.2024.115053

John D. Morrey, Venkatraman Siddharthan

The purpose of this study was to optimize the infectivity of four different orthoflaviviruses in mice for evaluating antiviral drugs by using wild-type mice with intact interferon responses, type 1 interferon alpha/beta receptor knockout mice, or by injecting wild type C57BL/6 mice with varying doses of anti-type 1 interferon receptor antibody (MAR1-5A3) to optimize the infectivity and lethality. West Nile virus productively infected wild-type C57BL/6 mice to cause lethality, whereas Usutu virus required a complete absence of type 1 interferon receptor function. Deer tick virus (lineage 2 Powassan virus) and Japanese encephalitis viruses required a dampening of type 1 interferon responses by adjusting the doses of MAR1-5A3 antibody injections. Challenge dose-responsive mortality, weight loss, and viral titers of these two viruses were observed if the type 1 interferon responses were dampened with MAR1-5A3. Conversely, without MAR1-5A3 injections, these disease phenotypes were not viral challenge dose-responsive. From these different interferon-responsive models, the appropriate lethality was identified to determine that 7-deaza-2’-C-methyladenosine has high efficacy for West Nile and Usutu viruses, and low efficacy for deer tick and Japanese encephalitis viruses.

本研究的目的是通过使用具有完整干扰素反应的野生型小鼠、1型干扰素α/β受体敲除小鼠或向野生型C57BL/6小鼠注射不同剂量的抗1型干扰素受体抗体（MAR1-5A3）来优化四种不同的正黄病毒在小鼠体内的感染性，以评估抗病毒药物。西尼罗河病毒能有效感染野生型 C57BL/6 小鼠并导致致死，而乌苏图病毒则要求 1 型干扰素受体功能完全缺失。鹿蜱病毒（2系Powassan病毒）和日本脑炎病毒需要通过调整MAR1-5A3抗体注射剂量来抑制1型干扰素反应。如果用 MAR1-5A3 抑制 1 型干扰素反应，就能观察到这两种病毒的挑战剂量反应死亡率、体重减轻和病毒滴度。相反，如果不注射 MAR1-5A3，这些疾病表型则不具有病毒挑战剂量反应性。通过这些不同的干扰素反应模型，确定了适当的致死率，从而确定 7-脱氮-2'-C-甲基腺苷对西尼罗河病毒和乌苏图病毒有很高的疗效，而对鹿蜱病毒和日本脑炎病毒的疗效较低。

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引用次数: 0

One-step TaqMan® RT-qPCR detection of sugar beet-infecting poleroviruses in Myzus persicae from yellow water pan traps opens up new possibilities for early risk assessment of virus yellows disease 一步式 TaqMan® RT-qPCR 检测黄水盘诱捕器中甜菜瘤蚜感染的多角体病毒，为病毒黄化病的早期风险评估提供了新的可能性。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-10-16 DOI: 10.1016/j.jviromet.2024.115052

Simon Borgolte , Wulf Menzel , Mark Varrelmann

Virus yellows disease (VY) is a major threat to sugar beet production in Europe. Beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV) are of particular economic importance and are both persistently transmitted by the aphid vector Myzus persicae. As part of integrated pest management strategies, M. persicae influx into sugar beet fields is recorded weekly using yellow water pan traps. To date, only ELISA and RT-PCR assays have been described for BChV and BMYV detection in individual aphids. In this study, we describe for the first time two one-step TaqMan® RT-qPCR assays designed for the specific detection of BChV and BMYV in M. persicae after 7d incubation in water pan trap medium. Both viruses were reproducibly detected in individual aphids. After 7d incubation in trap medium, both viruses were reproducibly detected in individual aphids, as well as in one viruliferous aphid in a pool of 99 non-viruliferous aphids. Significant correlations can be shown between different mixing ratios of viruliferous to non-viruliferous aphids and Ct values of total RNA templates, allowing the percentage of viruliferous aphids in yellow water pan traps to be estimated using a standard curve. The described methodology provides a high sensitivity combined with a high sample throughput and can be used, after evaluation in the field, for practical monitoring, risk modelling and development of decision support systems for VY.

