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 (molecular biology grade) for preparing crystal violet solution for plaque assay.</li>
	</ol>
	</li>
	<li>Vero cell growth media - DMEM whole media
	<ol>
		<li>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 &#176;C.</li>
	</ol>
	</li>
	<li>Plaque assay media - Stock of 5% methylcellulose media
	<ol>
		<li>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.</li>
		<li>Stir this at 4 &#176;C for 2 days using a magnetic stirrer. Refrigerate at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Media preparation for excised porcine cornea
	<ol>
		<li>Open a minimum essential media (MEM) bottle inside the tissue culture hood. Discard 10 mL of MEM from the bottle using a serological pipette.</li>
		<li>Add 5 mL of insulin-transferrin-selenium (ITS) and 5 mL of antimycotic-antibiotic (AA) to the media and refrigerate at 4&#176;C.</li>
	</ol>
	</li>
	<li>Master and working stock of crystal violet:
	<ol>
		<li>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.</li>
		<li>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). 
		&#8203;NOTE: Both these solutions need to be stored in the dark.</li>
	</ol>
	</li>
</ol>
2. Procedure
<ol>
	<li>Isolation of Porcine corneas from whole eyes17
	<ol>
		<li>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.</li>
		<li>Ensure that personal protection equipment is used and worn during this procedure to avoid contamination as well as accidents from spillage of vitreous humor.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#181;m) to avoid stromal involvement.</li>
		<li>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).</li>
		<li>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).</li>
		<li>Add 5 &#181;L 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.</li>
		<li>Spray any additional eyes not used in experiment with 10% bleach and securely disposed in biohazard bags.</li>
	</ol>
	</li>
	<li>Visualization 
	NOTE: The virus should be visualized every day prior to addition of drugs.
	<ol>
		<li>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.</li>
		<li>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</li>
		<li>Make sure to return the infected corneas back into the tissue culture incubator and save all the images that were taken.</li>
	</ol>
	</li>
	<li>Virus infection quantification 
	NOTE: Virus titers from porcine corneas should be evaluated every day to analyze the effect of drug treatment.
	<ol>
		<li>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.</li>
		<li>Aliquot 500 &#181;L 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Place the microcentrifuge tube containing the swabbed cotton tip on a vortex machine and vortex at high speed for 1 min.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Add 1 mL of 0.05% Trypsin to the cell monolayer. Incubate the flask at 37 &#176;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.</li>
		<li>Collect all the cells from the flask along with the whole media and place them in a 15 mL centrifuge tube.</li>
		<li>For every well of the 6 well plates that are used for plaque assay, use 300 &#181;L 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.</li>
		<li>In order to perform a plaque assay, perform a serial dilution of samples needs to be conducted before the quantification of virus.</li>
		<li>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 &#176;C incubator for 2 h.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<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>

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

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 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 &#181;M) 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.

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 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62195/62195fig01.jpg" /> 
Figure 1: Porcine eyes kept on ice until tissue is processed. <a href="https://www.jove.com/files/ftp_upload/62195/62195fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62195/62195fig02.jpg" /> 
Figure 2: Work bench setup. <a href="https://www.jove.com/files/ftp_upload/62195/62195fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62195/62195fig03.jpg" /> 
Figure 3: Porcine eyes placed on gauze. <a href="https://www.jove.com/files/ftp_upload/62195/62195fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62195/62195fig04.jpg" /> 
Figure 4: Porcine eye with 30G needle pictured. <a href="https://www.jove.com/files/ftp_upload/62195/62195fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62195/62195fig05.jpg" /> 
Figure 5: 30 G needle used; hole made at center of epithelial surface. <a href="https://www.jove.com/files/ftp_upload/62195/62195fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62195/62195fig06.jpg" /> 
Figure 6: Rotating action used to cut around corneal edge using sharp sterilized blade (A,B). <a href="https://www.jove.com/files/ftp_upload/62195/62195fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62195/62195fig07.jpg" /> 
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 <a href="https://www.jove.com/files/ftp_upload/62195/62195fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62195/62195fig08.jpg" /> 
Figure 8: Corneas placed in 12-well plate and incubated for 72 h. <a href="https://www.jove.com/files/ftp_upload/62195/62195fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62195/62195fig09.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/62195/62195fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Immunology and Infection

2.9K Views.  University of Illinois at Chicago. We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

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

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 malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

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 reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

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

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

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

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of 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 assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

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

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 during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

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

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

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 currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B 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 methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

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

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

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

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

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

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Chapters in this video

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

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

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

Assays for the Identification of Novel Antivirals against Bluetongue Virus

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

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

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

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy