Name: porcine corneal tissue explant to study the efficacy of herpes simplex virus-1 antivirals
Uploaded: 2021-09-20T15:00:00
Description: Watch this Scientific Journal Video about Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals at JoVE.com

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

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
<ol>
	<li>Reagents
	<ol>
		<li>Use following reagents for Plaque assay: powder methylcellulose, Dulbecco&#39;s modified eagle&#39;s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin (P/S) for Plaque assay.</li>
		<li>Use crystal violet tablets and ethanol (mol...

<ol>
	<li>Liesegang, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+epidemiology+and+ocular+importance.">Herpes simplex virus epidemiology and ocular importance.</a> Cornea. 20 (1), 1-13 (2001).</li><li>Farooq, A. V., Valyi-Nagy, T., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediators+and+mechanisms+of+herpes+simplex+virus+entry+into+ocular+cells.">Mediators and mechanisms of herpes simplex virus entry into ocular cells.</a> Current Eye Research. 35 (6), 445-450 (2010).</li><li>Farooq, A. V., Shah, A., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+herpesviruses+in+ocular+infections.">The role of herpesviruses in ocular infections.</a> Virus Adaptation and Treatment. 2 (1), 115-123 (2010).</li><li>Xu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seroprevalence+and+coinfection+with+herpes+simplex+virus+type+1+and+type+2+in+the+United+States,+1988-1994.">Seroprevalence and coinfection with herpes simplex virus type 1 and type 2 in the United States, 1988-1994.</a> Journal of Infectious Diseases. 185 (8), 1019-1024 (2002).</li><li>Xu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+herpes+simplex+virus+type+1+and+type+2+seroprevalence+in+the+United+States.">Trends in herpes simplex virus type 1 and type 2 seroprevalence in the United States.</a> Journal of the American Medical Association. 296 (8), 964-973 (2006).</li><li>Koganti, R., Yadavalli, T., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+emerging+therapies+for+ocular+herpes+simplex+virus+type-1+infections.">Current and emerging therapies for ocular herpes simplex virus type-1 infections.</a> Microorganisms. 7 (10), (2019).</li><li>Lobo, A. -., Agelidis, A. M., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+herpes+simplex+keratitis:+The+host+cell+response+and+ocular+surface+sequelae+to+infection+and+inflammation.">Pathogenesis of herpes simplex keratitis: The host cell response and ocular surface sequelae to infection and inflammation.</a> Ocular Surface. 17 (1), 40-49 (2019).</li><li>Koujah, L., Suryawanshi, R. K., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+processes+activated+by+herpes+simplex+virus-1+(HSV-1)+infection+in+the+cornea.">Pathological processes activated by herpes simplex virus-1 (HSV-1) infection in the cornea.</a> Cellular and Molecular Life Sciences. 76 (3), 405-419 (2019).</li><li>Lass, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiviral+medications+and+corneal+wound+healing.">Antiviral medications and corneal wound healing.</a> Antiviral Research. 4 (3), 143-157 (1984).</li><li>Burns, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterisation+of+resistant+Herpes+simplex+virus+after+acyclovir+therapy.">Isolation and characterisation of resistant Herpes simplex virus after acyclovir therapy.</a> Lancet. 1 (8269), 421-423 (1982).</li><li>Crumpacker, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+antiviral+drugs+of+herpes+simplex+virus+isolated+from+a+patient+treated+with+Acyclovir.">Resistance to antiviral drugs of herpes simplex virus isolated from a patient treated with Acyclovir.</a> New England Journal of Medicine. 306 (6), 343-346 (2010).</li><li>Yildiz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+kidney+injury+due+to+acyclovir.">Acute kidney injury due to acyclovir.</a> CEN Case Report. 2 (1), 38-40 (2013).</li><li>Fleischer, R., Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyclovir+nephrotoxicity:+a+case+report+highlighting+the+importance+of+prevention,+detection,+and+treatment+of+acyclovir-induced+nephropathy.">Acyclovir nephrotoxicity: a case report highlighting the importance of prevention, detection, and treatment of acyclovir-induced nephropathy.</a> Case Rep Med. 2010, 1-3 (2010).</li><li>Thakkar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+corneas+show+dendritic+spread+and+restrict+herpes+simplex+virus+infection+that+is+not+observed+with+cultured+corneal+cells.">Cultured corneas show dendritic spread and restrict herpes simplex virus infection that is not observed with cultured corneal cells.</a> Science Report. 7, 42559 (2017).</li><li>Pescina, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al+Development+of+a+convenient+ex+vivo+model+for+the+study+of+the+transcorneal+permeation+of+drugs:+Histological+and+permeability+evaluation.">et al Development of a convenient ex vivo model for the study of the transcorneal permeation of drugs: Histological and permeability evaluation.</a> Journal of Pharmaceutical Sciences. 104, 63-71 (2015).</li><li>Jaishankar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+off-target+effect+of+BX795+blocks+herpes+simplex+virus+type+1+infection+of+the+eye.">An off-target effect of BX795 blocks herpes simplex virus type 1 infection of the eye.</a> Science Translational Medicine. 10, 5861 (2018).</li><li>Duggal, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+oxide+tetrapods+inhibit+herpes+simplex+virus+infection+of+cultured+corneas.">Zinc oxide tetrapods inhibit herpes simplex virus infection of cultured corneas.</a> Molecular Vision. 23, 26-38 (2017).</li></ol>

