The authors would like to thank Dr. Meenhard Herlyn (Wistar Institute) and Dr. M. Celeste Simon (University of Pennsylvania) for providing reagents and technical support. We also thank the members of our laboratories for helpful discussion. This work was supported by the National Institutes of Health (NIH) grants (R01CA187718, R01CA148768 and R01CA142723), the NCI Cancer Center Support Grant (NCI P30 CA016520), and the Penn CFAR award (P30 AI 045008).

Human neonatal foreskins were obtained from Penn Skin Disease Research Center. Adult human fibroblasts were obtained from discarded normal skin after surgery. All the protocols were approved by the University of Pennsylvania Institutional Review Board.
1. Isolation of human dermal fibroblasts
<ol>
	<li>Use a pair of scissors to trim off fat and subcutaneous tissue from the human neonatal foreskin and cut the skin sample in halves or quarters.</li>
	<li>Incubate the tissue in 5 mL of 10 mg/mL dispase II in PBS supplemented with antibiotic-antimycotic at 4 &#176;C overnight.</li>
	<li>In a sterile 10 cm dish, carefully separate the epidermis from the dermal layers using microdissection forceps.</li>
	<li>Transfer the resulting dermal tissue to a 15 mL conical tube containing 5 mL of 2 mg/mL collagenase type IV in FBS-free medium supplemented with antibiotic-antimycotic.</li>
	<li>Incubate the sample at 37 &#176;C in 5% CO2 for 4-6 h with periodic shaking until just a few macroscopic tissue aggregates remain.</li>
	<li>Release single cells from the dermis by pipetting up and down (triturating) vigorously ten times with a 10 mL pipette.</li>
	<li>Centrifuge at 180 x g for 5 min, and discard the supernatant.</li>
	<li>Plate the dissociated cells in DMEM medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, and 1% L-glutamine at 37 &#176;C in 5% CO2.</li>
</ol>
2. Recombinant MCPyV virion preparation
<ol>
	<li>Digest 50 &#181;g of pR17b plasmid (carrying MCPyV genome) with 250 U of BamHI-HF in a 200 &#181;L volume (4 h at 37 &#176;C) to separate the viral genome from the vector backbone (Figure 2).</li>
	<li>Add 1200 &#181;L of buffer PB (supplemented with 10 &#181;L of 3 M NaAc, pH 5.2) to the digested DNA and purify over 2 miniprep spin columns (20 &#181;g DNA capacity). Elute the digested pR17b plasmid from each column with 200 &#181;L of TE buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA).</li>
	<li>Prepare the ligation reaction in a 50 mL centrifuge tube. Add 400 &#181;L of purified plasmid DNA from step 2.2, 8.6 mL of 1.05x T4 ligase buffer and 6 &#181;L of high concentration T4 ligase. Incubate at 16 &#176;C overnight.</li>
	<li>Add 45 mL of buffer PB (supplemented with 10 &#181;L of 3 M NaAc, pH 5.2) to the ligation and use a vacuum manifold to load through 2 miniprep spin columns. Elute each column with 50 &#181;L of TE buffer. Expect a yield of about 30 &#181;g of DNA.</li>
	<li>In the late afternoon/evening, seed 6 x 106 293TT28 cells into a 10 cm dish containing DMEM medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, and 1% L-glutamine without hygromycin B.</li>
	<li>The next morning, ensure that the cells are about 50% confluent. Transfect using 66 &#181;L of transfection reagent (1.1 &#181;L/cm2), 12 &#181;g of re-ligated MCPyV isolate R17b DNA from step 2.4, 8.4 &#181;g of ST expression plasmid pMtB and 9.6 &#181;g of LT expression plasmid pADL*29.</li>
	<li>When the transfected cells are nearly confluent, the following day, trypsinize the cells and transfer them to a 15 cm dish for continued expansion.</li>
	<li>Optionally take a small number of 293TT cells upon expansion and perform IF staining for MCPyV LT (CM2B4) and VP1 (MCV VP1 rabbit30) to determine transfection efficiency. At this stage, nuclear LT signal may be visible but VP1 expression probably will not be detectable.</li>
