Name: ex vivo infection of murine epidermis with herpes simplex virus type 1
Uploaded: 2015-08-24T15:00:00.000+00:00
Duration: 11 min 56 s
Description: Watch this Scientific Journal Video about Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1 at JoVE.com

We thank Peter Staeheli for providing B6.A2G-Mx1 mice and Semra &#214;zcelik for technical advice.
This work was supported by the German Research Foundation through SFB829 and KN536/16, and the K&#246;ln Fortune Program/Faculty of Medicine, University of Cologne.

Ethics statement.
The preparation of epidermal sheets from sacrificed animals is carried out in strict accordance with the recommendations of the Guide of Landesamt f&#252;r Natur, Umwelt and Verbraucherschutz, Northrhine-Westphalia (Germany). The study was approved by LANUV NRW (Number 8.84-02.05.20.13.018).
1. Preparation of Instruments and Culture Media
<ol>
	<li>Cultivate epidermal sheets in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)/high glucose containing glutamine dipeptide, 10% FCS, 100 IU/ml penicillin, and 100 &#181;g/ml streptomycin (hereinafter referred to as &#8220;DMEM&#8221;).</li>
	<li>For the preparation of epidermal sheets, carefully dissolve dispase II powder (5 mg/ml) in heated PBS (up to 37 &#176;C). Filter the solution through a 0.22 &#181;m filter and use it directly for treatment.</li>
	<li>For the preparation of epidermal whole mounts13, prepare blocking buffer with 0.5% milk powder, 0.25% gelatin from cold water fish skin and 0.5% Triton X-100 in TBS. Allow substances to dissolve by incubating the mixture for at least 2 hr&#160;at RT on a rotating mixer. Also prepare 0.2% Tween 20 in PBS as washing buffer. For fixation, prepare 3.4% and 4% formaldehyde in PBS.</li>
	<li>For the immunostaining of cryosections, prepare blocking buffer with 5% normal goat serum and 0.2% Tween 20 in PBS. For fixation, prepare 0.5% formaldehyde in PBS.</li>
</ol>
2. Preparation of Epidermal Sheets from Murine Skin
<ol>
	<li>Preparation of epidermis from newborn back skin for infection studies
	<ol>
		<li>Place a decapitated mouse (1 to 3 days after birth) into a dissection dish and cut off extremities and the tail close to the torso to allow efficient removal of the skin from the complete torso. During removal of the extremities, try to keep the cuts as small as possible in diameter.</li>
		<li>Fix the torso with curved fine forceps and gently move blunt tip scissors underneath the skin from cranial to caudal along the back to separate the skin from the torso. Slit the skin along the back.</li>
		<li>For FACS analysis, isolation of keratinocytes or preparation of RNA, peel off the complete skin from the torso using forceps to fix the tissue and the blunt tip scissors to push off the torso. For the preparation of cryosections or whole mounts take only pieces of the back skin.</li>
		<li>Chop the back skin with a scalpel into 1-2 pieces of approximately 10 x 10 mm. Put the pieces in one well of a 6-well plate with ~1 ml sterile PBS. Make sure that the dermal side faces the bottom.</li>
		<li>Replace PBS by 1.5 ml dispase II (5 mg/ml PBS) and incubate for either 30 min at 37 &#176;C (for whole mounts) or O/N at 4 &#176;C. Wash 3 times with PBS. Gently remove the epidermis as an intact sheet using one forceps to fix the underlying dermis and the other forceps to lift the epidermis. Use a binocular to make this step easy. Transfer the epidermal sheet into DMEM with its basal side towards the bottom. Use the sheets immediately for infection experiments. 
		NOTE: Use the preparation of epidermis from newborn skin mainly for FACS analysis, isolation of keratinocytes or preparation of RNA. Because of its fragility, the preparation of infected sheets for cryosections or whole mounts is less suitable.</li>
	</ol>
	</li>
	<li>Preparation of epidermis from adult tail skin for infection studies
	<ol>
		<li>Euthanize the mice at the age of 1 to 3 months by cervical dislocation. Take tail instead of back skin since the long embedded hair follicles on the back hamper separation of intact epidermal sheets. Cut off the tail from sacrificed mice and slit it lengthwise with a scalpel.</li>
		<li>Peel off the skin from the bone and chop it with a scalpel into pieces of about 5 x 5 mm. Put up to 4 pieces in one well and proceed with the incubation in dispase II as described under 2.1.5 
