病毒传播和基于细胞的比色定量
Summary
本方案描述了寨卡病毒（ZIKV）在Vero非洲绿猴肾细胞中的繁殖，以及使用基于细胞的比色免疫检测方法以24孔和96孔（高通量）形式对ZIKV进行定量。
Abstract
寨卡病毒（ZIKV）是一种蚊媒病毒，属于 黄病毒属。ZIKV感染与先天性脑部异常有关，并可能与成人的吉兰-巴雷综合征有关。研究寨卡病毒以了解疾病机制对于促进疫苗和治疗开发非常重要。在病毒学领域，量化病毒的方法至关重要。焦点形成测定 （FFA） 是一种病毒定量测定，它使用抗体检测病毒抗原并使用过氧化物酶免疫染色技术鉴定细胞的感染病灶。目前的研究描述了使用 24 孔和 96 孔（高通量）格式的病毒传播和定量方案。与其他类似研究相比，该协议进一步描述了病灶大小优化，可以作为扩大该测定对其他病毒使用范围的指南。首先，ZIKV在Vero细胞中繁殖3天。使用FFA收获并定量含有ZIKV的培养上清液。简而言之，将病毒培养物接种到Vero细胞上并孵育2-3天。然后，在优化的染色过程（包括细胞固定、透化、封闭、抗体结合和与过氧化物酶底物孵育）后确定病灶形成。使用体视显微镜（24孔格式的手动计数）或软件分析仪（96孔格式的自动计数）观察染色的病毒病灶。FFA 提供可重复的、相对较快的结果（3-4 天），适用于不同的病毒，包括非斑块形成病毒。随后，该方案可用于ZIKV感染的研究，并可用于检测其他临床上重要的病毒。
Introduction
寨卡病毒（ZIKV）感染是一种新出现的蚊媒病毒性疾病。1947 年在乌干达首次分离出 ZIKV 1,2;从 1947 年到 2007 年，它仍然被忽视，因为临床症状最常见的是无症状的，其特征是自限性发热性疾病。2007 年，寨卡病毒流行始于雅浦群岛 3,4，随后于 2013 年至 2014 年在太平洋地区（法属波利尼西亚、复活节岛、库克群岛和新喀里多尼亚）发生更大规模的流行病 5,6,7,8，其中首次在成人中报告了严重的神经系统并发症吉兰-巴雷综合征 （GBS）9.在2015年和2016年期间，早在2013年巴西出现亚洲寨卡病毒基因型后，寨卡病毒首次大范围流行席卷美洲10。在这次疫情期间，新生儿中报告了44万至130万例小头畸形和其他神经系统疾病病例11。目前尚无针对寨卡病毒感染的特异性治愈或治疗方法;因此，迫切需要能够预防感染的寨卡病毒疫苗，特别是在怀孕期间。
病毒定量是确定样品中存在的病毒数量的过程。它在研究中发挥着重要作用，学术实验室涉及许多领域，例如医学和生命科学。这一过程在商业领域也很重要，例如病毒疫苗、重组蛋白、病毒抗原或抗病毒药物的生产。许多方法或检测方法可用于病毒定量12。方法或检测方法的选择通常取决于病毒特征、所需的准确度以及实验或研究的性质。通常，定量病毒的方法可分为两类：检测病毒核酸（DNA或RNA）存在的分子测定和体外测量病毒感染性的测定12。定量聚合酶链反应（qPCR，用于DNA）或定量逆转录聚合酶链反应（qRT-PCR，用于RNA）13和数字液滴PCR14是用于定量给定样品中病毒核酸的常用分子技术的例子15。然而，这些高度灵敏的分子技术无法区分活病毒和非活病毒15。因此，使用上述分子技术无法完成需要生物学特征信息的研究，例如病毒对细胞的感染性;需要能够测量和确定活病毒存在的检测方法。测量病毒传染性的检测方法包括斑块形成试验 （PFA）、50% 组织培养感染剂量 （TCID50）、荧光焦点试验和透射电子显微镜 （TEM）12。
PFA 由 Renato Dulbecco 于 1952 年开发，是最常用的病毒滴定方法之一，包括 ZIKV16。它用于直接测定传染性裂解病毒粒子的病毒浓度。该方法基于裂解病毒在病毒感染后在接种的细胞单层中产生细胞病变效应（CPEs;细胞死亡区或斑块，被未感染细胞包围的感染区域）的能力。然而，该测定存在一些影响其效用的缺点。该检测非常耗时（大约需要 7-10 天，具体取决于病毒），依赖于 CPE，并且容易出错。在本研究中，我们报告了一种免疫比色技术，即焦点形成测定 （FFA），用于检测和定量 24 孔板和 96 孔板形式的 ZIKV。
Protocol
Representative Results
Discussion
有几种测定方法可以确定病毒滴度;PFA 具有与 FFA 相似的病毒定量方案，其中病毒接种物被稀释以区分单个斑块或病灶。染色后，每个斑块或病灶指示接种物19中的单个感染性颗粒。PFA用结晶紫染色，以可视化由细胞裂解或死亡引起的斑块形成。因此，PFA更耗时，因为病毒引起CPE需要更长的时间，并且仅限于诱导细胞裂解或死亡的病毒。许多实验室已成功使用 FFA 测定黄病毒的传?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了马来西亚高等教育部的长期研究资助计划（LRGS MRUN Phase 1： LRGS MRUN/F1/01/2018）的支持，并为高等院校卓越中心（HICoE）计划（MO002-2019）提供资金。本研究中的图 3 显示了病灶形成测定的染色工作流程，改编自 BioRender.com （2022） 的“DAB 免疫组织化学”。取自 https://app.biorender.com/biorender-templates/t-5f3edb2eb20ace00af8faed9-dab-immunohistochemistry。
Materials
| 0.22 µm Polyethersulfone syringe filter | Sartorius | S6534-FMOSK | |
| 1.5 mL microcentrifuge tube | Nest | 615601 | |
| 10 mL sterile serological pipette | Labserv | 14955156 | |
| 1x Dulbecco’s phosphate-buffered saline (dPBS) | Gibco | 14190-136 | |
| 2.0 mL Screw cap tube | Axygen | SCT-200-SS-C-S | |
| 24-well plate | Corning | 3526 | |
| 25 mL Sterile serological pipettes | Labserv | 14955157 | |
| 3,3'Diaminobenzidine (DAB) peroxidase substrate | Thermo Scientific | 34065 | |
| 37 °C incubator with 5% CO2 | Sanyo | MCO-18AIC | |
| 5 mL sterile serological pipette | Labserv | 14955155 | |