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 10&#181;M and using both western blot analysis and viral plaque analysis of key viral proteins, BX795 was shown to inhibit HSV-1 inf...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 G hypodermic needles.</td>
			<td>BD</td>
			<td>305128</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL glass bottle.</td>
			<td>Thomas Scientific</td>
			<td>844027</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimycotic and Antibiotic (AA)</td>
			<td>GIBCO</td>
			<td>15240096</td>
			<td>Aliquot into 5 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Benchtop vortexer.</td>
			<td>BioDot</td>
			<td>BDVM-3200</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet with a Bio-Safety Level-2 (BSL-2) certification.</td>
			<td>Thermofisher Scientific</td>
			<td>Herasafe 2030i</td>
			<td></td>
		</tr>
		<tr>
			<td>Calgiswab 6&#34; Sterile Calcium Alginate Standard Swabs.</td>
			<td>Puritan</td>
			<td>22029501</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper - 25 cm</td>
			<td>Biologix BE</td>
			<td>70-1180 70-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Sigma Aldrich</td>
			<td>C6158</td>
			<td>Store the powder in a dark place</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium - DMEM</td>
			<td>GIBCO</td>
			<td>41966029</td>
			<td>Store at 4 &#176;C until use</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum -FBS</td>
			<td>Sigma Aldrich</td>
			<td>F2442</td>
			<td>Aliquot into 50 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Flat edged tweezers &#8211; 2.</td>
			<td>Harward Instruments</td>
			<td>72-8595</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezers --80 &#176;C. -</td>
			<td>Thermofisher Scientific</td>
			<td>13 100 790</td>
			<td></td>
		</tr>
		<tr>
			<td>Fresh box of blades.</td>
			<td>Thomas Scientific</td>
			<td>TE05091</td>
			<td></td>
		</tr>
		<tr>
			<td>Guaze</td>
			<td>Johnson &#38; Johnson</td>
			<td></td>
			<td>108 square inch folder 12 ply</td>
		</tr>
		<tr>
			<td>HSV-1 17GFP</td>
			<td>grown in house</td>
			<td>-</td>
			<td>Original strain from Dr. Patricia Spears, Northwestern University. GFP expressing HSV-1 strain 17</td>
		</tr>
		<tr>
			<td>Insulin, Transferrin, Selenium - ITS</td>
			<td>GIBCO</td>
			<td>41400045</td>
			<td>Aliquot into 5 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Magnetic stirrer.</td>
			<td>Thomas Scientific</td>
			<td>H3710-HS</td>
			<td></td>
		</tr>
		<tr>
			<td>Metallic Scissors.</td>
			<td>Harward Instruments</td>
			<td>72-8400</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 1 to 1000 &#181;L.</td>
			<td>Thomas Scientific</td>
			<td>1159M37</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium - MEM</td>
			<td>GIBCO</td>
			<td>11095080</td>
			<td>Store at 4 &#176;C until use</td>
		</tr>
		<tr>
			<td>OptiMEM&#160;</td>
			<td>GIBCO</td>
			<td>31985047</td>
			<td>Store at 4 &#176;C until use</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin.</td>
			<td>GIBCO</td>
			<td>15140148</td>
			<td>Aliquot into 5 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline -PBS</td>
			<td>GIBCO</td>
			<td>10010072</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Porcine Corneas</td>
			<td>Park Packaging Co., Chicago, IL</td>
			<td>0</td>
			<td>Special order by request</td>
		</tr>
		<tr>
			<td>Procedure bench covers - as needed.</td>
			<td>Thermofisher Scientific</td>
			<td>S42400</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Thomas Scientific</td>
			<td>P7132, P7127, P7128, P7129, P7137</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipetting equipment.</td>
			<td>Thomas Scientific</td>
			<td>Ezpette Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>Carl Zeiss</td>
			<td>SteREO Discovery V20</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirring magnet.</td>
			<td>Thomas Scientific</td>
			<td>F37120</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks, T175 cm2.</td>
			<td>Thomas Scientific</td>
			<td>T1275</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture incubators which can maintain 5% CO2 and 37 &#176;C temperature.</td>
			<td>Thermofisher Scientific</td>
			<td>Forma 50145523</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture treated plates (6-well).</td>
			<td>Thomas Scientific</td>
			<td>T1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>GIBCO</td>
			<td>25-300-062</td>
			<td>Aliquot into 10 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>American Type Culture Collection ATCC</td>
			<td>CRL-1586</td>
			<td></td>
		</tr>
	</tbody>
</table>

porcine corneal tissue explant to study the efficacy of herpes simplex virus-1 antivirals

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.

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...

Watch this Scientific Journal Video about Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals at JoVE.com

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

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

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

Understanding the efficacy of drugs in animal models is cost and labor intensive. Our protocol which uses an ex vivo tissue explant model is significantly lower both in terms of cost and scientific personnel required to conduct the study. The main advantage of this technique is the use of porcine eyes, which have anatomy and physiology very similar to human eyes as opposed to some other small animal models.When we test drugs in vitro, they might show efficacy which might be absent in animal or human models. Ex vivo tissue systems are the best way to understand whether or not our in vitro systems can be translated into animal and human trials. The porcine cornea system, although less labor intensive, requires trainable skills to isolate, maintain, infect and treat with the drugs, especially the process of isolation of cornea from the entire porcine eye is tricky to express in words, but requires visual demonstration.Upon receiving porcine eyes from a suitable vendor, store the tissues on ice until processing and put on the appropriate personal protection equipment. Disinfect the work area with 70%ethanol and secure a bench cover onto the workspace. When the work area is ready, place the eyes onto a 50 millimeter piece of gauze and use the gauze to grasp the posterior section with one hand.Using a 30 gauge needle, carefully make a single puncture at approximately the center of the epithelial surface of the eye, taking care not to damage the stroma, and use a sharp sterilized blade to make a small incision on the sclera at a one millimeter distance from the cornea. Using a swift and smooth rotating action of the hand and taking care that the vitreous humor does not leak, cut the edge of the cornea and holding the cornea at the cornea-sclera edge with a flat tweezer, use the blade to cut off the remaining tethering membranes. As each cornea is isolated, place the tissues face up into individual wells of a 12-well plate containing two milliliters of cornea medium per well and add five microliters of a five times 10 to the fifth plaque-forming unit of 17 GFP virus solution to the debrided site on each corneal surface.When all of the corneas have been collected, place the plate into at 37 degree Celsius, 5%carbon dioxide incubator for 72 hours and spray any additional eyes not used in the experiment with 10%bleach for their secure disposal in biohazard bags. Every day before the addition of drugs, place the plate of corneas under a stereo microscope and select the GFP filter and a 500 microsecond exposure time. Capture the images at the lowest magnification before imaging the corneas at a series of increasing magnifications until all of the viral spread and dendrite formation can be visualized clearly.When all of the corneas have been imaged, return the plate to the tissue culture incubator. The day before the infection, wash a confluent vero cell culture two times with 10 milliliters of PBS per wash, and treat the cells with one milliliter of 0.05%trypsin per well for five to 10 minutes at 37 degrees Celsius. When the cells have detached, use nine milliliters of whole medium to ensure that the