	<li>When the 15 cm dish becomes nearly confluent (usually 5-6 days after initial transfection), transfer the cells into three 15 cm dishes. Harvest the cells from the 15 cm dishes when they become nearly confluent and follow the virus harvest protocol below. 
	NOTE: Optionally perform quality control IF as described in step 2.8. Most of the cells should be both MCPyV LT and VP1 positive at this stage.</li>
	<li>To harvest the virus, trypsinize cells, spin at 180 x g for 5 min at RT and remove the supernatant. Add one cell pellet volume of DPBS-Mg (DPBS with 9.5 mM MgCl2 and 1x antibiotic-antimycotic). Then add 25 mM ammonium sulfate (from a 1 M pH 9 stock solution) followed by 0.5% Triton X-100 (from a 10% stock solution), 0.1% Benzonase and 0.1% of an ATP-Dependent DNase. Mix well and incubate at 37 &#176;C overnight.</li>
	<li>Incubate the mixture for 15 min on ice and then add 0.17 volume of 5M NaCl. Mix and incubate on ice for another 15 min. Spin for 10 min at 12,000 x g in a 4 &#176;C centrifuge. If the supernatant is not clear, gently invert the tube and repeat the spinning step. Transfer the supernatant to a new tube.</li>
	<li>Resuspend the pellet using one volume of DPBS supplemented with 0.8 M NaCl, and spin again, as described in step 2.11.</li>
	<li>Combine the supernatants from the step 2.11 and 2.12, and spin one more time as described in step 2.11.</li>
	<li>Pour gradients of iodixanol in thin wall 5 mL polyallomer tubes by underlaying (27%, then 33%, then 39%) ~0.7 mL steps using a 3 mL syringe fitted with a long needle or a p1000 pipette.</li>
	<li>Load 3 mL of clarified virus-containing supernatant on the prepared iodixanol gradient.</li>
	<li>Spin for 3.5 h at 234,000 x g and 16 &#176;C in an SW55ti rotor. Set the acceleration and deceleration to slow.</li>
	<li>After ultracentrifugation, collect 12 fractions in siliconized tubes (each fraction is ~400 &#181;L).</li>
	<li>Analyze the fractions for the presence of virus by dsDNA reagent and/or Western blot for VP1 (MCV VP1 rabbit30). Pool gradient fractions with peak dsDNA and/or VP1 content and characterize the stock by quantitative PCR to calculate the viral genome equivalent31. Store MCPyV virion stock at -80 &#176;C.</li>
</ol>
3. Infection
<ol>
	<li>Maintain primary human dermal fibroblasts in DMEM with 10% fetal calf serum, 1% non-essential amino acids and 1% L-glutamine. Upon reaching confluence, split fibroblasts 1:4 without spinning down. 
	NOTE: For highest MCPyV infection efficiency, use primary fibroblasts between passages 5 and 12 that are actively dividing at the time of plating.</li>
	<li>To infect human dermal fibroblasts, aspirate the medium and wash the cells with DPBS.</li>
	<li>Add 1 mL of 0.05% Trypsin-EDTA to the dish and incubate at 37 &#176;C for 5-10 min.</li>
	<li>Check under the microscope to make sure that the cells are coming off the dish.</li>
	<li>Add 10 mL of DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF and 3 &#181;M CHIR99021, all of which stimulate MCPyV infection24, to the dish and transfer the cell solution into a 15 mL tube.</li>
	<li>Spin down the cells at 180 x g for 2 min, and discard the supernatant. Resuspend the cells in DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF and 3&#181;M CHIR99021 at 2-4 x 104 cells per mL.</li>
	<li>Seed 200 &#181;L of the cell suspension supplemented with 1 mg/mL collagenase type IV into each well of a 96-well plate. Thaw MCPyV virion stock on ice. Add 109 viral genome equivalents of MCPyV virions (calculated in step 2.18) per 1 &#181;L of iodixanol for each 2,500 to 5000 cells to be infected.&#160;&#160;Tap the side of the plate gently and place the plate in the incubator.</li>
	<li>After incubation at 37 &#176;C in 5% CO2 for 48-72 h, add 20% FBS to each well.</li>
	<li>Allow the infection to proceed at 37 &#176;C in 5% CO2 for 72-96 h.</li>
</ol>
4. Immunofluorescent staining
<ol>
	<li>Fix cells obtained in step 3.9 with 3% paraformaldehyde in PBS for 20 min.</li>
	<li>Wash the cells with PBS twice.</li>
