This work was supported by grants from NIAID (R01AI074951, U54AI057168) and the Penn Genome Frontiers Institute to SC; from NIH T32HG000046 to TSM; from NIH T32GM07229 and T32AI007324 to LRS.

Part 1: RNAi in 384 Well Plates
<ol>
 <li>Standardization of the reagents used for screening is essential. We test individual lots of Schneider's media, fetal bovine serum, and staining reagents. Then we aliquot and use the same lot for the entire screen.</li>
 <li>For any assay, it is best to test several different cell lines to determine which is most amenable to your biological read-out. We typically use the S2-DRSC line.</li>
 <li>Grow Drosophila cells at 25&deg;C in complete Schneider's media.
 <ol class="lalpha">
 <li>Schneider's media</li>
 <li>10% heat inactivated FBS</li>
 <li>1X L-glutamine</li>
 <li>1X Pen/strep antibiotics</li>
 </ol>
 </li>
 <li>Drosophila cells are semi-adherent and are passaged without trypsinization every four days by pipetting the cells off the flasks and diluting the cells 1:10 into a fresh flask. Cells should be in log phase for experiments. </li>
 <li>Plate-to-plate and day-to-day variability are minimized through the use of automated liquid handling for all liquid additions to multi-well plates. We use a WellMate (Matrix). </li>
 <li>Prepare WellMate tubing.
 <ol class="lalpha">
 <li>Spray hood and WellMate with 70% ethanol.</li>
 <li>Remove tubing from packaging and spray with 70% ethanol.</li>
 <li>Insert tubing cassette in dispensing position on the WellMate.</li>
 <li>Prime tubing with 25 mL of 70% ethanol.</li>
 <li>Sterilize tubing in ethanol for 15 minutes, then prime with another 25 mL of ethanol.</li>
 <li>Rinse tubing by priming with 50 mL of sterile PBS.</li>
 </ol>
 </li>
 <li>Dislodge cells from flask and count. Pellet cells so that you have 25% more than needed (300xg, 5 min). The number of cells seeded per well must be optimized according to assay length. When conducting automated image analysis, a single monolayer with no cell clumping is ideal for image segmentation.</li>
 <li>Resuspend pelleted cells in serum-free Schneider's media at 1.7x106 cells/mL.</li>
 <li>We perform the screen in 384 well plates pre-aliquoted with a commercially available library of dsRNA at a concentration of 250ng/well. Several wells of each plate are left empty for controls, which are added manually. We use dsRNA to luciferase as a negative control. We use dsRNA against Thread (dIAP) as a control for robust RNAi. Knock down of this anti-apoptotic factor results in dramatic cell death and is a quick visual way to assess the quality of the knock down. Lastly, we use a dsRNA against the viral reporter, in this case beta-galacosidase, as a positive control to validate that we can inhibit viral infection using our assay read-out. If spotting dsRNA into plate at the time of cell seeding, aliquot 1 &mu;l dsRNA into the corner of the well to allow for easier visualization, then centrifuge dsRNA onto bottom of well (300xg, 1 min) before adding cells.</li>
 <li>Use WellMate to add 10 &mu;L cells prepared above to each well.</li>
 <li>Spin cells onto plates by centrifuging 300xg, 1 min.</li>
 <li>Incubate in 25&deg;C incubator for 45 minutes.</li>
 <li>Add 20 &mu;L complete Schneider's media and centrifuge 300xg, 1 min.</li>
 <li>Place plates in humidified Tupperware containers lined with water-soaked paper towels. The volume of a 384 well is small; thus, evaporation can have large effects on the biology, which in turn can lead to artifactual changes in the read-outs of the edge wells. To overcome this, plates should be maintained under high humidity.</li>
 <li>Incubate for three days to allow for robust knockdown. There is no need to change the media or open humidified containers during this time.</li>
</ol>
Part 2: Infecting with Vaccinia Virus
<ol>
 <li>Sterilize multi-channel aspirator in Quatricide for 15 minutes, then rinse in 70% ethanol.</li>
 <li>Prepare WellMate as described above.</li>
 <li>Prepare Schneider's media with 2% FBS for infection by diluting complete media 1:5 in serum free media.</li>
 <li>Filter crude virus stock through 0.8 um syringe filter to remove cellular debris or use purified virus.</li>
 <li>Dilute vaccinia virus in 2% serum Schneider's media to obtain multiplicity of infection (MOI) of 2.</li>
 <li>Remove PBS from WellMate tubing, and prime with diluted virus until the tubing is free of air bubbles.</li>
 <li>Use sterilized multi-channel aspirator and vacuum manifold to gently remove media from the plates of dsRNA-treated Drosophila cells.</li>
 <li>Dispense 30 &mu;L virus per well onto plates using the WellMate.</li>
 <li>Centrifuge plates 1 minute at 300xg.</li>
 <li>Sterilize WellMate tubing. Prime with 25 mL of 10% bleach, wait 10 minutes, and prime with another 25 mL 10% bleach.</li>
 <li>Prime tubing with 50 mL water followed by 50 mL 70% ethanol. Return tubing to its packaging.</li>
 <li>Return plates to humidified Tupperware container. Incubate at 25&deg;C for 48 hours.</li>
</ol>
Part 3: Staining Plates
<ol>
 <li>Fix plates in 15 &mu;L 4% formaldehyde/PBS for 10 minutes. Liquid is removed using a multi-channel aspirator and vacuum manifold at each step. Once plates have been fixed, sterile equipment and reagents are no longer necessary.</li>
 <li>Remove fix and use WellMate to add 30 &mu;L PBST (PBS + 0.1% Triton-X). Incubate 10 minutes and repeat. Operate WellMate as described above, except tubing does not need to be sterilized.</li>
 <li>To detect vaccinia infection we use a &beta;-galactosidase reporter driven by an early/late vaccinia promoter.</li>
 <li>Remove liquid and incubate cells in block (PBST with 2% BSA) for 10 minutes.</li>
 <li>Dilute primary antibody in block, remove liquid and add 15 &mu;L antibody to each well using a multi-channel repeat pipettor (Matrix).</li>
 <li>Spin down plates (300xg, 1 min), cover with a clear sticker, and incubate at 4&deg;C overnight.</li>
 <li>Remove primary antibody, and use the WellMate to wash wells in PBST 3 times, 10 minutes each.</li>
 <li>Dilute fluorescently labeled secondary antibody (FITC) and nuclear stain in block, and add 15 &mu;L to wells using a multi-channel repeat pipettor.</li>
 <li>Incubate plates 1 hour at room temperature, protected from light.</li>
 <li>Wash wells in PBST 3 times, 10 minutes each using the WellMate.</li>
 <li>Seal plate with clear sticker, cover, and store at 4&deg;C up to three weeks.</li>
</ol>
Part 4: Imaging and Image Analysis
<ol>
 <li>Use an automated microscope at 20X magnification to image plates.</li>
 <li>Capture images of total nuclei (DAPI) and infected cells (FITC). Optimize exposure time for each channel individually in order to maximize dynamic range.</li>
 <li>Fine-tune the automatic focus function by taking a Z stack of images, spaced 1um apart, that span the planes above and below the expected plane of focus. Once the optimal focal plane is located, adjust autofocus settings accordingly.</li>
 <li>Image the entire plate, capturing at least three images per well in both the Hoechst channel (DAPI) and the virus channel (FITC). Choose sites from several different regions within one well in order to capture images that represent the well as a whole.</li>
 <li>Use MetaXpress software to write a journal to segment the image and use Count Nuclei modules to calculate the total number of cells positive for nuclear staining (total cells) and infection (FITC) for each plate.</li>
 <li>Calculate percent infection (100*FITC positive/total cells) and log transform.</li>
 <li>To identify candidates we use a robust Z score based on the median and interquartile range of each plate. This method is insensitive to outliers and nonsymmetric data, and thus provides greater power in identifying even weak or moderate hits 10. For each plate, the median is subtracted from the log-transformed data. Then the resulting values are divided by 0.74 times the interquartile range (the difference between the values of the 75th percentile and the 25th percentile). The 0.74 factor is used to approximate a standard normal distribution.</li>
 <li>The robust Z score is a measure of distance in standard deviations from the median of the plate. For example, a robust Z-score of 2 indicates the % infection of the well is ~2 standard deviations from the plate median or p&lt;0.05.</li>
 <li>Analyze cell number to identify cytotoxic candidates. Calculate the robust Z score of nuclear counts. Candidates with a robust Z&lt;-2 in duplicate screens are considered cytotoxic.</li>
 <li>Wells that have a robust Z score for percent infection of &gt;2 or &lt;-2 in duplicate screens (p&lt;0.0025) without cytotoxicity are considered potential candidates from the primary screen.</li>
 <li>Positive candidates are validated using independent reagents. dsRNAs are generated against the genes of interest from another region of the mRNA and tested as above.</li>
 <li>Those genes for which two independent dsRNAs have the same phenotype can be considered for further study.</li>
</ol>
Part 5: Representative Images and Interpretation of Infection Rates
<img src="/files/ftp_upload/2137/2137fig1.jpg" alt="Figure 1" /> Figure 1. Uninfected wells do not show any staining for vaccinia virus proteins, while a representative infected well contains cells staining positive for vaccinia-expressed beta-galactosidase (&beta;gal) protein, as measured by immunofluorescence microscopy. Virus=green, nuclei = blue.
<img src="/files/ftp_upload/2137/2137fig2.jpg" alt="Figure 2" /> Figure 2. Automated image analysis software quantifies infection in each image using parameters set by the user. In a representative image, the total number of cell nuclei and the number of cells expressing viral antigen are counted based on size and the intensity of staining above the local background staining.
<img src="/files/ftp_upload/2137/2137fig3.jpg" alt="Figure 3" /> Figure 3. Knockdown of thread (dIAP) serves as a positive control for robust RNAi depletion of target genes. Knockdown of this anti-apoptotic factor leads to dramatic cell death. Nuclei=blue.
<img src="/files/ftp_upload/2137/2137fig4.jpg" alt="Figure 4" /> Figure 4. Knockdown of luciferase serves as a negative control for the effect of double-stranded RNA treatment on infection. Depletion of luciferase has no effect on infection relative to cells left untreated. Virus=green, nuclei = blue.
<img src="/files/ftp_upload/2137/2137fig5.jpg" alt="Figure 5" /> Figure 5. Knockdown of &beta;gal protein serves as a positive control for a reduction in infection, since the assay uses &beta;gal protein levels as a read out of infection. Depletion of &beta;gal results in a decrease in the percentage of infected cells. Virus=green, nuclei = blue.
<img src="/files/ftp_upload/2137/2137fig6.jpg" alt="Figure 6" /> Figure 6. Knockdown of cellular factor Rab5 results in a decrease in the percentage of infected cells. Since Rab5 is known to participate in endocytosis, this factor likely contributes to vaccinia virus entry. This represents an example outlier from the screen. Virus=green, nuclei = blue.

