Varicella Zoster Virus Replication Assays

Experiment Summary

Varicella zoster virus (VZV) is a human herpesvirus which causes Varicella (chickenpox) upon primary infection and Zoster (shingles) following reactivation from latency. 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)
Minimal essential medium eagle with Earle's BBS, with L-glutamine
Fetal bovine serum (FBS)
Penicillin: streptomycin (5,000 units/ml each)
Non-essential amino acids (NEAA)
1x Trypsin-EDTA liquid (0.05% Trypsin, 0.53 mM EDTA-4Na)
Phosphate buffered saline without Magnesium or Calcium
Recombinant VZV-GFP cell-associated virus stock
MeWo growth medium

Equipment

75 cm² filter cap tissue culture flasks
96-well black tissue culture-treated plates (48/case)
50 ml centrifuge tube
Disposable hemocytometer (KOVA Glasstic slide 10 with counting grid)
Fluorescent plate reader
Class II Microbiological safety cabinet
Humidified cell culture incubator (37 °C, 5% CO₂)
Pipette aid
Centrifuge
Light microscope

Procedure

Take a semi-confluent (~50%) T75 flask of MeWo cells that has been split the previous day (see Note 1).
Remove media, rinse gently in sterile phosphate buffered saline (PBS), and discard wash.
Dislodge cells by adding 3 ml trypsin. Incubate for around 5 min at 37 °C or until cells are fully dislodged.
Inactivate trypsin by adding 7 ml MeWo growth medium.
Pipette up and down to create a single-cell suspension.
Remove 12 μl and count cell density in a disposable hemocytometer.
Remove media, rinse gently in sterile phosphate buffered saline (PBS), and discard wash (see Note 2).
Dislodge cells by adding 3 ml trypsin. Incubate for around 5 min at 37 °C or until cells are fully dislodged.
Inactivate trypsin by adding 7 ml MeWo growth medium (see Note 3).
Pipette up and down to create a single-cell suspension.
Remove 12 μl and count cell density in a disposable hemocytometer.
Remove media, rinse gently in sterile phosphate buffered saline (PBS), and discard wash.
Dislodge cells by adding 3 ml trypsin. Incubate for around 5 min at 37 °C or until cells are fully dislodged (see Note 4).
Inactivate trypsin by adding 7 ml MeWo growth medium.
Pipette up and down to create a single-cell suspension (see Note 5).
Remove 12 μl and count cell density in a disposable hemocytometer (see Note 6).
Remove media, rinse gently in sterile phosphate buffered saline (PBS), and discard wash. Dislodge cells by adding 3 ml trypsin. Incubate for around 5 min at 37 °C or until cells are fully dislodged. Inactivate trypsin by adding 7 ml MeWo growth medium. Pipette up and down to create a single-cell suspension. Remove 12 μl and count cell density in a disposable hemocytometer.

Fig. 1 Calculation of replication slopes.

Notes

Virus replication proceeds more efficiently in cells that have been freshly passaged before seeding into assay plates.
Using a black assay plate will help reduce background and crosstalk between wells.
When grown in culture VZV virus particles remain associated with the cell membrane. As such, VZV-infected MeWo cells are used as the inoculum for VZV replication assays. These cells are stored in liquid nitrogen and need to be thawed and washed before use. Cells are extremely sensitive to temperature changes. When thawing it is essential that they are thawed quickly at 37 °C until only just thawed (1-2 min). Do not leave cells for longer periods of time as this will considerably reduce cell viability and therefore titre of replication-competent virus.
It is important to do the infection with a known quantity of virus (multiplicity of infection; MOI) to generate growth curve over a reasonable time-scale. The titre [number of infectious units per ml of virus (IU/ml)] of VZV-GFP-infected MeWo cells is determined by a standard plaque assay, where a virus stock is added to a Mewo cell monolayer and overlaid with agarose. This results in the infection only of adjacent cells, which subsequently die to leave an empty patch within the cell monolayer. These patches, or 'plaques' can be counted to quantify the virus as 'plaque-forming units', or infectious units (IU), per ml inoculum. In our experience 100 IU per well of a 96-well plate produces a suitable growth curve for replication analyses and comparisons. For example, a virus stock with a titre of 6.6 x 104 IU/ml would contain 6,600 IU in 100 µl. A 1 ml aliquot of virus inoculum should therefore be resuspended in 66 ml media.
Tissue culture plasticware, cells and growth medium all have some level of fluorescence. When utilising fluorescent reporter genes, it is therefore essential to have appropriate controls to provide background fluorescence readings. For this assay, the fluorescence from mock-infected cells is used as background.
Replication is monitored over regular intervals to enable a complete growth curve (fluorescence over time) to be plotted. When comparing VZV replication between untreated and treated samples we use the slope of replication over the linear growth phase. It is therefore important to have as many measurements over this phase as possible (with a minimum of 6 for statistical reliability of the slope calculation). A test replication assay with your own equipment will allow the time of linear growth to be established, and assay timings can be adjusted to ensure measurements can be taken over this period.

* For research use only. Not intended for any clinical use.

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Varicella Zoster Virus Replication Assays

Experiment Summary

Materials and Reagents

Equipment

Procedure

Notes

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