	<li>Incubate the cells in blocking/permeabilization buffer (0.5% Triton X-100, 3% BSA in PBS filtered through 0.22 &#181;m filter) at RT for 1 h.</li>
	<li>Incubate the cells with the following primary antibodies: mouse monoclonal anti-MCPyV LT (CM2B4) (1:1000) and rabbit polyclonal anti-MCPyV VP1 antibody (1:2000), at RT for 3 h.</li>
	<li>Wash the cells with PBS three times.</li>
	<li>Incubate the cells with the secondary antibodies: Fluor 594 goat anti-mouse IgG (1:1000) and Fluor 488 goat anti-rabbit IgG (1:500) at RT for 1 h.</li>
	<li>Wash cells with PBS three times.</li>
	<li>Counterstain the cells with 0.5 &#181;g/mL DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride).</li>
	<li>Mount coverslips on glass slides and analyze using an inverted fluorescence microscope.</li>
</ol>
5. In situ DNA-HCR
NOTE: This technique requires that cells be seeded on coverslips. For this purpose, the infection conditions described above (step 3) may be scaled up to the 24-well plate format.
<ol>
	<li>Fix cells cultured on coverslips with 4% PFA in PBS for 10 min. Wash the coverslips twice with PBS, then treat them with 70% ethanol to permeabilize the cells at 4 &#176;C overnight. 
	NOTE: Fixed samples can remain in this state for several days.</li>
	<li>Pre-hybridize samples by replacing the ethanol with probe hybridization buffer and incubating for 60 min at RT.</li>
	<li>Dilute the probe(s) (1 &#181;M) (Table 1) in probe hybridization buffer solution at 1:500 dilution 30 min prior to the end of the pre-hybridization step and incubate at 45 &#176;C.</li>
	<li>At the end of the incubations, pipette ~10 &#956;L of the diluted probe mix per coverslip on microscope slides. Place the coverslips, cell-side down, on their respective droplets of hybridization probe mix. Seal the edges and backs of the coverslips to the slides with a liberal amount of rubber cement.</li>
	<li>Heat the slides with added probes at 94 &#176;C for 3 min by setting the slides on the flat side of a heat block. Transfer the slides to a humidified chamber and incubate at 45 &#176;C overnight. To make a simple humidified chamber, lightly dampen sterile paper towels with dH2O and place in the bottom of a plastic container that seals with a rubber gasket. 
	NOTE: During heating the rubber cement can bubble briefly. However, ensure that the seal does not break.</li>
	<li>Carefully peel away the rubber cement with forceps and place the coverslips cell-side up back into wells of a 24-well plate. Wash the coverslips with probe wash buffer at RT three times.</li>
	<li>Incubate the coverslip in the amplification buffer (200 &#181;L in 24-well plates) at RT 30-60 min.</li>
	<li>Meanwhile, anneal each of the two labeled oligonucleotide hairpins recognizing the probes (Table 1) in separate PCR tubes by heating to 94&#160;&#176;C for 90 s and cooling to RT for 30 min while protecting from light. Mix the two hairpins in amplification buffer, each at a dilution of 1:50.</li>
	<li>Make a surface for the amplification reaction by stretching paraffin film over the open face of a 24-well plate lid. Pipette 50-100 &#181;L droplets of the hairpin/amplification buffer mixture onto the paraffin film for each coverslip. Use forceps to carefully remove the coverslips from the pre-amplification solution. Dry the coverslip by touching the edge to a porous disposable wipe, and place cell-side down onto the amplification droplet.</li>
	<li>Place the plate lid holding the coverslips into a humidified chamber and incubate overnight at RT in the dark.</li>
	<li>Return the coverslips to wells once more. Incubate the samples with 5x SSCT [5x sodium chloride sodium citrate (SSC) 0.1% Tween 20 filtered through a 0.22 &#181;m filter] containing 0.5 &#181;g/mL DAPI for 1 h at RT. Wash the samples twice with 5x SSCT at RT.</li>
	<li>Mount the coverslips on microscope slides. Analyze the cells and image the samples using an inverted fluorescence microscope.</li>
</ol>

