VZV Replication Assays
水痘带状疱疹复制分析
发布: 2014年08月20日第4卷第16期 DOI: 10.21769/BioProtoc.1210 浏览次数: 11977
评审: Pinchas TsukermanAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2013
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Abstract
Varicella zoster virus (VZV) is a human herpesvirus which causes Varicella (chickenpox) upon primary infection and Zoster (shingles) following reactivation from latency (von Bokay, 1909). Whilst VZV is extensively studied, inherent features of VZV replication, such as cell-association of virus particles during in vitro culture and a restricted host range (limited to humans and some other primates) mean the cellular and viral mechanisms underlying VZV reactivation and pathogenesis remain largely uncharacterised. Much remains to be learnt about VZV, interactions with its host, and the development of disease. This protocol describes a basic VZV replication assay using a recombinant VZV-GFP reporter virus. As VZV is highly cell-associated in tissue culture, the reporter virus inoculum described here is a preparation of infected cells. This reporter virus-infected cell line can be used in combination with siRNA gene depletion or cDNA overexpression transfection protocols to determine the effect of individual cellular genes on virus replication.
Materials and Reagents
- VZV-permissive human cells (e.g. MeWo cells) (ATCC, catalog number: HTB-65 )
- Minimal essential medium eagle with Earle’s BBS, with L-glutamine (Lonza, catalog number: 12-611F )
- Fetal bovine serum (FBS) (LabTech, catalog number: FCS-SA-10454 )
- Penicillin: streptomycin (5,000 units/ml each) (Lonza, catalog number: DE17-603E )
- Non-essential amino acids (NEAA) (Life Technologies, catalog number: 11140035 )
- 1x Trypsin-EDTA liquid (0.05% Trypsin, 0.53 mM EDTA-4Na) (Life Technologies, catalog number: 25300096 )
- Phosphate buffered saline without Magnesium or Calcium (Lonza, catalog number: 17-516F )
- Recombinant VZV-GFP cell-associated virus stock (Zerboni, 2000)
- MeWo growth medium (see Recipes)
Equipment
- 75 cm2 filter cap tissue culture flasks (Sigma-Aldrich, catalog number: C7106-120EA )
- 96-well black tissue culture-treated plates (48/case) (VWR International, catalog number: 734-1609 )
- 50 ml centrifuge tube (e.g. Corning, catalog number: 430290 )
- Disposable hemocytometer (KOVA Glasstic slide 10 with counting grid) (HYCOR Biomedical, catalog number: 87144E )
- Fluorescent plate reader (e.g. POLARstar OPTIMA, BMG LABTECH)
- Class II Microbiological safety cabinet
- Humidified cell culture incubator (37 °C, 5% CO2)
- Pipette aid (Alpha laboratories, catalog number: 4-131-201-E )
- Centrifuge (e.g. Eppendorf, catalog number: 5811-000.010 )
- Light microscope
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Griffiths, S. J. and Haas, J. (2014). VZV Replication Assays. Bio-protocol 4(16): e1210. DOI: 10.21769/BioProtoc.1210.
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分类
微生物学 > 微生物-宿主相互作用 > 体外实验模型 > 细胞系
微生物学 > 微生物-宿主相互作用 > 病毒
微生物学 > 微生物细胞生物学 > 细胞分离和培养
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