This research was supported by grants NIH/NIAID AI112423 and NIH/NIGMS GM113885 to Benjamin K. Chen and NIH/NIAID K08-AI120806 to Talia H. Swartz. We would like to thank the Icahn School of Medicine at Mount Sinai Dean&#39;s flow Cytometry CORE.

1. Generation of Target Cell Lines
NOTE: This step is optional; if using an existing RG target cell line, start at step 2.
<ol>
	<li>Cotransfect a 70% confluent 10 cm plate of 293T cells11 [in 10 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 10% fetal bovine serum (FBS)] with pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE10 and pCL-10A112 (packaging plasmid) in a 1:1 ratio (20 &#181;g total) using calcium-based transfection13.</li>
	<li>Harvest 10 mL of viral supernatant 48 h post-transfection by pipetting the media from the plate into a 15 mL conical tube and centrifuging the tube at 3,000 x g for 5 min at 23 &#176;C to pellet any cell debris.</li>
	<li>Attach a 0.45 &#181;m filter to a 10 mL syringe. After centrifugation, take the entire supernatant and load the syringe. Run the sample through into a clean 15 mL tube.</li>
	<li>Aliquot the filtered viral supernatant into appropriate sizes (usually, 0.5&#8211;1 mL aliquots). These can be used immediately in the next step or can be frozen at -80 &#176;C and stored.</li>
	<li>Use 50&#8211;100 &#181;L of viral supernatant to infect a cell line of choice in a 96-well plate. Culture the cells at 37 &#176;C with 5% CO2. The RFP signal should appear in as soon as 24 h under a fluorescence microscope (40X magnification, 532 nm excitation, 588 nm emission) but may take up to 72 h. 
	NOTE: In these experiments, Jurkat cells were used, but other types may be used as well.</li>
	<li>Select transduced cells with puromycin by setting up a series of 10 wells and adding puromycin to each, leaving at least 1 well untreated as a control (use a range of concentrations from 0.5 &#181;g/mL to 5 &#181;g/mL; this may vary per cell type). Monitor the cell viability (cell lysis will occur in cells without puromycin resistance) over the course of several days compared to the untreated control and use a concentration of puromycin where only RFP-expressing cells survive. 
	NOTE: Select the concentration where untransfected cells are killed while transfected cells survive.</li>
	<li>Using either single-cell flow cytometry sorting or a limiting dilution (see steps 1.8&#8211;1.10), grow&#160;cultures derived from single cells1 to develop a clonal cell line (this may take several weeks to grow).</li>
	<li>If using the dilution method, count the cells using a hemocytometer and dilute the sample to approximately 500 cells/mL.</li>
	<li>In a 96-well plate, pipette 50 &#181;L of the cell dilution into 50 &#181;L of media [Roswell Park Memorial Institute (RPMI) medium with 10% FBS and 2% penicillin-streptomycin, if using Jurkat cells] and perform 1:1 serial dilutions into 11 wells containing 50 &#181;L of media. For the best results, perform at least 10 replicates.</li>
	<li>Monitor the cell growth via microscopy (40X) of the plate in a tissue culture incubator (37 &#176;C with 5% CO2) or&#160;approximately 4 weeks. Choose cultures from the lowest puromycin concentration with growth (as seen via microscope, 40X) for the clonal cultures.</li>
	<li>Freeze the aliquots by centrifuging 20 mL of the culture (500,000 cells/mL, counted with a hemocytometer) at 800 x g for 5 min at 23 &#176;C and resuspending it in 500 &#181;L of FBS with 10% DMSO. Place it at -80 &#176;C using a cell-freezing container and then store it in liquid nitrogen.</li>
</ol>
2. Cell-to-cell Virus Transmission
<ol>
	<li>Preparation of target cells
	<ol>
		<li>Thaw one 500 &#181;L vial of Jurkat RG reporter cells by placing it into a 37 &#176;C water bath. Pipette the cells from the vial into 10 mL of RPMI complete medium and then centrifuge the mixture at 800 x g for 5 min at 23 &#176;C. Resuspend the pellet in 20 mL of RPMI complete medium in a T-75 flask. Incubate the flask overnight (37 &#176;C and 5% CO2). 
		NOTE: RPMI complete medium contains RPMI 1640, 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin.</li>
		<li>The next day, add 0.5 &#181;g/mL of puromycin (1 &#181;L of a 2 mg/mL stock per 8 mL of media). Culture the cells, maintaining a density of 200,000&#8211;800,000 cells/mL (counted via hemocytometer).</li>
		<li>The day before setting up the transfer assay, split the cells down to 200,000&#8211;400,000 cells/mL in fresh RPMI medium containing 0.5 &#181;g/mL of puromycin.</li>
	</ol>
	</li>
	<li>Preparation of donor cells
	<ol>
		<li>Thaw untransduced Jurkat cells and culture them in a T-75 flask with 20 mL of RPMI complete medium (as described in step 2.1), keeping a density of 200,000&#8211;800,000 cells/mL.</li>
		<li>Centrifuge 7,500,000 cells (800 x g for 5 min) and resuspend them in 120 &#181;L of nucleofection solution V with supplement (see Table of Materials). Transfer the cells to an electroporation cuvette and add 4.5 &#181;g of Gag-iCre1 DNA. Transfect the cells (via an electroporation-based approach, see the Table of Materials) using an appropriate program (e.g., S-18) and then immediately transfer them to 3 mL of RPMI medium (with 10% FBS).</li>
		<li>Allow the cells to recover by incubating them overnight at 37 &#176;C with 5% CO2.</li>
		<li>The next morning, centrifuge the cells at 800 x g for 5 min at 23 &#176;C and resuspend them in 3 mL of RPMI complete medium, allowing them 2 h to recover at 37 &#176;C (5% CO2) before proceeding with the assay set-up.</li>
	</ol>
	</li>
	<li>Co-culture
	<ol>
		<li>Count the cells using a hemocytometer then spin down 50,000 cells (800 x g for 5 min at 23 &#176;C) per well to be assayed (of both donor and target cells). Resuspending 1 x 106 cell/mL in RPMI complete without puromycin.</li>
		<li>In one plate, add 25 &#181;L of donor cell suspension to each well; in another plate, add 25 &#181;L of target cells.</li>
		<li>To each well, add 25 &#181;L of the test compound diluted in RPMI complete medium at the appropriate concentration. Incubate the donor and target cells with the compound for 30 min. 
		NOTE: The concentration will vary per drug. If it has a known IC50, use this as a starting point, titrating above and below. If the IC50 is unknown, prepare a 200 &#181;M stock for a final concentration of 10 &#181;M.</li>
		<li>After the pretreatment, combine the donor cells with the target cells by pipetting the contents of one plate into the other and incubate the cells for 40 h in a tissue culture incubator at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Add paraformaldehyde (PFA) to each well to a final concentration of 2%. 
		NOTE: For a 16% stock solution, this is 14 &#181;L per 100 &#181;L well.</li>
		<li>CAUTION: PFA is harmful to exposed skin and eyes. Wear a lab coat, gloves, and safety goggles, and handle the stock solution in a safety hood.</li>
		<li>Read the cells on a flow cytometer with mCherry and FITC channels. 
		NOTE: It is possible to see 5&#8211;20% of the cells expressing GFP above background in the infected cells vs. uninfected cells.</li>
		<li>Visualize the GFP signal via fluorescence microscopy (40X) on GFP-specific channels (with an excitation filter of 400 nm and an emission filter of 508 nm).</li>
	</ol>
	</li>
</ol>
3. Alternative Protocol for a Cell-free Virus Infection
<ol>
	<li>Preparation of target cells
	<ol>
		<li>Thaw one 500 &#181;L vial of Jurkat RG reporter cells by placing into a 37 &#176;C water bath. Pipette the cells from the vial into 10 mL of RPMI complete medium and then centrifuge the RPMI complete medium at 800 x g for 5 min at 23 &#176;C. Resuspend the pellet in 20 mL of RPMI complete medium in a T-75 flask. Incubate the flask overnight (37 &#176;C and 5% CO2).</li>
		<li>The next day, add 0.5 &#181;g/mL of puromycin (1 &#181;L of a 2 mg/mL stock per 8 mL of media). Culture the cells, maintaining a density of 200,000&#8211;800,000 cells/mL (counted via hemocytometer).</li>
		<li>The day before setting up the transfer assay, split the cells down to 200,000&#8211;400,000 cells/mL in fresh RPMI medium containing 0.5 &#181;g/mL of puromycin.</li>
	</ol>
	</li>
	<li>Production of a cell-free virus
	<ol>
		<li>Transfect a 70% confluent 10 cm plate of 293T cells11 (in 10 mL of DMEM with 10% FBS) with 5 &#181;g of Gag-iCre1 plasmid using calcium-based transfection13. 
