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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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. Drug....

Protokół

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 medi....

Wyniki

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.......

Dyskusje

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.......

Materiały

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	0	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