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The authors have nothing to disclose.

The methods described above , including isolation of dermal fibroblasts from human skin tissue, preparation of recombinant MCPyV virions, infection of cultured cells, immunofluorescent staining, and a sensitive FISH method adapted from HCR technology, which should enable researchers to analyze MCPyV infection27. One of the most critical steps to achieving MCPyV infection in vitro is the production of high-titer virion preparations. Using the protocol for preparation of recombinant MCPyV virions de...

Merkel cell polyomavirus (MCPyV) is a small, double-stranded DNA virus that has been associated with a rare but aggressive skin cancer, Merkel cell carcinoma (MCC)1,2. The mortality rate of MCC, around 33%, exceeds that of melanoma3,4. MCPyV has a circular genome of ~5 kb1,5 bisected by a non-coding regulatory region (NCRR) into early and late coding regions1. The NCRR contains the viral origin of replication (Ori) and bidirectional promoters for viral transcription6,7. The early region encodes tumor antigen proteins called large T (LT), small T (sT), 57kT, alternative LT ORF (ALTO), as well as an autoregulatory miRNA1,8,9,10. The late region encodes the capsid proteins VP1 and VP211,12,13. LT and sT are the best-studied MCPyV proteins and have been shown to support the viral DNA replication and MCPyV-induced tumorigenesis5. Clonal integration of MCPyV DNA into the host genome, which has been observed in up to 80% of MCCs, is likely a causal factor for virus-positive tumor development14,15.
The incidence of MCC has tripled over the past twenty years16. Asymptomatic MCPyV infection is also widespread in the general population17,18,19. With the increasing number of MCC diagnoses and the high prevalence of MCPyV infection, there is a need to improve our understanding of the virus and its oncogenic potential. However, many aspects of MCPyV biology and oncogenic mechanisms remain poorly understood20. This is largely because MCPyV replicates poorly in established cell lines11,12,21,22,23 and, until recently, skin cells capable of supporting MCPyV infection had not been discovered22. Mechanistic studies to fully investigate MCPyV and its interaction with host cells have been hampered by a lack of cell culture system for propagating the virus5.
We discovered that primary human dermal fibroblasts (HDFs) isolated from neonatal human foreskin support robust MCPyV infection both in vitro and ex vivo24. From this study, we established the first cell culture infection model for MCPyV24. Building on this model system, we showed that the induction of matrix metalloproteinase (MMP) genes by the WNT/&#946;-catenin signaling pathway and other growth factors stimulates MCPyV infection. Moreover, we found that the FDA-approved MEK antagonist trametinib effectively inhibits MCPyV infection5,25. From these studies, we also established a set of protocols for isolating human dermal fibroblasts24,25, preparing MCPyV virions11,12, performing MCPyV infection on human dermal fibroblasts24,25 and detecting MCPyV proteins by IF staining26. In addition, we adapted the in situ DNA hybridization chain reaction (HCR) technology27 to develop a highly sensitive FISH technique (HCR-DNA FISH) for detecting MCPyV DNA in infected human skin cells. These new methods will be useful for studying the infectious cycle of MCPyV as well as the cellular response to MCPyV infection. The natural host reservoir cells that maintain MCPyV infection and the cells that give rise to MCC tumors remain unknown. The techniques we describe in this manuscript could be applied to examine various types of human cells to identify both the reservoir cells and origin of MCC tumors. Our established methods, such as HCR-DNA FISH, could also be employed in the detection of other DNA tumor viruses and the characterization of host cell interactions.