		NOTE: This scale will produce 10 mL of virus, which is enough for approximately two to four 96-well plates, depending on the efficiency of the transfection.</li>
		<li>Harvest the entire viral supernatant at 48 h by pipetting the media from the plate into a 15 mL conical tube and centrifuging the tube at 3,000 x g for 5 min to pellet all cell debris.</li>
		<li>Attach a 0.45 &#181;m filter to a 10 mL syringe. After centrifugation, take the entire supernatant and load the syringe. Run the sample through into a clean 15 mL tube.</li>
		<li>Use the virus to infect target cells immediately (step 3.3) or freeze and store the virus at -80 &#176;C (thaw the sample as needed before use).</li>
	</ol>
	</li>
	<li>Infection
	<ol>
		<li>Count the RG Jurkat target cells (from step 3.1.3) using a hemocytometer and spin down 50,000 target cells per well at 800 x g for 5 min, resuspending the cells at 1 x 106 cells/mL in RPMI complete medium without puromycin.</li>
		<li>In one plate, add 25 &#181;L of RG Jurkat target cells to each well; in a separate plate, add 25 &#181;L (2 ng) virus to each well.</li>
		<li>To each well, add 25 &#181;L of the test compound, diluted in media at the appropriate concentration. Incubate the cells and the virus with the compound for 30 min. 
		NOTE: The test concentration will vary per drug. If it has a known IC50, use this as a starting point, titrating above and below. If unknown, prepare a 200 &#181;M stock medium for a final concentration of 10 &#181;M.</li>
		<li>After the pretreatment, add the entire content (50 &#181;L) of the virus/compound mix from the wells to the target cells and incubate everything for 40 h in a tissue culture incubator at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Add PFA to each well to a final concentration of 2%. 
		NOTE: For a 16% stock solution, this is 14 &#181;L per 100 &#181;L well.</li>
		<li>Read the cells on a flow cytometer with mCherry and FITC channels. The GFP signal can also be visualized via fluorescence microscopy on GFP channels. 
		NOTE: Once can expect to see 5&#8211;20% of the cells expressing GFP above background in the infected cells vs. the uninfected cells.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Esposito, A. M., 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=A+high+throughput+Cre-lox+activated+viral+membrane+fusion+assay+identifies+pharmacological+inhibitors+of+HIV+entry.">A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.</a> Virology. 490, 6-16 (2016).</li><li>Cavrois, M., De Noronha, C., Greene, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+specific+enzyme-based+assay+detecting+HIV-1+virion+fusion+in+primary+T+lymphocytes.">A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes.</a> Nature Biotechnology. 20, 1151-1154 (2002).</li><li>Huerta, L., Lamoyi, E., Baez-Saldana, A., Larralde, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+envelope-dependent+cell-cell+fusion:+a+quantitative+fluorescence+cytometric+assay.">Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay.</a> Cytometry. 47, 100-106 (2002).</li><li>Sakamoto, T., 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=Establishment+of+an+HIV+cell-cell+fusion+assay+by+using+two+genetically+modified+HeLa+cell+lines+and+reporter+gene.">Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene.</a> Journal of Virological Methods. 114, 159-166 (2003).</li><li>Herschhorn, 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=An+inducible+cell-cell+fusion+system+with+integrated+ability+to+measure+the+efficiency+and+specificity+of+HIV-1+entry+inhibitors.">An inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of HIV-1 entry inhibitors.</a> PLoS One. 6, e26731 (2011).</li><li>Swartz, T. H., Esposito, A. M., Durham, N. D., Hartmann, B. M., Chen, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X-selective+purinergic+antagonists+are+strong+inhibitors+of+HIV-1+fusion+during+both+cell-to-cell+and+cell-free+infection.">P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection.</a> Journal of Virology. 88, 11504-11515 (2014).</li><li>Marin, M., 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=High-Throughput+HIV-Cell+Fusion+Assay+for+Discovery+of+Virus+Entry+Inhibitors.">High-Throughput HIV-Cell Fusion Assay for Discovery of Virus Entry Inhibitors.</a> Assay and Drug Development Technologies. 