nav emailalert searchbtn searchbox tablepage yinyongbenwen piczone journalimg qikanlogo popupnotification paper
2024 03 v.40 492-499
检测痘苗病毒体外感染的病灶形成方法的优化与应用
基金项目(Foundation): 国家重点研发计划(项目号:2023YFD1800405),题目:抗猴痘药物筛选与阻断技术研究;国家重点研发计划(项目号:2022YFC2303401),题目:潜在高危新病毒风险识别的新技术体系研究~~
邮箱(Email): dengyao@ivdc.chinacdc.cn;tanwj@ivdc.chinacdc.cn;
DOI: 10.13242/j.cnki.bingduxuebao.004508
中文作者单位:

中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会生物安全重点实验室;温州医科大学检验医学院生命科学学院,浙江省医学遗传学重点实验室;内蒙古医科大学基础医学院;新乡医学院公共卫生学院;

摘要(Abstract):

本研究旨在建立一种快速、通用、高通量的病灶形成测定法(Focus-forming assay, FFA),用于痘苗病毒(Vaccinia virus, VACV)的研究。首先采用经典噬斑染色法与免疫噬斑法对不同VACV代表性毒株(Western Reserve strain, WR; Tiantan strain, VTT; Modified Ankara strain, MVA)在BHK-21细胞和Vero细胞上的噬斑特性进行评估,通过实验条件(包括显色液、非特异性染色、细胞培养板和病毒培养时间)的优化,提高病灶的可视化质量和测定的准确性。并基于改进优化的FFA法研究了VACV在BHK-21细胞和Vero细胞上的生长曲线,同时对FFA法的重复性进行了评估。研究发现BHK-21细胞对VACV的感染更加敏感,适宜于多种VACV滴度测定。实验条件的优化显著提高了病灶的可视化质量和测定的准确性。应用FFA法发现WR、VTT和MVA毒株在BHK-21细胞上能够有效复制,而MVA在Vero细胞上的复制受限,且FFA法在同一实验内具有良好的重复性,变异系数在0.17%到3.50%之间。改进后的FFA法是一种快速、通用、高通量的方法,适用于痘苗病毒滴度的测定,为痘苗病毒的研究和应用提供了可靠的技术支持。

