Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Summary

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

Abstract

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

Introduction

People suffering from ocular infections often incur vision loss¹. With a high seroprevalence worldwide, HSV infected individuals suffer from recurring eye infections which lead to corneal scarring, stromal keratitis and neovascularization^2,3,4,5. HSV infections have also shown to cause less frequently, a range of serious conditions among immunocompromised, untreated patients like encephalitis and systemic morbidity^6,7,8. Drugs like Acyclovir (ACV) and its nucleoside analogs have shown consistent success in curbing HSV-1 infection and even control reactivation yet the prolonged use of these drugs is associated with renal failure, fetal abnormalities and failure to restrict the emergence of drug-resistance to evolving viral strains^{9,10,11,12,13}. Complexities associated with HSV-1 ocular infections, have been previously studied in vitro using monolayers and 3D cultures of human corneal cells and in vivo using murine or rabbit ocular infections. While these in vitro models provide significant data on the cellular biological components of HSV-1 infections, they, however, fail to mimic the intricate complexity of corneal tissue and do little to illuminate the dendritic spread of the virus¹⁴. In contrast, although in vivo systems are more insightful in showing infection spread in corneas and immune activation responses during HSV-1 infection, they do come with the caveat that they require trained investigators and large facilities for animal care to overlook the experiments.

Here we use porcine corneas as an ex vivo model to examine the HSV-1 infection induced wound system. Both the potential pharmacology of certain drugs as well as the cell and molecular biology of the wound system caused by the infection can be studied through tissue explant cultures. This model may also be amended for the use for other viral and bacterial infections as well. In this study, porcine corneas were used to test the antiviral efficacy of a preclinical small molecule, BX795. The use of porcine corneas was preferred due to ease of access and cost effectiveness. Additionally, porcine corneal models are good models of human eyes with the corneas being easy to isolate, adequately sized for infection and visualization and robust to handle¹⁵. Porcine corneas are also comparable to the complexity of human corneal models in both trans corneal permeability and systemic absorption¹⁵. By using this model for the study, we were able to elucidate how BX795 is worthy of further investigation as a competent inhibitor of HSV-1 virus infection and adds to the literature of classifying it as a potential small-molecule antiviral compound¹⁶.

Protocol

All the porcine tissue used in this study was provided by a third-party private organization and none of the animal handling was performed by University of Illinois at Chicago personnel.

1. Materials

1. Reagents
   1. Use following reagents for Plaque assay: powder methylcellulose, Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin (P/S) for Plaque assay.
   2. Use crystal violet tablets and ethanol (molecular biology grade) for preparing crystal violet solution for plaque assay.
2. Vero cell growth media - DMEM whole media
   1. Open the DMEM media bottle inside the tissue culture hood. Remove 55 mL of media from the bottle using a serological pipette and discard it. Add 50 mL of FBS of P/S to DMEM. Refrigerate at 4 °C.
3. Plaque assay media - Stock of 5% methylcellulose media
   1. Measure 2.5 g of methylcellulose powder with a stirring magnet inside a 500 mL glass bottle and autoclave it. After the bottle cools to room temperature, take 500 mL of DMEM whole media containing 50 mL of FBS and 5 mL of P/S and add it to glass bottle containing methylcellulose.
   2. Stir this at 4 °C for 2 days using a magnetic stirrer. Refrigerate at 4 °C.
4. Media preparation for excised porcine cornea
   1. Open a minimum essential media (MEM) bottle inside the tissue culture hood. Discard 10 mL of MEM from the bottle using a serological pipette.
   2. Add 5 mL of insulin-transferrin-selenium (ITS) and 5 mL of antimycotic-antibiotic (AA) to the media and refrigerate at 4°C.
5. Master and working stock of crystal violet:
   1. To make the crystal violet stock solution, add 1 g of crystal violet powder to 100 mL of 20% ethanol in water. This stock (1% w/v crystal violet) can be stored and used for up to year when stored in a place that is dark.
   2. To make a working stock from this, add 50 mL of original stock solution to 350 mL of water. Add 100mL of ethanol to this solution to make a 500 mL working crystal violet solution (0.1% w/v crystal violet).
      ​NOTE: Both these solutions need to be stored in the dark.

