水痘带状疱疹复制分析

Samantha J.  Griffiths; Jürgen  Haas

doi:10.21769/BioProtoc.1210

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Peer-reviewed

VZV Replication Assays

水痘带状疱疹复制分析

Samantha J. Griffiths email

Jürgen Haas

发布: 2014年08月20日第4卷第16期 DOI: 10.21769/BioProtoc.1210 浏览次数: 11215

评审: Pinchas TsukermanAnonymous reviewer(s)

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The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

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