		NOTE: Use adult tail skin to prepare cryosections or whole mounts. Alternatively to the immediate use, tails can be stored or transported for approximately 24 hr&#160;at 4 &#176;C without losing significant infection efficiency. If tails are kept up to 48 hr, epidermal sheets could be hardly infected with HSV-1.</li>
	</ol>
	</li>
	<li>Dissociation of epidermis for FACS analysis 
	NOTE: Detection of surface protein expression by FACS analysis requires appropriate cell dissociation techniques that do not harm the cell surface and the protein of interest.
	<ol>
		<li>Incubate newborn epidermal sheets in a 6-well plate with the basal side on 1.5 ml/well recombinant trypsin-based cell dissociation solution for 15 min at RT and for 15 min at 37 &#176;C.</li>
		<li>Stop the incubation by adding 3 ml DMEM. Dissociate keratinocytes by using a curved fine forceps to gently move the epidermis in the 6-well plate so that the keratinocytes get dissolved. Collect the cell suspension and repeat this step 3 times always using fresh DMEM. 
		NOTE: This dissociation procedure is appropriate to detect nectin-1 on the surface of keratinocytes.</li>
		<li>Alternatively, incubate the epidermal sheets on enzyme-free cell dissociation solution (CDS) for 15 min at RT and 15 min at 37 &#176;C. Incubate another 15 min at RT to gently flush out the keratinocytes by pipetting CDS up and down.</li>
		<li>Collect the cell suspension and repeat the flushing step 3 times using fresh DMEM. 
		NOTE: This dissociation is suitable to detect surface expression of HVEM. The disadvantage of the CDS solution is that fewer cells are dissociated than with recombinant trypsin-based cell dissociation solution. In addition to surface expression, nuclear or cytoplasmic expression such as ICP0 expression can be analyzed by FACS after dissociation of the infected epidermal sheets with recombinant trypsin-based cell dissociation solution.</li>
	</ol>
	</li>
	<li>Dissociation of epidermis to isolate keratinocytes or extract RNA
	<ol>
		<li>Put newborn epidermal sheets in a 6-well plate with the basal side towards the bottom of the dish which contains 1.5 ml/well recombinant trypsin-based cell dissociation solution. Incubate for 30 min at RT. Add 3 ml DMEM and dissociate keratinocytes by using a curved fine forceps to gently move the epidermis in the dish.</li>
		<li>Collect the cell suspension and repeat this step 3 times using fresh DMEM. Use this cell suspension to extract RNA or to culture primary murine keratinocytes.</li>
	</ol>
	</li>
</ol>
3. Infection of Epidermal Sheets with HSV-1
<ol>
	<li>Prepare a solution of HSV-1 wt strain 1711 in not less than 500 &#181;l DMEM and replace the medium by the virus solution on which the epidermal sheets are floating. This time point is defined as time 012. Perform infection of an epidermal sheet in one well of a 24-well plate at 37 &#176;C. The calculation of the virus titer is based on the estimated number of cells in the basal layer per epidermal sheet (approximately 2 x 105 cells per 5 x 5 mm sheet).</li>
	<li>Infect with HSV-1 at approximately 100 plaque forming units (PFU)/cell and replace the virus-containing medium by DMEM at 1 hr p.i. &#160; 
	NOTE: In case of epidermal sheets from newborn skin, keep in mind that sheets start to dissociate during incubation.</li>
</ol>
4. Visualization of Infected Cells
<ol>
	<li>Epidermal Whole Mounts13
	<ol>
		<li>Fix epidermal sheets with 3.4% formaldehyde either for 2 hr&#160;at RT or O/N&#160;at 4 &#176;C. Wash two times with PBS and block with 0.5% milk powder, 0.25% gelatin from cold water fish skin and 0.5% Triton X-100 in TBS for 1 hr at RT14.</li>
		<li>Incubate the sheets O/N with mouse anti-ICP0 (monoclonal antibody 11060)15 diluted 1:60 in blocking buffer at RT. To visualize the intermediate filament network incubate simultaneously with rabbit polyclonal anti-mouse keratin 14 (100 ng/ml).</li>
		<li>Wash 4 to 5 times with PBS-0.2% Tween 20 during 4 hr at RT. Incubate O/N with AF488-conjugated anti-mouse IgG (1 &#181;g/ml), AF555-conjugated anti-rabbit IgG (1 &#181;g/ml), and DAPI (4&#8217;, 6-diamidino-2-phenylindole) (100 ng/ml) in blocking buffer at RT.</li>
		<li>Wash with PBS-0.2% Tween 20 for 4 hr&#160;at RT. Mount epidermal sheets with their basal side on top of a specimen slide, embed in fluorescent mounting medium and cover with coverslips.</li>
