This article describes a method for quantifying the infectious titer of herpes simplex virus using a plaque assay. The procedure involves infecting a monolayer of epithelial cells with serial dilutions of the virus and visualizing the resulting plaques.
To begin, prepare serial dilutions of herpes simplex virus, an enveloped infectious virus, in buffer.
Obtain a multi-well plate containing a monolayer of virus-susceptible epithelial cell culture. Replace specific volume of media with diluted viral suspensions, ensuring even monolayer coverage. Allow the virus to adsorb onto the cell surface.
Remove any unadsorbed viruses. Cover the cell monolayer with methylcellulose-containing media, forming a semisolid overlay.
Upon incubation, the viral envelope and cell membrane fuse, releasing the viral nucleocapsid enclosing double-stranded genome into the cytoplasm. Further, viral genome gets released into the cell nucleus.
Using the host's cellular machinery and virion protein, viral genome expresses immediate early viral proteins, which regulate the expression of early genes. Early viral proteins mediate viral DNA replication.
Then, late viral genes express structural proteins, which along with viral genome assemble to produce progeny virion particles. The virus-infected cells lyse, releasing virions.
Methylcellulose restricts virus movement, causing virions to only infect the neighboring cells and subsequent lysis, creating holes in the monolayer.
Post-incubation, remove methylcellulose overlay. Add ethanolic crystal violet solution. Ethanol fixes cells and inactivates viruses, while crystal violet stains the cells. As a result, clear lysed zones or plaques appear on the purple cell monolayer.
Count the plaques in each well at each virus dilution to determine the infectious virus titer or concentration.
Remove the cell culture medium from one or two wells using a pipette. Carefully, add the diluted virus sample drop-wise to each monolayer of cells, drop-wise through the side of each well.
Repeat the process for every one or two wells until the entire plate is filled with the virus samples being plaqued. Gently rock the entire plate by hand.
Then, incubate the plate at 37 degrees Celsius for 1 hour to allow the virus adsorption. After every 10 minutes, remove the plate from the incubator, gently rock it again to spread the virus more evenly across each well, and then, place it back in the incubator.
To diminish the possibility of inadvertently counting unabsorbed inoculum, remove the virus sample from the wells using a 1,000-microliter pipette tip, and discard the sample in a waste beaker.
Place 2.5 milliliters of a methylcellulose overlay on the plate, and place it back in the incubator. Disinfect the waste beaker, typically with the addition of bleach, an iodophor, or a similar virucide, before removing it from the biosafety cabinet.
After the two-day incubation, remove the methylcellulose overlay from one or two wells at a time, using a pipette, and place it in a waste beaker. Add approximately 2 milliliters of 1% crystal violet and 50% ethanol to each well. Incubate the plate with all wells filled with the stain for 30 minutes at 37 degrees Celsius.
At this point, disinfect the waste beaker, as demonstrated previously. Wash the plates vigorously with tap water, until the runoff is clear.
Ensure that there are approximately 10-fold differences in the number of plaques across each dilution series.
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