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JoVE Journal Immunology and Infection

Immunology and Infection

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

Published: September 20, 2021 doi: 10.3791/62195
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1, 1, 1,2
1Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 2Department of Microbiology and Immunology, University of Illinois at Chicago

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 neovascularization2,3,4,5. HSV infections have also shown to cause less frequently, a range of serious conditions among immunocompromised, untreated patients like encephalitis and systemic morbidity6,7,8. 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 strains9,10,11,12,13. 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.

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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.

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Representative 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 in PBS. BX795, the experimental preclinical drug was the test group. A total of 4 corneas were assigned to each group and the drugs were added 3-times every day to the porcine corneas. Our results using stereoscopic fluorescence imaging show that the antiviral efficacy of BX795 is similar to TFT in controlling viral spread. Viral spread in our studies can be visualized by imaging the green fluorescence channel in the stereoscope. We observed that vehicle only treated negative control group corneas showed spread of the virus from the central infection zone to its periphery by 6 days post initial viral inoculation, while both drugs (BX795 and TFT) clear the infection in day 4 - 6 images (Figure 9A). Similarly, the ocular swabs taken on days 2 and 4 post infection show a complete inhibition of virus in the positive control and BX795 treated samples while a sharp increase in infectious virus titer is observed in the negative control group (Figure 9B).

Figure 1
Figure 1: Porcine eyes kept on ice until tissue is processed. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Work bench setup. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Porcine eyes placed on gauze. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Porcine eye with 30G needle pictured. Please click here to view a larger version of this figure.

Figure 5
Figure 5: 30 G needle used; hole made at center of epithelial surface. Please click here to view a larger version of this figure.

Figure 6
Figure 6: Rotating action used to cut around corneal edge using sharp sterilized blade (A,B). Please click here to view a larger version of this figure.

Figure 7
Figure 7: Cornea images. (A,B) The cornea finally cut using sharp scissors (C) Isolated cornea is held by tweezers (D) Completely isolated cornea, ready for use Please click here to view a larger version of this figure.

Figure 8
Figure 8: Corneas placed in 12-well plate and incubated for 72 h. Please click here to view a larger version of this figure.

Figure 9
Figure 9: Progression of viral spread taken during course of infection. Freshly excised porcine corneas were infected with HSV-1 17-GFP and (A) imaged using microscope on days 2, 4 and 6 post infection. Topical treatment with DMSO, TFT or BX795 was started on day 2 post infection. (B) Plaque assays were performed using swabs taken from the porcine corneas on Vero Cells. Two-way ANOVA test was performed to understand significant differences between the treatment groups. n=4, ****p=0.0001. Please click here to view a larger version of this figure.

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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 infection comparable to the activity of TFT16. The study on porcine corneas followed the same analysis and obtained similar results as demonstrated above; BX795 is shown to be just as effective as TFT in inhibiting infection - images taken of porcine corneas at day 4 and 6 of infection show comparable results in both TFT and BX795. Plaque assays performed to quantify secreted virions also reinforce these findings16. BX795 has also shown to have antiviral effects in vitro and its use topically in mouse models in vivo has also shown suppression of corneal HSV-1 infection16.

The study contributes to establishing BX795 as an effective and leading compound for broad-spectrum antiviral application against HSV-1 infection. By showing its efficacy in porcine models as comparable to TFT, BX795 stands to be successful in multiple infection models16. Additionally, BX795 is important as it has been shown to be effective in multiple virus strains, even HSV-1 (KOS)tk12 which is resistant to another widely used drug Acyclovir (ACV)16. BX975 treated cells show very little expression of HSV-1 viral protein gB which certify its inhibition efficacy of HSV-1 virus. BX795 is also demonstrates better anti-viral efficacy at lower doses compared to other treatments and anti-herpesvirus therapies. Furthermore, therapeutic concentrations of BX795 (even proposed concentration of 10 µM) show no adverse cytotoxicity towards cells - no apoptosis inducement and cell death, which is again comparable to the TFT control16.

Critical steps within the protocol section include isolation of porcine cornea from the whole eye. This involves the debridement of the cornea at the center of the eye using a needle and requires very little force to ensure no stromal involvement followed by excising the cornea from the ocular surface gently without disturbing the iris. Another set of critical steps include parts involving pre-wetting cotton tips and swabbing the corneal surface. The motion should be gentle to ensure corneal epithelium is not being dislodged from the ocular surface.

Modification can be done to the epithelial debridement step. Instead of using a 30 G needle to make a single poke at the center of the cornea, the experimenter can use a sterile blade or 30 G needle to gently make grid shaped scratches to the corneal epithelium. This ensures robust infection to the epithelium.

Porcine corneas should be used on the same day of procurement and should not be held for longer than 24 h. Porcine corneas kept in ice for longer than 4 h will result in the iris sticking to cornea. This makes it harder to separate the cornea from the rest of the eye during excision. Not all porcine corneas will get infected alike. The experimenter should infect a minimum of 5 eyes per group and then pick equally infected corneas on day 2 post infection to proceed with the experiment.

The significance of the current technique involves the cost effectiveness of using porcine over human corneas. The technique is also significant because of the relative freshness of the corneas being used when compared to human corneas.

Future applications of the current technique include but not limited to its use in testing drug permeability studies, ex vivo pharmacokinetic and pharmacodynamic studies. Porcine corneas can also be used to test antibacterial and antifungal drugs in addition to antiviral drugs. The corneas can also be used for ex vivo wound healing assays to study diabetic wounds.

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Disclosures

The authors declare no conflict of interest and no competing financial interests.

Acknowledgments

This study was supported by NIH grants (R01 EY024710, RO1 AI139768, and RO1 EY029426) to D.S. A.A. was supported by an F30EY025981 grant from the National Eye Institute, NIH.Study was conducted using the porcine corneas obtained from Park Packing company, 4107 Ashland Avenue, New City, Chicago, IL-60609

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

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References

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  9. Lass, J. H., et al. Antiviral medications and corneal wound healing. Antiviral Research. 4 (3), 143-157 (1984).
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Tags

Porcine Corneal Tissue Explant Model Herpes Simplex Virus-1 Antivirals Drug Efficacy Ex Vivo Tissue System In Vitro Systems Animal Models Human Trials Porcine Eyes Anatomy And Physiology Isolate Cornea Maintain Tissue Infect Tissue Treat With Drugs Porcine Eye Isolation Process
Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals
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Cite this Article

Yadavalli, T., Volety, I., Shukla,… More

Yadavalli, T., Volety, I., Shukla, D. Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals. J. Vis. Exp. (175), e62195, doi:10.3791/62195 (2021).

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