cells dislodge completely from the flask surface and transfer the resulting cell suspension to a 15 milliliter centrifuge tube.Add 300 microliters of cells to each well of a new six well-plate, followed by the addition of two milliliters of medium to each well, then place the plate in the cell culture incubator overnight. The next morning, to collect the virus from each cornea for quantification, soak sterile cotton swab tips in 500 microliters of serum-free medium per swab in multiple microcentrifuge tubes in a biosafety cabinet for at least five minutes. At the end of the incubation, carefully place the infected porcine cornea cultures into the cabinet.Using the wet cotton swabs, make three revolutions clockwise and three revolutions anti-clockwise five millimeters from the center of each infected porcine cornea without applying excessive pressure. At the end of the series of revolutions, rotate each swab in its serum-free medium filled microcentrifuge tube clockwise and anticlockwise five times before cutting the swabs so that the tips fit within each tube with the lid closed. When all of the corneas have been swabbed, vortex the tubes at high speed for one minute.To quantify the amount of virus collected from each cornea, prepare a log10 one-fold dilution of the virus in serum-free medium in microcentrifuge tubes until a dilution of one times 10 to the negative eight is reached. After aspirating the growth medium from each well of cells, transfer one milliliter of each virus dilution starting at one times 10 to the negative three to the plated cell monolayers and place the infected cells in the cell culture incubator for two hours. At the end of the incubation, gently wash each well two times with PBS before adding two milliliters of methylcellulose-laden medium to each well for a 72-hour incubation or until the formation of plaques can be observed.Upon plaque observation, slowly add one milliliter of methanol to the corner of each well for a 15-minute incubation at room temperature. At the end of the incubation, slowly aspirate the contents from each well without disturbing the cell monolayer. Next, label each well with one milliliter of crystal violet working solution, taking care that all of the cells are covered for a 30-minute incubation protected from light.At the end of the incubation, discard the solution and dry the wells on a sheet of absorbent paper, then count the number of plaques at the highest dilution well to quantify the total virus content in the starting solution three times. As observed in this stereoscopic fluorescence imaging analysis, the antiviral efficacy of BX795 is similar to that of TFT in controlling viral spread, while corneas treated with vehicle only demonstrate spread of the virus from the central infection zone to the periphery by six days post-initial viral inoculation. Similarly, the ocular swabs taken on days two and four post-infection exhibit 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 samples.The isolation of the porcine cornea from the whole porcine eye is the most important step. Steps 2.3 to 2.6 are the most crucial in this process. Our model can be used to understand the dendritic spread of the virus in porcine corneas.This method can be extended towards bacterial and fungal infections as well. This method has helped in visualizing dendritic spread of the virus in corneas outside a human model. This model is helpful in testing the efficacy of drugs prior to moving towards animal and human trials.

Research

JoVE Journal

Immunology and Infection

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...