| 50 mL centrifuge tube | Falcon | LAB352070 | |
| 75 cm2 tissue culture flask | Corning | 430725U | |
| 96-well plate | Falcon | 353072 | |
| Anti-flavivirus monoclonal antibody, 4G2 (clone D1-4G2-4-15) | MilliporeSigma | MAB10216 | |
| Autoclaved 20x Phosphate buffered saline (PBS) | N/A | N/A | 22.8 g of 8 mM Na2HPO4, 4.0 g of 1.5 mM KH2PO4, 160 g of 0.14 M NaCl, 4.0 g of 2.7 mM KCl, 1 L of MilliQ H2O |
| Biological safety cabinet, Class II | Holten | HB2448 | |
| CTL S6 Universal ELISpot/FluoroSpot Analyzer | ImmunoSpot, Cellular Technology Limited (CTL) | CTL-S6UNV12 | Commercial software analyzer |
| Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 12800-017 | |
| Fetal bovine serum (FBS) | Bovogen | SFBS | |
| Goat anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (HRP) | MilliporeSigma | 12-349 | |
| Hemacytometer | Laboroptik LTD | Neubauer improved | |
| IGEPAL CA-630 detergent | Sigma-Aldrich | I8896 | Octylphenoxy poly(ethyleneoxy)ethanolIGEPAL |
| Inverted microscope | ZEISS | TELAVAL 31 | |
| Laboratory rocker | FINEPCR | CR300 | |
| L-Glutamine | Gibco | 25030-081 | |
| Low viscosity carboxymethyl cellulose (CMC) | Sigma-Aldrich | C5678 | |
| Multichannel micropipette (10 – 100 µL) | Eppendorf | 3125000036 | |
| Multichannel micropipette (30 – 300 µL) | Eppendorf | 3125000052 | |
| Paraformaldehyde | Sigma-Aldrich | P6148 | |
| Penicillin-streptomycin | Gibco | 15140-122 | |
| Single channel pipettes (10 – 100 µL) | Eppendorf | 3123000047 | |
| Single channel pipettes (100 – 1000 µL) | Eppendorf | 3123000063 | |
| Single channel pipettes (20 – 200 µL) | Eppendorf | 3123000055 | |
| Skim milk | Sunlac Low Fat | N/A | Prepare 3% Skim milk in 1x PBS for blocking stage in staining |
| Sodium Hypochlorite | Clorox | N/A | To disinfect any discarded infectious liquid waste from flasks/plates |
| Stereomicroscope | Nikon | SMZ1000 | |
| Syringe disposable, Luer Lock, 10 mL with 21 G Needle | Terumo | SS10L21G | |
| Vero African green monkey kidney cells | – | ECACC 88020401 | Received from collaborator. However, Vero cells obtained from other suppliers should be able to be used with some optimization. |
References
- Dick, G. W. A., Kitchen, S. F., Haddow, A. J. Zika virus. I. Isolations and serological specificity. Transactions of the Royal Society of Tropical Medicine and Hygiene. 46 (5), 509-520 (1952).
- Dick, G. W. A., Kitchen, S. F., Haddow, A. J. Zika virus. II. Pathogenicity and physical properties. Transactions of the Royal Society of Tropical Medicine and Hygiene. 46 (5), (1952).
- Duffy, M. R., et al. Zika virus outbreak on Yap Island, Federated States of Micronesia. The New England Journal of Medicine. 360 (24), 2536-2543 (2009).
- Musso, D., Nilles, E. J., Cao-Lormeau, V. M. Rapid spread of emerging Zika virus in the Pacific area. Clinical Microbiology and Infection. 20 (10), O595-O596 (2014).