	</ol>
	</li>
	<li>Epidermal Cryosections
	<ol>
		<li>Adult epidermal sheets were fixed in 4% formaldehyde in PBS for 20 min at RT and washed 2 times with PBS. Embed fixed sheets in tissue freezing medium and freeze in liquid nitrogen. Cut 8-10 &#181;m sections with a cryomicrotome.</li>
		<li>Fix the sections again with 0.5% formaldehyde in PBS for 10 min at RT, wash 2 times with PBS, block with 5% normal goat serum and 0.2% Tween 20 in PBS for 30 min at RT, and then stain for 60 min with mouse anti-ICP0 (monoclonal antibody 11060)15 diluted 1:60 in blocking buffer, followed by 3 times washing with the blocking buffer.</li>
		<li>Then incubate with AF488-conjugated anti-mouse IgG (1 &#181;g/ml) and DAPI (100 ng/ml) in blocking buffer for 45 min at RT and wash 3 times. Embed sections in fluorescent mounting medium and cover with coverslips.&#160;</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Koelle, D. M., Corey, L. <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:+insights+on+pathogenesis+and+possible+vaccines.">Herpes simplex: insights on pathogenesis and possible vaccines.</a> Annu. Rev. Med. 59, 381-395 (2013).</li><li>Roizman, B., Knipe, D. M., Whitley, R. 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+Viruses.">Herpes simplex Viruses.</a> Fields Virology. , 1823-1897 (2013).</li><li>Syrj&#228;nen, S., Mikola, H., Nyk&#228;nen, M., Hukkanen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+establishment+of+lytic+and+nonproductive+infection+by+herpes+simplex+virus+type+1+in+three-dimensional+keratinocyte+culture.">In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture.</a> J. Virol. 70, 6524-6528 (1996).</li><li>Visalli, R. J., Courtney, R. J., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+and+replication+of+herpes+simplex+virus+type+1+in+an+organotypic+epithelial+culture+system.">Infection and replication of herpes simplex virus type 1 in an organotypic epithelial culture system.</a> Virology. 230, 236-243 (1997).</li><li>Hukkanen, V., Mikola, H., Nyk&#228;nen, M., Syrj&#228;nen, S. <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+type+1+infection+has+two+separate+modes+of+spread+in+three-dimensional+keratinocyte+culture.">Herpes simplex virus type 1 infection has two separate modes of spread in three-dimensional keratinocyte culture.</a> J. Gen. Virol. 80, 2149-2155 (1999).</li><li>Gescher, K., 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=Inhibition+of+viral+adsorption+and+penetration+by+an+aqueous+extract+from+Rhododendron+ferrugineum+L.+as+antiviral+principle+against+herpes+simplex+virus+type-1.">Inhibition of viral adsorption and penetration by an aqueous extract from Rhododendron ferrugineum L. as antiviral principle against herpes simplex virus type-1.</a> Antiviral Res. 82, 408-413 (2010).</li><li>Petermann, P., Haase, I., Knebel-M&#246;rsdorf, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Rac1+and+Cdc42+signaling+during+early+herpes+simplex+virus+type+1+infection+of+keratinocytes.">Impact of Rac1 and Cdc42 signaling during early herpes simplex virus type 1 infection of keratinocytes.</a> J. Virol. 83, 9759-9772 (2009).</li><li>Everett, R. D., Maul, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSV-1+IE+protein+Vmw110+causes+redistribution+of+PML.">HSV-1 IE protein Vmw110 causes redistribution of PML.</a> EMBO J. 13, 5062-5069 (1994).</li><li>Chrostek, A., 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=Rac1+is+crucial+for+hair+follicle+integrity+but+is+not+essential+for+maintenance+of+the+epidermis.">Rac1 is crucial for hair follicle integrity but is not essential for maintenance of the epidermis.</a> Mol. Cell. Biol. 26, 6957-6970 (2006).</li><li>Petermann, P., 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=Entry+mechanisms+of+Herpes+Simplex+Virus+Type+1+into+murine+epidermis:+Involvement+of+nectin-1+and+HVEM+as+cellular+receptors.">Entry mechanisms of Herpes Simplex Virus Type 1 into murine epidermis: Involvement of nectin-1 and HVEM as cellular receptors.</a> J. Virol. 89, 262-274 (2015).</li><li>McGeoch, D. J., 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=The+complete+DNA+sequence+of+the+long+unique+region+in+the+genome+of+herpes+simplex+virus+type+1.">The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1.</a> J. Gen. Virol. 69, 1531-1574 (1988).