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. 2019 Dec 5;9(23):e3441.
doi: 10.21769/BioProtoc.3441.

Herpes Simplex Virus Type 1 Propagation, Titration and Single-step Growth Curves

Linda Grosche  1 , Katinka Döhner  2 , Alexandra Düthorn  1 , Ana Hickford-Martinez  2 , Alexander Steinkasserer  1 , Beate Sodeik  2
Affiliations

Herpes Simplex Virus Type 1 Propagation, Titration and Single-step Growth Curves

Linda Grosche et al. Bio Protoc. .

Abstract

Given the endemic seroprevalence of herpes simplex viruses (HSV), its associated human diseases, and the emergence of acyclovir-resistant strains, there is a continuous need for better antiviral therapies. Towards this aim, identifying mechanistic details of how HSV-1 manipulates infected cells, how it modulates the immune responses, and how it causes diseases are essential. Measuring titers and growth kinetics of clinical isolates and viral mutants are important for a thorough characterization of viral phenotypes in vitro and in vivo. We provide protocols for the preparation as well as titration of HSV-1 stocks, and explain how to perform single-step growth curves to characterize the functions of viral proteins or host factors during infection. In particular, we describe methods to prepare and characterize high-titer HSV-1 stocks with low genome to titer ratios that are required for infection studies in cell culture and animal experiments.

Keywords: Herpes simplex virus type 1; Plaque assay; Single-step growth curve; Virus propagation.

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Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Subconfluent BHK-21 cells for HSV-1 inoculation.
Scale bar = 0.4 mm.
Figure 2.
Figure 2.. BHK-21 cells infected with HSV-1 strain 17+CMV-GFP/UL43 at 1 dpi (upper panel) and 4 dpi (lower panel).
Scale bar= 0.4 mm.
Video 1.
Video 1.. Visualization of the interphase formation during preparation of a step gradient.
The tube contains 10% sucrose (transparent). We underlay with the 40% sucrose (blue) using a syringe. To better visualize the interphase formation, we stained the 40% sucrose solution here with a blue dye. If preparing gradients for virus preparations we do not add any dye. It is crucial to add the heavy solution smoothly without pressing the needle to the bottom of the tube and without pushing the plunger too abruptly. An interphase between the 10% and 40% solution forms and migrates up.
Figure 3.
Figure 3.. Gradient purification of HSV-1 virions.
Exemplary result of a linear nycodenz sedimentation gradient with a light-scattering virus band (arrow) slightly beneath the middle of the tube (red line).
Figure 4.
Figure 4.. Pipetting scheme of a serial dilution for plaque assays
Figure 5.
Figure 5.. HSV-1 plaques in a Vero cell monolayer at 2 dpi (left) or 3 dpi (right).
Scale bar = 0.4 mm.
Figure 6.
Figure 6.. Crystal violet stained HSV-1 plaque assay.
Vero cells cultured in a 12-well plate were inoculated with 10-fold serial dilutions of a virus suspension, fixed at 3 dpi and stained with crystal violet.
Figure 7.
Figure 7.. α-gD stained HSV-1 plaque assay.
Vero cells were inoculated with serial dilutions of HSV-1 suspensions and infected for 3 days. Cells were fixed and subsequently labeled with an α-gD antibody followed by labeling with a secondary alkaline phosphatase antibody.
See this image and copyright information in PMC

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