<table><tbody><tr><td>Fetal calf serum</td><td>HyClone</td><td>SH30071.03</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution, 100X</td><td>Thermo Fisher Scientific</td><td>11140050</td><td></td></tr><tr><td>GLUTAMAX I, 100X</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>L-Glutamine</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fisher Scientific</td><td>14190136</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25300-054</td><td></td></tr><tr><td>DMEM/F12 medium</td><td>Thermo Fisher Scientific</td><td>11330-032</td><td></td></tr><tr><td>Recombinant Human EGF Protein, CF</td><td>R&amp;amp;D systems</td><td>236-EG-200</td><td>Store at -80 degree celsius</td></tr><tr><td>CHIR99021</td><td>Cayman Chemical</td><td>13122</td><td>Store at -80 degree celsius</td></tr><tr><td>CHIR99021</td><td>Sigma</td><td>SML1046</td><td>Store at -80 degree celsius</td></tr><tr><td>Collagenase type IV</td><td>Thermo Fisher Scientific</td><td>17104019</td><td></td></tr><tr><td>Dispase II</td><td>Roche</td><td>4942078001</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Thermo Fisher Scientific</td><td>15240-062</td><td>Protect from light</td></tr><tr><td>DMEM medium</td><td>Thermo Fisher Scientific</td><td>11965084</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-mouse IgG</td><td>Thermo Fisher Scientific</td><td>A11032</td><td>Protect from light</td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit IgG</td><td>Thermo Fisher Scientific</td><td>A11034</td><td>Protect from light</td></tr><tr><td>OptiPrep Density Gradient Medium</td><td>Sigma</td><td>D1556</td><td>Protect from light</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>anti-MCPyV LT (CM2B4)</td><td>Santa Cruz</td><td>sc-136172</td><td>Lot # B2717</td></tr><tr><td>MCV VP1 rabbit</td><td></td><td>Rabbit polyclonal serum #10965</td><td>https://home.ccr.cancer.gov/lco/BuckLabAntibodies.htm</td></tr><tr><td>Hygromycin</td><td>Roche</td><td>10843555001</td><td></td></tr><tr><td>Basic Fibroblast Growth Factors (bFGF), Human Recombinant</td><td>Corning</td><td>354060</td><td>Store at -80 degree celsius</td></tr><tr><td>Benzonase Nuclease</td><td>Sigma</td><td>E8263</td><td></td></tr><tr><td>Plasmid-Safe ATP-Dependent DNase</td><td>EPICENTRE</td><td>E3101K</td><td></td></tr><tr><td>Probe hybridization buffer</td><td>Molecular technologies</td><td></td><td></td></tr><tr><td>Probe wash buffer</td><td>Molecular technologies</td><td></td><td></td></tr><tr><td>Amplification buffer</td><td>Molecular technologies</td><td></td><td></td></tr><tr><td>Alexa 594-labeled hairpins</td><td>Molecular technologies</td><td>B4</td><td>Protect from light</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Reagent</td><td>Thermo Fisher Scientific</td><td>P7581</td><td></td></tr><tr><td>BamHI-HF</td><td>NEB</td><td>R3136</td><td></td></tr><tr><td>Buffer PB</td><td>Qiagen</td><td>19066</td><td></td></tr><tr><td>blue miniprep spin column</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>50mL Conical Centrifuge Tubes</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>T4 ligase</td><td>NEB</td><td>M0202T</td><td></td></tr><tr><td>MagicMark XP</td><td>Thermo Fisher Scientific</td><td>LC5602</td><td></td></tr></tbody></table>