</li><li>Schelhaas, M., Jansen, M., Haase, I., Knebel-M&#246;rsdorf, D. <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+type+1+exhibits+a+tropism+for+basal+entry+in+polarized+epithelial+cells.">Herpes simplex virus type 1 exhibits a tropism for basal entry in polarized epithelial cells.</a> J. Gen. Virol. 84, 2473-2484 (2003).</li><li>Braun, K. M., Niemann, C., Jensen, U. B., Sundberg, J. P., Silva-Vargas, V., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+stem+cell+proliferation+and+lineage+commitment:+visualisation+of+label-retaining+cells+in+wholemounts+of+mouse+epidermis.">Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis.</a> Development. 130, 5241-5255 (2003).</li><li>Rahn, E., Petermann, P., Hsu, M. J., Rixon, F. J., Knebel-M&#246;rsdorf, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+pathways+of+herpes+simplex+virus+type+1+into+human+keratinocytes+are+dynamin-+and+cholesterol-dependent.">Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent.</a> PLoS One. 6, e25464 (2011).</li><li>Everett, R. D., Cross, A., Orr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+truncated+form+of+herpes+simplex+virus+type+1+immediate-early+protein+Vmw110+is+expressed+in+a+cell+type+dependent+manner.">A truncated form of herpes simplex virus type 1 immediate-early protein Vmw110 is expressed in a cell type dependent manner.</a> Virology. 197, 751-756 (1993).</li><li>Horisberger, M. A., Staeheli, P., Haller, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+induces+a+unique+protein+in+mouse+cells+bearing+a+gene+for+resistance+to+influenza+virus.">Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus.</a> Proc. Natl. Acad. Sci. USA. 80, 1910-1914 (1983).</li><li>Christian, A. E., Haynes, M. P., Phillips, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+cyclodextrins+for+manipulating+cellular+cholesterol+content.">Use of cyclodextrins for manipulating cellular cholesterol content.</a> J. Lipid Res. 38, 2264-2272 (1997).</li><li>Ilangumaran, S., Hoessli, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cholesterol+depletion+by+cyclodextrin+on+the+sphingolipid+microdomains+of+the+plasma+membrane.">Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane.</a> Biochem. J. 335, 433-440 (1998).</li><li>Yancey, P. G., 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=Cellular+cholesterol+efflux+mediated+by+cyclodextrins:+Demonstration+of+kinetic+pools+and+mechanism+of+efflux.">Cellular cholesterol efflux mediated by cyclodextrins: Demonstration of kinetic pools and mechanism of efflux.</a> J. Biol. Chem. 271, 16026-16034 (1996).</li><li>Montgomery, R. I., Warner, M. S., Lum, B. J., Spear, P. G. <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-1+entry+into+cells+mediated+by+a+novel+member+of+the+TNF/NGF+receptor+family.">Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family.</a> Cell. 87, 427-436 (1996).</li><li>Geraghty, R. J., Krummenacher, C., Cohen, G. H., Eisenberg, R. J., Spear, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+alphaherpesviruses+mediated+by+poliovirus+receptor-related+protein+1+and+poliovirus+receptor.">Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.</a> Science. 280, 1618-1620 (1998).</li><li>Taylor, J. M., Lin, E., Susmarski, N., Yoon, M., Zago, A., Ware, C. F., Pfeffer, K., Miyoshi, J., Takai, Y., Spear, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+entry+receptors+for+herpes+simplex+virus+and+their+roles+in+disease.">Alternative entry receptors for herpes simplex virus and their roles in disease.</a> Cell Host Microbe. 2, 19-28 (2007).</li><li>Barron, M. J., Brookes, S. J., Draper, C. E., Garrod, D., Kirkham, J., Shore, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+adhesion+molecule+nectin-1+is+critical+for+normal+enamel+formation+in+mice.">The cell adhesion molecule nectin-1 is critical for normal enamel formation in mice.</a> Hum. Mol. Genet. 17, 3509-3520 (2008).</li><li>Wang, Y., Subudhi, S. K., Anders, R. A., Lo, J., Sun, Y., Blink, S., Wang, Y., Liu, X., Mink, K., Degrandi, D., Pfeffer, K., Fu, Y. X. <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+herpesvirus+entry+mediator+as+a+negative+regulator+of+T+cell-mediated+responses.">The role of herpesvirus entry mediator as a negative regulator of T cell-mediated responses.</a> J. Clin. Invest. 115, 711-717 (2005).</li></ol>