<ol>
	<li>Ramadan, N., Flockhart, I., Booker, M., Perrimon, N., Mathey-Prevot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+high-throughput+RNAi+screens+in+cultured+Drosophila+cells.">Design and implementation of high-throughput RNAi screens in cultured Drosophila cells.</a> Nat Protoc. 2, 2245-2264 (2007).</li><li>Chotkowski, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus+infection+of+Drosophila+melanogaster+induces+a+protective+RNAi+response.">West Nile virus infection of Drosophila melanogaster induces a protective RNAi response.</a> Virology. 377, 197-206 (2008).</li><li>Blondel, D., Petitjean, A. M., Dezelee, S., Wyers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+in+Drosophila+melanogaster+cells:+regulation+of+viral+transcription+and+replication.">Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication.</a> J Virol. 62, 277-284 (1988).</li><li>Sessions, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+insect+and+human+dengue+virus+host+factors.">Discovery of insect and human dengue virus host factors.</a> Nature. 458, 1047-1050 (2009).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+RNAi+screening+in+Drosophila+S2+cells:+what+have+we+learned+about+host-pathogen+interactions?.">Genomic RNAi screening in Drosophila S2 cells: what have we learned about host-pathogen interactions?.</a> Curr Opin Microbiol. 11, 262-270 (2008).</li><li>Hao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+RNAi+screen+identifies+host+genes+important+for+influenza+virus+replication.">Drosophila RNAi screen identifies host genes important for influenza virus replication.</a> Nature. 454, 890-893 (2008).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+activity+coupled+with+fatty+acid+biosynthesis+is+required+for+viral+replication.">COPI activity coupled with fatty acid biosynthesis is required for viral replication.</a> PLoS Pathog. 2, e102-e102 (2006).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+reveals+a+specific+sensitivity+of+IRES-containing+RNA+viruses+to+host+translation+inhibition.">Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition.</a> Genes Dev. 19, 445-452 (2005).</li><li>Moser, T. S., Jones, R. G., Thompson, C. B., Coyne, C. B., Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Kinome+RNAi+Screen+Identified+AMPK+as+Promoting+Poxvirus+Entry+through+the+Control+of+Actin+Dynamics.">A Kinome RNAi Screen Identified AMPK as Promoting Poxvirus Entry through the Control of Actin Dynamics.</a> PLoS Pathog. 6, e1000954-e1000954 (2010).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4, 67-73 (1999).</li><li>Echeverri, C. J., Perrimon, N. <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+RNAi+screening+in+cultured+cells:+a+user's+guide.">High-throughput RNAi screening in cultured cells: a user's guide.</a> Nat Rev Genet. 7, 373-384 (2006).</li></ol>