13, 155-166 (2015).</li><li>Giroud, 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=Screening+and+Functional+Profiling+of+Small-Molecule+HIV-1+Entry+and+Fusion+Inhibitors.">Screening and Functional Profiling of Small-Molecule HIV-1 Entry and Fusion Inhibitors.</a> Assay and Drug Development Technologies. 15, 53-63 (2017).</li><li>Hubner, W., 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=Sequence+of+human+immunodeficiency+virus+type+1+(HIV-1)+Gag+localization+and+oligomerization+monitored+with+live+confocal+imaging+of+a+replication-competent,+fluorescently+tagged+HIV-1.">Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1.</a> Journal of Virology. 81, 12596-12607 (2007).</li><li>Koo, B. 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=Controlled+gene+expression+in+primary+Lgr5+organoid+cultures.">Controlled gene expression in primary Lgr5 organoid cultures.</a> Nature Methods. 9, 81-83 (2011).</li><li>Pear, W. 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=Production+of+high-titer+helper-free+retroviruses+by+transient+transfection.">Production of high-titer helper-free retroviruses by transient transfection.</a> Proceedings of the National Academy of Sciences. 90, 8392-8396 (1993).</li><li>Naviaux, R. K., Costanzi, E., Haas, M., Verma, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pCL+vector+system:+Rapid+production+of+helper-free,+high+titer,+recombinant+retroviruses.">The pCL vector system: Rapid production of helper-free, high titer, recombinant retroviruses.</a> Journal of Virology. 70, 5701-5705 (1996).</li><li>Kingston, R. E., Chen, C. A., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Phosphate+Transfection.">Calcium Phosphate Transfection.</a> Current Protocols in Immunology. 10, 10-13 (2001).</li></ol>

The authors have nothing to disclose.

The Gag-iCre assay has proven to be very useful for screening drug candidates which may inhibit the fusion step of viral replication. When performing this assay, the most important steps to get a good signal are similar to most viral infection assays. The first critical step is producing high titers of good-quality virus. This step requires that the 293T cells are passaged frequently (at least 1x every 48 h) so they do not become overconfluent and clump together. Additionally, it may be worth experimenting with different transfection methods, as some researchers find that calcium-phosphate transfection gives better results, while others favor lipid-based reagents. Similarly, the other critical step in this protocol is to not let the target cells overgrow but to keep them in the exponential growth phase right before the infection, as the stationary phase reduces their ability to be infected.
Compared to the well-established BlaM-Vpr assay2, the Gag-iCre assay offers a substrate-free assay with no need to label target cells. This makes it well suited for certain high-throughput applications1. Additionally, the assay provides a permanent marker for viral entry even if the cell becomes latent, offering some potential to study latent infection. Future studies for latency may utilize this system in mice where RG cells which have been switched to green due to Gag-iCre exposure, yet are not productively infected, could be isolated to study the cell populations with a latent virus or nonproductive infection. In addition to latency, the assay has other potential applications. It has been successfully utilized for pseudo-typing with other viral envelope proteins such as Ebola and VSV (unpublished data), opening up the potential for this assay to identify fusion inhibitors for a variety of viruses in a safer manner than working with a live virus.
One potential limitation of the Gag-iCre assay is that it does take an additional day for the signal to develop, compared to BlaM-Vpr. In addition, the Gag-iCre virus in its current state only replicates for a single cycle, which works for drug screening under the conditions we have described but would not be suitable for long-term studies or growth curves. In the future, new versions of the assay will be made with different reporter signals, such as luciferase, allowing for a wider variety of uses. Further, the screening of larger compound libraries will likely yield novel inhibitors specific to the fusion step of viral replication.