- Cao-Lormeau, V. -. M., et al. Zika virus, French polynesia, South pacific, 2013. Emerging Infectious Diseases. 20 (6), 1085-1086 (2014).
- Dupont-Rouzeyrol, M., et al. Co-infection with Zika and dengue viruses in 2 patients, New Caledonia, 2014. Emerging Infectious Diseases. 21 (2), 381-382 (2015).
- Roth, A., et al. Concurrent outbreaks of dengue, chikungunya and Zika virus infections-an unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012-2014. Euro Surveillance. 19 (41), 20929 (2014).
- Tognarelli, J., et al. A report on the outbreak of Zika virus on Easter Island, South Pacific, 2014. Archives of Virology. 161 (3), 665-668 (2016).
- Oehler, E., et al. Zika virus infection complicated by Guillain-Barre syndrome-case report, French Polynesia, December 2013. Euro Surveillance. 19 (9), 20720 (2014).
- Faria, N. R., et al. Zika virus in the Americas: early epidemiological and genetic findings. Science. 352 (6283), 345-349 (2016).
- Rapid risk assessment: Zika virus epidemic in the Americas: potential association with microcephaly and Guillain-Barré syndrome – 4th update. European Centre for Disease Prevention and Control Available from: https://www.ecdc.europa.eu/en/publications-data/rapid-risk-assessment-zika-virus-epidemic-americas-potential-association (2015)
- Payne, S. Methods to study viruses. Viruses. , 37-52 (2017).
- Bustin, S. A., Mueller, R. Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clinical Science. 109 (4), 365-379 (2005).
- Sedlak, R. H., Jerome, K. R. Viral diagnostics in the era of digital polymerase chain reaction. Diagnostic Microbiology and Infectious Disease. 75 (1), 1-4 (2013).
- Bae, H. -. G., Nitsche, A., Teichmann, A., Biel, S. S., Niedrig, M. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay. Journal of Virological Methods. 110 (2), 185-191 (2003).
- Dulbecco, R. Production of plaques in monolayer tissue cultures by single particles of an animal virus. Proceedings of the National Academy of Sciences. 38 (8), 747-752 (1952).
- Nahapetian, A. T., Thomas, J. N., Thilly, W. G. Optimization of environment for high density Vero cell culture: effect of dissolved oxygen and nutrient supply on cell growth and changes in metabolites. Journal of Cell Science. 81, 65-103 (1986).
- Agbulos, D. S., Barelli, L., Giordano, B. V., Hunter, F. F. Zika virus: quantification, propagation, detection, and storage. Current Protocols in Microbiology. 43, (2016).
- Brien, J. D., Lazear, H. M., Diamond, M. S. Propagation, quantification, detection, and storage of West Nile virus. Current Protocols in Microbiology. 31, (2013).
- Bolívar-Marin, S., Bosch, I., Narváez, C. F. Combination of the focus-forming assay and digital automated imaging analysis for the detection of dengue and Zika viral loads in cultures and acute disease. Journal of Tropical Medicine. 2022, 2177183 (2022).
- Dangsagul, W., et al. Zika virus isolation, propagation, and quantification using multiple methods. PLoS One. 16 (7), e0255314 (2021).
- Brien, J. D., et al. Genotype-specific neutralization and protection by antibodies against dengue virus type 3. Journal of Virology. 84 (20), 10630-10643 (2010).
- Lazear, H. M., et al. A mouse model of Zika virus pathogenesis. Cell Host & Microbe. 19 (5), 720-730 (2016).
- Miner, J. J., et al. Zika virus infection during pregnancy in mice causes placental damage and fetal demise. Cell. 165 (5), 1081-1091 (2016).
- Kang, W., Shin, E. -. C. Colorimetric focus-forming assay with automated focus counting by image analysis for quantification of infectious hepatitis C virions. PLoS One. 7 (8), e43960 (2012).
- Brien, J. D., et al. Isolation and quantification of Zika virus from multiple organs in a mouse. Journal of VIsualized Experiments. (150), e59632 (2019).
- Payne, A. F., Binduga-Gajewska, I., Kauffman, E. B., Kramer, L. D. Quantitation of flaviviruses by fluorescent focus assay. Journal of Virological Methods. 134 (1-2), 183-189 (2006).
- Moser, L. A., et al. Growth and adaptation of Zika virus in mammalian and mosquito cells. PLoS Neglected Tropical Diseases. 12 (11), e0006880 (2018).
- Ishimine, T., Tadano, M., Fukunaga, T., Okuno, Y. An improved micromethod for infectivity assays and neutralization tests of dengue viruses. Biken Journal. 30 (2), 39-44 (1987).
- Leiva, S., et al. Application of quantitative immunofluorescence assays to analyze the expression of cell contact proteins during Zika virus infections. Virus Research. 304, 198544 (2021).
- Lee, E. M., Titus, S. A., Xu, M., Tang, H., Zheng, W. High-throughput Zika viral titer assay for rapid screening of antiviral drugs. ASSAY and Drug Development Technologies. 17 (3), 128-139 (2019).