merkel cell polyomavirus infection and detection

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully investigate MCPyV molecular biology and oncogenic mechanisms have been hampered by a lack of adequate cell culture models. Here, we describe a set of protocols for performing and detecting MCPyV infection of primary human skin cells. The protocols describe the isolation of human dermal fibroblasts, preparation of recombinant MCPyV virions, and detection of virus infection by both immunofluorescent (IF) staining and in situ DNA-hybridization chain reaction (HCR), which is a highly sensitive fluorescence in situ hybridization (FISH) approach. The protocols herein can be adapted by interested researchers to identify other cell types or cell lines that support MCPyV infection. The described FISH approach could also be adapted for detecting low levels of viral DNAs present in the infected human skin.

The protocol described in this manuscript allowed isolation of a nearly homogenous population of HDFs (Figure 1). As demonstrated by immunofluorescent staining, almost 100% of the human dermal cells isolated using the conditions described in this protocol were positively stained for dermal fibroblast markers, vimentin, and collagen I24, but negative for human foreskin keratinocyte marker K14 (Figure 1). <s...

Watch this Scientific Journal Video about Merkel Cell Polyomavirus Infection and Detection at JoVE.com

Merkel Cell Polyomavirus Infection and Detection

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully ...

merkel-cell-polyomavirus-infection-and-detection

Research

JoVE Journal

Immunology and Infection

9.9K Views.  University of Pennsylvania. We presenteerden het eerste celkweekmodel voor Merkel Cell Polyomavirus infectie. Het protocol omvat isolatie van de huidfibroblasten, voorbereiding van de MCPyV-virusinfectie, immunofluorescente kleuring en de fluorescerende in situ-hybridisatie. Dit protocol maakt het mogelijk om de MCPyV infectieuze cyclus en interacties met gastheercellen te bestuderen, terwijl artefacten in verband met overexpressie van virale genen worden vermeden.Om te beginnen, gebruik maken van een schaar om h...

Video: Merkel Cell Polyomavirus Infection and Detection

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Cancer Research

Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals are currently not available, methods measuring the impact of potential antiviral agents are required. Here, a dual fluorescence reporter that allows the analysis of the BKPyV non-coding control-region (NCCR) driven early and late promoter activity was constructed to quantify the impact of potential antiviral drugs on viral gene expression via tdTomato and eGFP expression. In addition, by cloning BKPyV-NCCR amplicons which in this protocol have been exemplarily obtained from the blood-derived DNA of immunocompromised renal transplanted patients, the impact of NCCR-rearrangements on viral gene expression can be determined. Following cloning of the patient derived amplicons, HEK293T cells were transfected with the reporter-plasmids, and treated with potential antiviral agents. Subsequently, cells were subjected to FACS-analysis for measuring mean fluorescence intensities 72 h post transfection. To also test the analysis of drugs that have a potential cell cycle inhibiting effect, only transfected and thus fluorescent cells are used. Since this assay is performed in large T Antigen expressing cells, the impact of early and late expression can be analyzed in a mutually independent manner.

In this manuscript, a protocol is presented to perform FACS-based measurement of BK-polyomavirus transcriptional activity by using HEK293T cells transfected with a bidirectional reporter plasmid expressing tdTomato and eGFP. This method further allows to quantitatively determine the influence of novel compounds on viral transcription.

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals ...

Genetics

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 ...

Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line

The Epstein-Barr virus (EBV), formally designated as Human herpesvirus 4 (HHV-4), is the first isolated human tumor virus. Nearly 90-95% of the world&#39;s adult population is infected by EBV. With the recent advancements in molecular biology and immunology, the application of both in vitro and in vivo experimental models has provided deep and meaningful insight into the pathogenesis of EBV in many diseases as well as into EBV-associated tumorigenesis. The aim of this visualized experiment paper is to provide an overview of the isolation of EBV viral particles from cells of the P3HR1 cell line, followed by quantification of the viral preparation. P3HR1 cells, originally isolated from a human Burkitt lymphoma, can produce a P3HR1 virus, which is a type 2 EBV strain. The EBV lytic cycle can be induced in these P3HR1 cells by treatment with phorbol 12-myristate 13-acetate (PMA), yielding EBV viral particles.
Using this protocol for the isolation of EBV particles, P3HR1 cells are cultured for 5 days at 37 &#176;C and 5% CO2 in complete RPMI-1640 medium containing 35 ng/mL PMA. Subsequently, the culture medium is centrifuged at a speed of 120 x g for 8 min to pellet the cells. The virus-containing supernatant is then collected and spun down at a speed of 16,000 x g for 90 min to pellet the EBV particles. The viral pellet is then resuspended in a complete RPMI-1640 medium. This is followed by DNA extraction and quantitative real-time PCR to assess the concentration of EBV particles in the preparation.

This protocol permits the isolation of Epstein-Barr virus particles from the human P3HR1 cell line upon inducing the viral lytic cycle with phorbol 12-myristate 13-acetate. DNA is subsequently extracted from the viral preparation and subjected to real-time PCR to quantify the viral particle concentration.

The Epstein-Barr virus (EBV), formally designated as Human herpesvirus 4 (HHV-4), is the first isolated human tumor virus. Nearly 90-95% of the ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Biology

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

HCR-DNA FISH: A Fluorescence In Situ Hybridization Technique to Detect Viral DNA in Infected Cells

Source: Wei Liu&#160;et al., <a href="https://www.jove.com/v/58950"> Merkel Cell Polyomavirus Infection and Detection</a>, J. Vis. Exp. (2019).
In this video, the cells infected with MCPyV virions were subjected to in situ hybridization chain reaction to detect MCPyV specific genomes. Further, the DNA-HCR technique was combined with FISH to visualize the MCPyV DNA in infected human skin cells.

Source: Wei Liu&#160;et al.,  Merkel Cell Polyomavirus Infection and Detection, J. Vis. Exp. (2019).
In this video, the cells infected with MCPyV virions ...

JoVE Encyclopedia of Experiments

Skin Cancer

Assays and techniques

Recombinant Merkel Cell Polyomavirus (MCPyV) Virion Preparation: A Technique to Produce High-titer Recombinant MCPyV Virion

Source:&#160;Wei Liu&#160;et al. <a href="https://www.jove.com/v/58950"> Merkel Cell Polyomavirus Infection and Detection</a>, J. Vis. Exp. (2019)
In this video, we show the preparation of Merkel cell polyomavirus or MCPyV virions using 273TT cells. This technology helps to produce high-titer of the recombinant MCPyV virions.

Source:&#160;Wei Liu&#160;et al.  Merkel Cell Polyomavirus Infection and Detection, J. Vis. Exp. (2019)
In this video, we show the preparation of Merkel ...

An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

This video demonstrates an immunofluorescence assay designed to measure the neutralization of Human Cytomegalovirus (HCMV). Virus-specific antibodies are mixed with the virus and incubated with human fibroblast cells. The antibodies neutralize the virus, rendering it unable to infect the host cells, while non-neutralized viruses infect the cells. After incubation, the extent of neutralization is determined by quantifying the virus-infected cells using immunofluorescence.

1. Preparation of target cells for infection

	Seed target cells the day prior to performing the experiment in a 96-well plate (the &#39;target&#39; ...

Merkel Cell Polyomavirus Infection and Detection

Samenvatting

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