病毒性黄化病（VY）是欧洲甜菜生产的主要威胁。甜菜萎黄病病毒（BChV）和甜菜轻度黄化病毒（BMYV）具有特别重要的经济意义，这两种病毒都是由蚜虫载体持久蚜（Myzus persicae）传播的。作为虫害综合防治战略的一部分，每周都会使用黄水盘诱捕器记录甜菜田中的蚜虫数量。迄今为止，只有 ELISA 和 RT-PCR 检测法可用于检测单个蚜虫体内的 BChV 和 BMYV。在本研究中，我们首次介绍了两种一步式 TaqMan® RT-qPCR 检测方法，这两种方法设计用于在水盘诱捕培养基中培养 7 天后特异性检测柿蚜中的 BChV 和 BMYV。在单个蚜虫体内均可重复检测到这两种病毒。在诱捕培养基中培养 7 天后，在单个蚜虫以及 99 个无病毒蚜虫池中的一个带毒蚜虫体内都能重复检测到这两种病毒。带病毒蚜虫和不带病毒蚜虫的不同混合比例与总 RNA 模板的 Ct 值之间存在显著的相关性，因此可以利用标准曲线估算黄水盘诱捕器中带病毒蚜虫的百分比。所述方法灵敏度高、样品处理量大，经过实地评估后，可用于实际监测、风险建模和开发 VY 决策支持系统。

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引用次数: 0

Analysis of the antigenic and immunogenic properties of the native rabies virus glycoprotein purified by Lens culinaris lectin affinity chromatography 用 Lens culinaris 凝集素亲和层析法纯化的原生狂犬病病毒糖蛋白的抗原性和免疫原性分析。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-10-14 DOI: 10.1016/j.jviromet.2024.115044

Cíntia Pinto da Silva, Talita Gonçalves Aires de Queiroz, Keila Iamamoto Nogi, Iana Suly Santos Katz, Fernanda Guedes, Elaine Raniero Fernandes, Karina Ribeiro Silva, Sandriana Ramos Silva

Rabies virus glycoprotein (RABV-G) is responsible for the recognition of specific cell surface receptors and induces the production of neutralizing antibodies (VNA). Since RABV-G is a glycoprotein, this work aimed to evaluate Lens culinaris (LCA) chromatography as a simple and effective purification method. The purity and identification of the protein obtained were analyzed by SDS-PAGE, ELISA and lectin-binding assay. The antigenic properties of the purified RABV-G were evaluated by direct ELISA using human serum samples from individuals who had received rabies pre-exposure vaccination. For the immunogenicity study, Swiss Webster mice were immunized with purified RABV-G and the specific antibodies were measured by direct ELISA and RFFIT. As results, it was observed that the purified protein reveled a molecular mass of 55 kDa and the presence of carbohydrate; additionally, it was recognized by anti-rabies virus glycoprotein monoclonal antibody. Purified RABV-G induced high VNA titers (>50.0 IU/ml) in vivo, as detected by RFFIT, as well as RABV-G specific IgG1 (0.8 mean OD±SD) and IgG2a (0.3 mean OD±SD) antibodies, with a predominance of IgG1 (p< 0.001). In addition, it was observed that RABV-G was efficient in selectively detecting anti- RABV-G IgG in the sera of vaccinated individuals compared to the negative control. Therefore, LCA chromatography was efficient in preserving the native properties of RABV-G that are essential in inducing an adequate humoral immune response. In addition, the purified RABV-G presented analytical potential as an ELISA reagent.