The authors declare that they have no competing financial interests.

When epidermal sheets of adult skin from C57BL/6 are infected with HSV-1 at approximately 100 PFU/cell, we observe infection in nearly all cells of the basal layer in the interfollicular epidermis while lower virus doses correlate with less infected cells and a slower progress of infection. In general, hair follicles show a variable number of infected cells; while most of the rather small keratinocytes lining the developing hair follicles are infected, only the hair germ of the adult hair follicle is completely infected....

Herpes simplex virus (HSV) can cause a range of diseases in humans from mild uncomplicated mucocutaneous lesions to life-threatening infections. HSV type 1 (HSV-1) is dominantly associated with orofacial infections and encephalitis, whereas HSV type 2 (HSV-2) more likely causes genital infections1. While there is significant progress in understanding how HSV enters cells in culture, initiates infection and produces viral progeny, we know little about the viral invasion pathway(s) into tissue at the cellular level2. For studies of HSV skin or mucosal infections, mice, rabbits and guinea pigs have been used as animal models. Skin infection was established by intradermal injection or by scratching the skin in the presence of virus, and disease development was correlated with virus production. These methods helped to understand various aspects of disease pathogenesis, and are used to evaluate antiviral drugs. To study HSV infection at the tissue level, organotypic human skin models have been applied. As the rate of infection is restricted in these raft cultures, only a limited number of studies investigating infection, viral spread and the effects of antiviral components have been published3-6.
In order to characterize cellular determinants that play a role during HSV-1 infection in the intact epithelium, we established a protocol for ex vivo infection studies of murine epidermis7. Skin was prepared from newborns or from the tails of adult mice. Since HSV-1 could not infect complete skin samples, which were submerged in virus-containing medium, we separated the epidermis from the dermis by dispase II treatment. After floating of the epidermal sheets on virus-containing medium, infected cells can be visualized in the epidermal basal layer at various times postinfection (p.i.)7. To visualize the initiation of infection in individual cells prior to viral replication and virus production, we stained with an antibody against the infected-cell protein&#160;0 (ICP0), which is one of the first proteins expressed during HSV-1 infection. The cellular localization of ICP0 passes through distinct phases during early infection. While ICP0 is present in nuclear foci during an early stage of viral gene expression, relocalization of ICP0 to the cytoplasm indicates a subsequent phase of infection8.
We used the ex vivo infection assay of epidermal sheets from different mouse models to test the potential role of various cellular factors during infection. To address the impact of Rac1 as a key regulator of actin dynamics, we infected the epidermis of mice with a keratinocyte-specific deletion of the rac1 gene9. This model allowed us to study the consequences of deficient Rac1 on the efficiency of HSV-1 infection in epidermal keratinocyte layers. The comparison to infected epidermis of control littermates revealed no significant difference, indicating that the absence of Rac1 had no effect on the initiation of infection in the basal layer of the epidermis7. The use of further mouse models allowed us to address which cellular receptors mediate entry into the epidermis. Infecting epidermal sheets from either nectin-1- or HVEM-deficient mice with HSV-1 revealed that the initial viral entry into tissue strongly depends on the presence of nectin-110. Furthermore, our results demonstrate that HVEM can also serve as receptor in murine epidermis, although less efficiently than nectin-110.
To address the spatial distribution of infected cells in the epidermal layers, we visualize ICP0 expression in tissue sections and epidermal whole mounts (Figure 1). In cryosections of complete skin, no ICP0-expressing cells are detected (Figure 1). In contrast, cryosections of epidermal sheets demonstrate cytoplasmic ICP0 expression in the basal layer already at 3 hours p.i. (Figure 1). At later times, viral spreading to suprabasal layers can be visualized. The spatial distribution of infected cells in the basal layer can be easily followed in epidermal whole mounts (Figure 1). Upon infection with HSV-1 at 100 PFU/cell, approximately 50% of the basal keratinocytes in the interfollicular epidermis show ICP0 expression at 1.5 hr p.i. At this time point most infected cells express nuclear ICP0. The relocalization of ICP0 to the cytoplasm indicating a later stage of early gene expression is present in nearly all cells at 3 hr p.i. (Figure 1). These modes of visualizing infected cells upon ex vivo HSV-1 infection provide a powerful assay to study the effect of inhibitors or of deleted/mutated cellular components on viral entry and spread in tissue.