The need for novel antiretroviral therapies has prompted the developement of&#160;high-throughput screens for&#160;inhibitors of HIV-1 entry. The Gag-iCre reporter assay is developed to identify inhibitors of viral entry at the fusion step in a cell-to-cell infection system by specifically measuring viral membrane fusion with the host cell membrane1. An assay was developed to&#160;screen for novel inhibitors that acted specifically at the early stages of HIV-1 infection up to the point of viral membrane fusion. One challenge to measuring cell-cell infection is that the initial inoculum contains infected donor cells and ininfected&#160;target cells, so measuring new infection is difficult to discriminate from the input cells. The ideal system would involve a heterologous gene marker that can be induced by the initiation of a target cell infection and is not present in the donor cell. A popular viral contents mixing assay based on a viral protein-enzyme fusion,&#160;BlaM-Vpr, can be effective, but has&#160;limitations for high throughput, cell-cell screening2. This assay involves a beta-lactamase (BlaM) reporter gene that is fused to HIV-1 Vpr, packaged into virions, and delivered into target cells upon viral membrane fusion. The substrate CCF2-AM is loaded into the cytoplasm of target cells and undergoes a fluorescence shift upon cleavage. The CCF2-AM substrate is expensive and can be prohibitive for high-throughput screens. In order to distinguish donor and target cells, it is necessary to dye-label the target populations. Finally, the protocol requires several wash and incubation steps which can be burdensome and costly when testing large numbers of compounds.
The Gag-iCre assay was developed as a solution to these issues, to develop a high-throughput screening for inhibitors of fusion in cell-to-cell transmission. This system does not require&#160;substrate to be loaded into cells. The assay can also be used for cell-free studies and is adaptable to other viral fusion studies using pseudo-typed HIV-1 Gag-iCre virus particles. HIV Env mediated cell-cell fusion assays can measure virus Env protein-mediated cell fusion, however these do not use actual virus particles as the HIV-1 Gag-iCre assay does3,4,5. This assay can be read with&#160;flow cytometry or fluorescence microscopy. It has been successfully used to screen the FDA library as well as a small library of purinergic inhibitors1,6. Other investigators have also identified purinergic inhibitors in HIV-1 fusion using an adapted and optimized high-throughput fusion assay that reports the transfer of virus-encapsulated beta-lactamase into the cytoplasm7,8.
The Gag-iCre virus was designed with a similar approach to the Gag-iGFP virus, inserting Cre recombinase between matrix and capsid, flanked by HIV-1 protease sites9. The Cre enzyme is made as a precursor inserted within the Gag polyprotein that matures when HIV-1 protease is activated within the nascent virus particle. The Cre delivery is, thus, dependent upon the delivery of the contents of a protease-activated HIV-1 particle into a target cell via a viral membrane fusion process. Two versions of the protocol are provided here. The first uses an HIV-1 infected population to infect target cells, to study direct cell-to-cell transmission of HIV. The second version uses a cell-free virus to study a cell-free viral infection. The cell-to-cell transmission assay takes 7 days to complete from the day the cells are thawed, or 5 days if the cells are already thawed and passaged. The cell-free infection assay can be performed&#160;in 5 days if the cells need to be thawed and 3 days if the cells are thawed and passaged. Instructions are also given to generate an RG (red-to-green) target cell line in the desired cell type (if a preexisting RG target cell line is not being used) using a plasmid created by the Clevers Lab10. It is recommended that appropriate biosafety precautions are taken with the virus and the virus-expressing cells in this assay. We conduct the infectious portion of this assay in a BSL2+ tissue culture facility. After the cells are fixed, they can be analyzed in standard flow cytometry and microscopy facilities.
Here we describe the application of this assay to screen for novel compounds that inhibit HIV-1 viral membrane fusion (Figure 1). Purinergic receptors are pro-inflammatory mediators. Our laboratory has demonstrated that non-selective inhibitors of purinergic receptors act as inhibitors of HIV-1 viral membrane fusion6. We report that the utilization of this assay in high throughput can identify novel HIV-1 viral membrane fusion inhibitors. We demonstrate that an inhibitor of the purinergic class of receptors represents a novel class of HIV-1 viral membrane fusion inhibitors.