2. Procedure

1. Isolation of Porcine corneas from whole eyes¹⁷
   1. Upon receiving the porcine eyes from a suitable vendor, store on ice if there is a delay with tissue processing as pictured in Figure 1.
   2. Ensure that personal protection equipment is used and worn during this procedure to avoid contamination as well as accidents from spillage of vitreous humor.
   3. Spray the working area with 70% ethanol to clean and disinfect. To ensure the working space is stable, spread a bench cover and tape down the sides securely as pictured in Figure 2.
   4. Place porcine eyes on gauze (Figure 3). Using 50 mm gauze, hold posterior section (Figure 4A) of porcine eyes as shown in Figure 4B with one hand.
   5. With a 30 G needle, gently make a single poke at approximately the center of epithelial surface of the eye carefully and ensure that there no damage to stroma (Figure 5). The poke should be limited to the epithelium (~100-200 µm) to avoid stromal involvement.
   6. Using a sharp sterilized blade, make a small incision on the sclera at 1 mm distance from the cornea. Cut the edge of the cornea ensuring that the vitreous humor does not leak using a swift and smooth rotating action of the hand (Figure 6A). By holding the cornea at the cornea-sclera edge with a flat tweezer, cut off the remaining tethering membranes of eye using the blade (Figure 6B).
   7. Take the cornea and place it in a 12-well plate overlaid with 2 mL of cornea medium. The cornea should be placed facing up, demonstrated in a series of steps in Figures 7A-D, Figure 8).
   8. Add 5 µL of the virus solution containing 5 x 10⁵ plaque forming units (PFU) of 17 GFP to the debrided site on the corneal surface. Place the 12-well plate containing infected porcine corneas in an incubator with 5% CO₂ for 72 h.
   9. Spray any additional eyes not used in experiment with 10% bleach and securely disposed in biohazard bags.
2. Visualization
   NOTE: The virus should be visualized every day prior to addition of drugs.
   1. Turn on the stereo microscope and LED light source and allow lamp of machine to warm up before imaging the corneas. Carefully carry the plate of corneas to instrument without disturbing the solution. Change the filter so that GFP (380-460 nm) is used to look at specimens. Set the exposure time to 500 ms to capture images.
   2. First, place the cornea plate under the stereoscope and capture the images at the lowest magnification of 7.5x. Follow this up with a series of increasing magnification images (e.g., 15x, 25x, 35x) such that all the viral spread and dendrite formation is visualized clearly
   3. Make sure to return the infected corneas back into the tissue culture incubator and save all the images that were taken.
3. Virus infection quantification
   NOTE: Virus titers from porcine corneas should be evaluated every day to analyze the effect of drug treatment.
   1. To quantify virus, seed Vero cells at a density of 5 x 10⁵ of cells per well if using a 6-well plate as done in this experiment. Do this a day prior to the infection. Incubate the plated cells overnight to ensure they are confluent for virus quantification.
   2. Aliquot 500 µL of serum free media into multiple microcentrifuge tubes. Insert sterile cotton tipped swab dipped into the serum free media filled tubes. The cotton swabs need to be dipped and wetted for at least 5 min prior to the use.
   3. Without disturbing the underlying media, transport the infected porcine cornea plate into a biosafety cabinet. Using the wet cotton swabs, make 3 revolutions clockwise and 3 revolutions anti-clockwise at a diameter of 5 mm from the center of the infected porcine cornea without applying excessive pressure.
   4. Insert back the cotton swab into the serum free media filled microcentrifuge tube and rotate it clockwise and anti-clockwise 5 times. The metal tip of the cotton swab should be cut short so that it fits entirely into microcentrifuge tube and the lid can be closed.
   5. Place the microcentrifuge tube containing the swabbed cotton tip on a vortex machine and vortex at high speed for 1 min.
   6. Perform virus quantification via a plaque assay on these samples.
      NOTE: This quantification step needs to be performed on days 2, 4 and optionally on 6 days post infection.
4. Virus quantification by Plaque assay
   NOTE: To perform a plaque assay, grow and plate Vero cells into a 6 well plate and ensure 90% confluency of cells before start of assay. Use a confluent 75 cm² flask of these cells. All the steps below need to be performed inside a biosafety cabinet.
   1. Wash the confluent monolayer of Vero cells in the flask with 10 mL of fresh phosphate buffer saline (PBS) after aspirating the culture medium. Repeat the wash step once again with PBS after aspirating the first set of wash solution.
   2. Add 1 mL of 0.05% Trypsin to the cell monolayer. Incubate the flask at 37 °C for 5 min. With the naked eye, ensure that the cells are detached from the inside surface of the flask. If not, wait for another 5 min before examining the flask again. When cells appear to be detached, add 9 mL of whole media to the monolayer to ensure that the cells dislodge completely from the flask surface.
   3. Collect all the cells from the flask along with the whole media and place them in a 15 mL centrifuge tube.
   4. For every well of the 6 well plates that are used for plaque assay, use 300 µL from the centrifuge tube. Top up each well of the 6 well plate with 2 mL of whole media. Leave the plates in the incubator overnight to allow them to grow and form a confluent monolayer in each well.
   5. In order to perform a plaque assay, perform a serial dilution of samples needs to be conducted before the quantification of virus.
   6. Perform a log₁₀1 fold dilution of the virus in micro-centrifuge tubes using serum free media until a dilution of 10^-8 is reached. When at a 10^-3 dilution, transfer 1 mL of the dilution to the monolayer of plated cells after aspirating the growth media on the cells from the 6 well plate, this will be the infection step. Incubate the infected plate at 37 °C incubator for 2 h.
   7. Aspirate the existing infection media, wash with PBS twice gently to coat cells and add 2 mL of methylcellulose laden media per well to all 6 wells. Incubate for 72 h or until formation of plaques can be seen. Plaques can be identified by the formation of small gaps between cells in the cell monolayer.
   8. Add 1 mL of methanol slowly to the corner of each well, using the wall as guiding tip. Incubate the 6 well plate at room temperature for 15 min. Slowly aspirate the contents of each well from the plate without disturbing or agitating the cell monolayer.
   9. Add 1 mL of crystal violet working solution to each well of 6 well plate, ensuring all cells are covered. Incubate the 6 well plate in the dark for 30 min. Discard the crystal violet solution by aspirating it and dry the wells on a sheet of absorbent paper.
   10. Count the number of plaques at the highest dilution well to quantify the total virus content in the starting solution. Repeat the process twice to confirm the viral titer.

Results

To understand the efficacy of the experimental antivirals, they need to be tested extensively before they are sent for in vivo human clinical trials. In this regard, positive control, negative control and test groups have to be identified. Trifluorothymidine (TFT) has long been used as the preferred treatment to treat herpes keratitis topically¹⁶. Used as a positive control, the TFT treated corneal groups show lower infection spread. As a negative control, we used DMSO or vehicle control dissolved...

Discussion

Prior research has shown BX795 to have a promising role as an antiviral agent against HSV-1 infection; by inhibiting the TANK-binding kinase 1 (TBK1)¹⁶. Both TBK1 and autophagy have played a role in helping inhibit HSV-1 infection as demonstrated on human corneal epithelial cells. BX795 was shown to be maximally effective with antiviral activity at a concentration of 10µM and using both western blot analysis and viral plaque analysis of key viral proteins, BX795 was shown to inhibit HSV-1 inf...

Materials

Name	Company	Catalog Number	Comments
30 G hypodermic needles.	BD	305128
500 mL glass bottle.	Thomas Scientific	844027
Antimycotic and Antibiotic (AA)	GIBCO	15240096	Aliquot into 5 mL tubes and keep frozen until use
Benchtop vortexer.	BioDot	BDVM-3200
Biosafety cabinet with a Bio-Safety Level-2 (BSL-2) certification.	Thermofisher Scientific	Herasafe 2030i
Calgiswab 6" Sterile Calcium Alginate Standard Swabs.	Puritan	22029501
Cell scraper - 25 cm	Biologix BE	70-1180 70-1250
Crystal violet	Sigma Aldrich	C6158	Store the powder in a dark place
Dulbecco’s modified Eagle’s medium - DMEM	GIBCO	41966029	Store at 4 °C until use
Ethanol	Sigma Aldrich	E7023
Fetal bovine serum -FBS	Sigma Aldrich	F2442	Aliquot into 50 mL tubes and keep frozen until use
Flat edged tweezers – 2.	Harward Instruments	72-8595
Freezers --80 °C. -	Thermofisher Scientific	13 100 790
Fresh box of blades.	Thomas Scientific	TE05091
Guaze	Johnson & Johnson		108 square inch folder 12 ply
HSV-1 17GFP	grown in house	-	Original strain from Dr. Patricia Spears, Northwestern University. GFP expressing HSV-1 strain 17
Insulin, Transferrin, Selenium - ITS	GIBCO	41400045	Aliquot into 5 mL tubes and keep frozen until use
Magnetic stirrer.	Thomas Scientific	H3710-HS
Metallic Scissors.	Harward Instruments	72-8400
Micropipettes 1 to 1000 µL.	Thomas Scientific	1159M37
Minimum Essential Medium - MEM	GIBCO	11095080	Store at 4 °C until use
OptiMEM	GIBCO	31985047	Store at 4 °C until use
Penicillin/streptomycin.	GIBCO	15140148	Aliquot into 5 mL tubes and keep frozen until use
Phosphate Buffer Saline -PBS	GIBCO	10010072	Store at room temperature
Porcine Corneas	Park Packaging Co., Chicago, IL		Special order by request
Procedure bench covers - as needed.	Thermofisher Scientific	S42400
Serological Pipettes	Thomas Scientific	P7132, P7127, P7128, P7129, P7137
Serological Pipetting equipment.	Thomas Scientific	Ezpette Pro
Stereoscope	Carl Zeiss	SteREO Discovery V20
Stirring magnet.	Thomas Scientific	F37120
Tissue culture flasks, T175 cm2.	Thomas Scientific	T1275
Tissue culture incubators which can maintain 5% CO2 and 37 °C temperature.	Thermofisher Scientific	Forma 50145523
Tissue culture treated plates (6-well).	Thomas Scientific	T1006
Trypsin-EDTA (0.05%), phenol red	GIBCO	25-300-062	Aliquot into 10 mL tubes and keep frozen until use
Vero cells	American Type Culture Collection ATCC	CRL-1586