<table><tbody><tr><td>DMEM/high glucose/GlutaMAX</td><td>Life Technologies</td><td>31966047</td><td>needed for cultivation of epidermal sheets</td></tr><tr><td>dispase II powder</td><td>Roche</td><td>4942078001</td><td>has to be solved in heated PBS</td></tr><tr><td>enzyme-free cell dissociation solution</td><td>Sigma</td><td>C5914</td><td>needed for very gentle dissociation of epidermal sheets</td></tr><tr><td>TrypLE select cell dissociation solution</td><td>Life Technologies</td><td>12563-029</td><td>needed for dissociation of epidermal sheets</td></tr><tr><td>chelex 100 resin</td><td>Bio-Rad</td><td>142-2832</td><td>needed for chelation of polyvalent metal ions from the fetal calf serum</td></tr><tr><td>gelatin from cold water fish skin&amp;nbsp;</td><td>Sigma</td><td>G7765</td><td>needed for minimization of non-specific antibody binding</td></tr><tr><td>Keratin 14 Polyclonal Antibody (AF64) (conc.: 1 mg/ml)</td><td>Covance</td><td>PRB-155P</td><td>used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis</td></tr><tr><td>Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (conc.: 2 mg/ml)</td><td>Life Technologies</td><td>A-11029</td><td>used as secondary antibodies</td></tr><tr><td>Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate (conc.: 2 mg/ml)</td><td>Life Technologies</td><td>A-21429</td><td>used as secondary antibodies</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (conc.: 0.1 mg/ml)</td><td>Sigma</td><td>36670</td><td>used to counterstain the nucleus</td></tr></tbody></table>

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.

The challenge of the method is to prepare epidermal sheets into which HSV-1 can penetrate from the basal layer. The critical step is the separation of the epidermis from the dermis by dispase II treatment, which, depending on the mouse strain, needs to be adapted. The concentration of dispase II can range from 2.5 to 5 mg/ml, and the time of incubation from 20 to 45 min. The staining of the intermediate filament protein keratin 14 readily allows predicting whether the basal epidermal layer can be infected or whether it w...

Watch this Scientific Journal Video about Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1 at JoVE.com

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.

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 ...

ex-vivo-infection-of-murine-epidermis-with-herpes-simplex-virus-type-1

Research

JoVE Journal

Immunology and Infection

11.1K Views.  University of Cologne. 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.

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

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

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 ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

Neuroscience

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

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.

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 ...

Plaquing of Herpes Simplex Viruses

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can ...

siRNA-Based Inhibition of Autophagy in Herpes Simplex Virus-Infected Immature Dendritic Cells

Source: D&#252;thorn, A., et al. <a href="https://app.jove.com/v/60190">siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells.</a> J. Vis. Exp. (2019).
This video demonstrates a small interfering RNA (siRNA)-based technique to interfere with autophagic flux. A siRNA targeting FIP200 mRNA is introduced into monocyte-derived immature dendritic cells (iDCs) using electroporation, causing the knockdown of FIP200 expression and leading to the inhibition of nuclear lamin disruption via autophagy. Upon adding herpes simplex virus (HSV), the nuclear egress of viral progeny is inhibited due to the absence of lamin disruption via autophagic flux.

Monocyte-derived DCs were generated from leukapheresis products of healthy donors. For this, a positive vote from the local ethics committee has been ...

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

Summary

Explore More Videos

Chapters in this video

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

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

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

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

Plaquing of Herpes Simplex Viruses

siRNA-Based Inhibition of Autophagy in Herpes Simplex Virus-Infected Immature Dendritic Cells