狂犬病毒糖蛋白（RABV-G）负责识别特定的细胞表面受体并诱导产生中和抗体（VNA）。由于 RABV-G 是一种糖蛋白，本研究旨在评估 Lens culinaris（LCA）色谱法是否是一种简单有效的纯化方法。通过 SDS-PAGE、ELISA 和凝集素结合试验分析了所获得蛋白质的纯度和鉴定。使用接种过狂犬病暴露前疫苗的人血清样本，通过直接 ELISA 方法评估了纯化的 RABV-G 的抗原特性。在免疫原性研究中，用纯化的 RABV-G 对瑞士韦伯斯特小鼠进行免疫，并通过直接 ELISA 和 RFFIT 测定特异性抗体。结果显示，纯化蛋白的分子质量为 55 KDa，且含有碳水化合物；此外，抗狂犬病毒糖蛋白单克隆抗体也能识别该蛋白。通过 RFFIT 检测，纯化的 RABV-G 在体内诱导出高 VNA 滴度（>50.0 IU/ml），以及 RABV-G 特异性 IgG1（0.8 平均 OD±SD）和 IgG2a（0.3 平均 OD±SD）抗体，其中 IgG1 占主导地位（p< 0.001）。此外，与阴性对照相比，RABV-G 能有效地选择性检测接种者血清中的抗 RABV-G IgG。因此，LCA 色谱法能有效保留 RABV-G 的原生特性，而这些特性对于诱导适当的体液免疫反应至关重要。此外，纯化的 RABV-G 还具有作为 ELISA 试剂的分析潜力。

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引用次数: 0

Infectivity titers and aggregation states of intracellular and extracellular nervous necrosis virus in cell lines with cytolytic and persistent infections 细胞溶解性和持续性感染细胞系中细胞内和细胞外神经坏死病毒的感染滴度和聚集状态。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-10-10 DOI: 10.1016/j.jviromet.2024.115043

Han Sol Lee , Toyohiko Nishizawa

Nervous necrosis virus (NNV) in the genus Betanodavirus (Nodaviridae) is highly lethal to a wide range of fish species. Although striped snakehead (SSN-1) cell lines have been widely used for culturing NNV, cell lines persistently infected (PI) with NNV have only recently been established. This study investigated the infectivity titers of intracellular and extracellular NNV and the associated aggregation states. The intracellular NNV infectious doses were higher than those of extracellular NNV, irrespective of the cell lines. In SSN-1 cells, the intracellular-to-extracellular-NNV ratio was approximately 50–60-fold on days 1 and 2 after NNV infection, although it decreased following the onset of the cytopathic effect (CPE), reaching 3.5-fold on day 4. In the PI-cell lines, both the intracellular and extracellular NNV were in a nearly monomeric state. While the extracellular NNV were in a monomeric state in the SSN-1 cells, more than 92 % of the intracellular virus were in an aggregated state. When the NNV accumulated intracellularly at a median tissue culture infectious dose (TCID₅₀)/cell of 10⁴ to 10^4.5, SSN-1 cells appeared to exhibit CPE and eventually died. We believe that the aggregates of intracellularly accumulated NNV particles may be related to the cellular CPE onset and/or cell death.

神经坏死病毒（NNV）属 Betanodavirus（Nodaviridae），对多种鱼类具有高度致命性。虽然条纹乌鳢（SSN-1）细胞系已被广泛用于培养 NNV，但持续感染（PI）NNV 的细胞系最近才建立起来。本研究调查了细胞内和细胞外 NNV 的感染滴度以及相关的聚集状态。无论哪种细胞系，细胞内 NNV 感染剂量都高于细胞外 NNV 感染剂量。在 SSN-1 细胞中，细胞内 NNV 与细胞外 NNV 的比率在 NNV 感染后的第 1 天和第 2 天约为 50-60 倍，但在细胞病理效应（CPE）开始后有所下降，在第 4 天达到 3.5 倍。在 PI 细胞系中，细胞内和细胞外的 NNV 几乎都处于单体状态。在 SSN-1 细胞中，细胞外的 NNV 处于单体状态，而细胞内则有 92% 以上的病毒处于聚集状态。当 NNV 在细胞内聚集的中位组织培养感染剂量（TCID50）为 104 至 104.5 时，SSN-1 细胞出现 CPE 并最终死亡。我们认为，细胞内积累的 NNV 粒子聚集体可能与细胞 CPE 的发生和/或细胞死亡有关。

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