关键词(KeyWords): 痘苗病毒;;噬斑实验;;病灶形成实验;;滴度测定
参考文献 [1] Travieso T, Li J, Mahesh S, et al. The use of viral vectors in vaccine development[J]. NPJ Vaccines,2022, 7(1):75. DOI:10. 1038/s41541-022-00503-y.
[2] Deng Y, Chuai X, Chen P, et al. Recombinant vaccinia vector-based vaccine(Tiantan)boosting a novel HBV subunit vaccine induced more robust and lasting immunity in rhesus macaques[J]. Vaccine, 2017, 35(25):3347-3353. DOI:10. 1016/j.vaccine. 2017. 04. 059.
[3] Zhang Z, Dong L, Zhao C, et al. Vaccinia virus-based vector against infectious diseases and tumors[J]. Hum Vaccin Immunother, 2021, 17(6):1578-1585. DOI:10. 1080/21645515. 2020. 1840887.
[4] Colby D J, Sarnecki M, Barouch D H, et al. Safety and immunogenicity of Ad26 and MVA vaccines in acutely treated HIV and effect on viral rebound after antiretroviral therapy interruption[J]. Nat Med, 2020,26(4):498-501. DOI:10. 1038/s41591-020-0774-y.
[5] Wang S V, Schneeweiss S, Franklin J M, et al.Emulation of randomized clinical trials with nonrandomized database analyses:results of 32 clinical trials[J]. Jama, 2023, 329(16):1376-1385. DOI:10. 1001/jama. 2023. 4221.
[6] Park B H, Hwang T, Liu T C, et al. Use of a targeted oncolytic poxvirus, JX-594, in patients with refractory primary or metastatic liver cancer:a phase I trial[J].Lancet Oncol, 2008, 9(6):533-542. DOI:10. 1016/s1470-2045(08)70107-4.
[7] Heo J, Reid T, Ruo L, et al. Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer[J]. Nat Med, 2013, 19(3):329-336. DOI:10. 1038/nm. 3089.
[8] Hwang T H, Moon A, Burke J, et al. A mechanistic proof-of-concept clinical trial with JX-594, a targeted multi-mechanistic oncolytic poxvirus, in patients with metastatic melanoma[J]. Mol Ther, 2011, 19(10):1913-1922. DOI:10. 1038/mt. 2011. 132.
[9] Ye M, Keicher M, Gentschev I, et al. Efficient selection of Recombinant Fluorescent Vaccinia Virus Strains and Rapid Virus Titer Determination by Using a Multi-Well Plate Imaging System[J]. Biomedicines,2021, 9(8):1032. DOI:10. 3390/biomedicines9081032.
[10]Case J B, Bailey A L, Kim A S, et al. Growth,detection, quantification, and inactivation of SARS-CoV-2[J]. Virology, 2020, 548:39-48. DOI:10. 1016/j.virol. 2020. 05. 015.
[11]Bailly B, Richard C A, Sharma G, et al. Targeting human respiratory syncytial virus transcription antitermination factor M2-1 to inhibit in vivo viral replication[J]. Scientific Reports, 2016, 6(1):25806. DOI:10. 1038/srep25806.
[12]Cruz D J M, Shin H-J. Application of a focus formation assay for detection and titration of porcine epidemic diarrhea virus[J]. J Virol Methods, 2007, 145(1):56-61. DOI:10. 7554/eLife. 81869.
[13]Waris G, Kang W, Shin E-C. Colorimetric FocusForming Assay with Automated Focus Counting by Image Analysis for Quantification of Infectious Hepatitis C Virions[J/OL]. PLoS ONE, 2012, 7(8):e43960.DOI:10. 1371/journal. pone. 0043960.
[14]Keiser P T, Anantpadma M, Staples H, et al.Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID50 Assays[J]. Microorganisms, 2021, 9(1):156. DOI:10. 3390/microorganisms9010156.
[15]Elahi S M, Nazemi-Moghaddam N, Gadoury C, et al.A rapid Focus-Forming Assay for quantification of infectious adenoviral vectors[J]. J Virol Methods,2021, 297:114267.DOI:10. 1016/j.jviromet. 2021. 114267.
[16]Ye C, Wang D, Liu H, et al. An Improved EnzymeLinked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection[J]. Front Pharmacol, 2019, 10:1203. DOI:10. 3389/fphar. 2019. 01203.
[17]Stone E T, Hirsch A J, Smith J L, et al. Titration and neutralizing antibody quantification by focus forming assay for Powassan virus[J]. STAR Protocols, 2022, 3(3):101473. DOI:10. 1016/j. xpro. 2022. 101473.DOI:10. 1016/j. xpro. 2022. 101473.
[18]Earl P L, Cooper N, Wyatt L S, et al. Preparation of cell cultures and vaccinia virus stocks[J]. Curr Protoc Mol Biol, 2001, Chapter 16:Unit16. 16. DOI:10. 1002/0471140864. ps0512s13.
[19]Wiebe M S, Peng C, Wyatt L S, et al. Zinc-finger antiviral protein(ZAP)is a restriction factor for replication of modified vaccinia virus Ankara(MVA)in human cells[J/OL]. PLoS Pathogens, 2020, 16(8):e1008845. DOI:10. 1371/journal. ppat. 1008845.
[20]Liu R, Americo J L, Cotter C A, et al. MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease[J]. bioRxiv, 2021,2020. 12. 30. 424878.DOI:10. 1101/2020. 12. 30. 424878.
[21]Le?o T L, Louren?o K L, de Oliveira Queiroz C, et al.Vaccinia virus induces endoplasmic reticulum stress and activates unfolded protein responses through the ATF6αtranscription factor[J]. Virol J, 2023, 20(1):145.DOI:10. 1186/s12985-023-02122-y.
[22]Kubinski M, Beicht J, Zdora I, et al. A recombinant Modified Vaccinia virus Ankara expressing prME of tickborne encephalitis virus affords mice full protection against TBEV infection[J]. Front Immunol, 2023, 14:1182963. DOI:10. 3389/fimmu. 2023. 1182963.
[23]Ramírez J C, Gherardi M M, Esteban M. Biology of attenuated modified vaccinia virus Ankara recombinant vector in mice:virus fate and activation of B-and T-cell immune responses in comparison with the Western Reserve strain and advantages as a vaccine[J]. J Virol,2000, 74(2):923-933. DOI:10. 1128/jvi. 74. 2. 923-933. 2000.

基本信息:

DOI:10.13242/j.cnki.bingduxuebao.004508

中图分类号:R373

引用信息:

[1]楚巧鸿,冯霞,李佳等.检测痘苗病毒体外感染的病灶形成方法的优化与应用[J].病毒学报,2024,40(03):492-499.DOI:10.13242/j.cnki.bingduxuebao.004508.

基金信息:

国家重点研发计划(项目号:2023YFD1800405),题目:抗猴痘药物筛选与阻断技术研究;国家重点研发计划(项目号:2022YFC2303401),题目:潜在高危新病毒风险识别的新技术体系研究~~

检 索 高级检索

引用

GB/T 7714-2015 格式引文
MLA格式引文
APA格式引文