No conflicts of interest declared.

Genome-wide RNAi screening in Drosophila provides a robust and efficient method for examining the cellular component of viral infection. A number of mammalian viruses infect Drosophila cells, which can be used to identify components of the host intrinsic immune response, and host protein factors required for viral replication that may represent novel therapeutic targets. Before performing a screen, the assay must be carefully optimized for cell type, cell number, and infection level, and must be well controlled, including both positive and negative viral and cellular targets11. It is important to ensure sufficient separation between positive and negative controls prior to screening to maximize the dynamic range of the assay. Once the screen has been performed, candidates can be divided into functional categories to determine which host mechanisms contribute to infection. For mammalian viruses such as vaccinia, the contribution of mammalian homologs by RNAi should be determined as part of secondary analysis. Taken together, these robust screening methods will allow us to gain insight into complex mechanisms by which viruses interact with their host cells.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell culture</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Schneider&#x2019;s media</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11720</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX 100X</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>35050</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin (pen/strep)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>F6178</td>
<td>&nbsp;</td>
<tr>
<td>Screening</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Genome-wide dsRNA library</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>384 well plates, black with clear bottom</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3712</td>
<td>&nbsp;</td>
<tr>
<td>Aluminum seals</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>6569</td>
<td>&nbsp;</td>
<tr>
<td>Clear seals</td>
<td>&nbsp;</td>
<td>Denville</td>
<td>B1212-4</td>
<td>&nbsp;</td>
<tr>
<td>Well Mate</td>
<td>&nbsp;</td>
<td>Matrix</td>
<td>201-10001</td>
<td>&nbsp;</td>
<tr>
<td>24 pin manifold</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>3-000-101</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B2261</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescently-labeled secondary antibodies</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ImageXpressMicro automated microscope</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MetaXpress image analysis software</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

rnai screening for host factors involved in vaccinia virus infection using drosophila cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

Watch this Scientific Journal Video about RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells at JoVE.com

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

rnai-screening-for-host-factors-involved-vaccinia-virus-infection

Research

JoVE Journal

Immunology and Infection

9.4K vues.  University of Pennsylvania. Facteurs de l'hôte roman impliqués dans l'infection virale peuvent être identifiés par la cellule basée échelle du génome perte de fonction ARNi dépistage. Une Drosophile Modèle de culture cellulaire est prête particulièrement bien à cette approche en raison de la facilité et l'efficacité de l'ARNi. Ici nous montrer cette technique en utilisant Vaccine Virus comme un exemple.

Vidéo: RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

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

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

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

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

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...