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Sigma Aldrich</td>
			<td>D5546</td>
			<td>Media for 293 Cells</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma Aldrich</td>
			<td>R0883</td>
			<td>Media for Jurkats</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Albumin</td>
			<td>Gibco</td>
			<td>16140-071</td>
			<td>Serum for 293 Cells</td>
		</tr>
		<tr>
			<td>Cosmic Calf Serum</td>
			<td>Hyclone</td>
			<td>SH30087.03</td>
			<td>Serum for Jurkat Cells</td>
		</tr>
		<tr>
			<td>Hyclone Pennecillin Streptomycin solution</td>
			<td>GE Healthcare Life sciences</td>
			<td>SV30010</td>
			<td>Penn/Strep used in both media</td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>Corning</td>
			<td>3073</td>
			<td>Used for Culture of Jurkat Cells</td>
		</tr>
		<tr>
			<td>10cm Tissue Culture Plates</td>
			<td>Corning</td>
			<td>430167</td>
			<td>Used for Culture of 293 Cells</td>
		</tr>
		<tr>
			<td>96 Well Plates (tissue culture Treated)</td>
			<td>Corning</td>
			<td>3595</td>
			<td>Used for fusion assay</td>
		</tr>
		<tr>
			<td>Polyjet Transfection Reagent</td>
			<td>Signagen</td>
			<td>SL100688</td>
			<td>Used to transfect 293 Cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537-100ML</td>
			<td>Used in wash steps</td>
		</tr>
		<tr>
			<td>Hyclone Trypsin Protease</td>
			<td>GE Healthcare Life sciences</td>
			<td>SH30042.01</td>
			<td>For Trypsinization of 293 cells</td>
		</tr>
		<tr>
			<td>Amaxa Cell Line Nucleofector Kit V</td>
			<td>Lonza</td>
			<td>VACA-1003</td>
			<td>For Nucleofection of Jurkats</td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>GE Healthcare Life sciences</td>
			<td>17144002</td>
			<td>For Nucleofection of Jurkats</td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Fisher Brand</td>
			<td>13-678-11E</td>
			<td>For all tissue culture</td>
		</tr>
		<tr>
			<td>Pipettor Tips</td>
			<td>Denville Scientific</td>
			<td>P3020-CPS</td>
			<td>For all tissue culture and liquid handling steps</td>
		</tr>
		<tr>
			<td>Millex Syringe Filter (0.45 micron)</td>
			<td>Millipore</td>
			<td>SLHA033</td>
			<td>For filtration of virus</td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile syringes</td>
			<td>BD Diagnostics</td>
			<td>309656</td>
			<td>For filtration of virus</td>
		</tr>
		<tr>
			<td>Amaxa Nucleofector</td>
			<td>Lonza</td>
			<td>2b</td>
			<td>for Nucleofection of Jurkats (various models available)</td>
		</tr>
		<tr>
			<td>BD Fortessa Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>for flow cytometry analyss of samples</td>
		</tr>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>Various models</td>
			<td>Fortessa 2</td>
			<td></td>
		</tr>
		<tr>
			<td>pMSCV-loxp-dsRed-loxp-eGFP-Puro-W PRE</td>
			<td>Addgene</td>
			<td>32702</td>
			<td>Koo BK et al. Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods 9:81-83 (2011).</td>
		</tr>
		<tr>
			<td>pCL10a1</td>
			<td>Novus Bio</td>
			<td>NBP2-29542</td>
			<td>Naviaux, RK, Costanzi, E, Haas, M and Verma, I. The pCL vector system: Rapid production of helper-free, high titer, recombinant retroviruses. Journal of Virology 70: 5701-5705 (1996)</td>
		</tr>
		<tr>
			<td>Gag-iCre</td>
			<td>Benjamin Chen Lab</td>
			<td></td>
			<td>Esposito AM et al. A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry. Virology 490:6-16 (2016).</td>
		</tr>
	</tbody>
</table>

a high-throughput cre-lox activated viral membrane fusion assay to identify inhibitors of hiv-1 viral membrane fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

Uninfected RG Jurkat Cells exhibit a low level of background GFP signal (0.3%) with a very strong RFP signal (Figure 2A, uninfected column). The infection with Gag-iCre causes an increase in GFP signal (24.9%,) while the presence of the HIV-1 fusion inhibitor AMD3100 (20 &#181;M) inhibits the development of this signal, bringing it down to uninfected background levels (0.34%). When an inhibitor of a post-fusion event such as the reverse transcription inhibitor AZT (2 &#181;M) is used, the signal is not affected significantly (23.5%), indicating that the GFP signal produced by the assay is specific to HIV-1 fusion. Figure 2B indicates a dose-dependent inhibition of Gag-iCre fusion with PPADS (100 &#181;M), a non-selective purinergic inhibitor.
Figure 3 indicates a comparison of cell-free HIV-1 viral membrane fusion using the Gag-iCre assay (Figure 3A) and the convention BlaM-Vpr assay (Figure 3B). A titration of Gag-iCre and BlaM-Vpr viruses was used to spinoculate&#8212;a 2 h long 1,200 x g spin of the 96-well infection plate at the beginning of the infection to increase its efficiency&#8212;RG Jurkat cells with Gag-iCre and Jurkat cells with the BlaM-Vpr virus as previously described2. At 3 ng of the virus, both assays offered a robust signal of 64.7% (Gag-iCre) and 47.2% (BlaM-Vpr) of the cells were fused. At 0.3 ng of the virus, the signals were 1.18% (Gag-iCre) and 2% (BlaM-Vpr). These data suggest that Gag-iCre can offer a comparable signal for viral fusion to the well-established BlaM-Vpr assay at a range of virus concentrations.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58074/58074fig1.jpg" /> 
Figure 1: Gag-iCre assay overview. RG Jurkat cells are co-incubated with donor Jurkat cells transfected with HIV Gag-iCre. Upon viral membrane fusion, the Cre is released into the target cells, which can cleave the dsRed gene at the loxP sites to allow EGFP expression, resulting in the cells fluorescing green. <a href="https://www.jove.com/files/ftp_upload/58074/58074fig1large.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/58074/58074fig2.jpg" /> 
Figure 2: Representative results from a cell-free and a cell-to-cell Gag-iCre infection. (A) Cell-free Gag-iCre fusion is inhibited by the fusion inhibitor AMD3100, but not by the reverse transcription inhibitor AZT. These panels show uninfected RG Jurkat cells, untreated RG Jurkat cells 48 h after the infection, and RG Jurkat cells 48 h after the infection but treated with AMD3100 or AZT, as indicated. The upper panels represent fluorescent micrographs on the GFP channel, the middle panels represent fluorescent micrographs on the RFP channel, while the lower panels represent fluorescence-activated cell sorting (FACS) data for samples showing GFP positive of a percentage indicative of fusion. Scale bar = 100 &#181;m. This panel has been modified with permission from Esposito et al.1. (B) The HIV-1 viral membrane fusion following cell-to-cell infection is inhibited by PPADS. RG Jurkat cells were mixed with Jurkat cells transfected with HIV-1 Gag-iCre, and the inhibitors AMD3100 and PPADS were tested for their ability to block viral membrane fusion at 100 &#181;M, as shown in representative fluorescence-activated cell sorter plots. This figure has been modified from Swartz et al.6. <a href="https://www.jove.com/files/ftp_upload/58074/58074fig2large.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/58074/58074fig3.jpg" /> 
Figure 3: HIV Gag-iCre reporter signal is comparable to BlaM-Vpr. These panels show the results of a FACS analysis of (A) RG cells spinoculated with a Gag-iCre virus and (B) Jurkat cells spinoculated with NL43 BlaM-Vpr, 16 h after a 6 h infection at the indicated concentration of the virus. This panel has been modified with permission from Esposito et al.1. <a href="https://www.jove.com/files/ftp_upload/58074/58074fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion at JoVE.com

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.3K Views.  Icahn School of Medicine at Mount Sinai, Immunology Institute. We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

Video: A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

Summary

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Chapters in this video

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins