Name: Vaccinia Virus Infection and Temporal Analysis of Virus Gene Expression: Part 1 (Video) | JoVE Journal
Uploaded: 2009-04-08T00:00:00.000+00:00
Duration: 12 min
Description: Learn vaccinia virus infection and RNA extraction from HeLa cells for temporal gene expression analysis. This protocol establishes methods to study viral-host interactions using microarray hybridization techniques.

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5 minutes

Whitehead Institute Fellows Funds

Part 1: Setting up the infection
<ol>
<li>Grow HeLa cells in flasks and wait until the cells are approximately 80% confluent.</li>
<li>Prepare enough viral growth medium for the experiment: regular DMEM with 2% FBS and no added antibiotics.</li>
<li>The infection can be performed with either a crude stock of vaccinia virus with a known titer, or sucrose purified virus with a known titer if you're concerned about host gene expression.</li>
<li>If you are using sucrose purified virus, proceed directly to part 2.</li>
<li>If you are using a crude vaccinia virus stock, just prior to the infection, sonicate an aliquot of the virus in a cup sonicator with a bath of mostly ice and a little water.
<ul style="list-style-type:lower-alpha;">
<li>Sonicate at around 30W for 20 seconds.</li>
<li>Vortex the tube and repeat the sonication 3 times, adding more ice if needed to keep the cup packed with ice and chilled.</li>
<li>Vortex in between each sonication step.</li>
<li>Proceed to part 2.</li>
</ul>
</li>
<li>Alternatively, if you do not possess a cup sonicator, mix an equal volume of the crude virus stock and 0.25mg/mL trypsin.
<ul style="list-style-type:lower-alpha;">
<li>Vortex vigorously.</li>
<li>Incubate the virus stock/trypsin mix in a 37&deg;C waterbath for 30 minutes.</li>
<li>Vortex at 5-10 minute intervals.</li>
<li>Proceed to part 2.</li>
</ul>
</li>
</ol>
Part 2: Infecting cells
<ol>
<li>Calculate the amount of viral particles needed to infect the monolayer at the desired multiplicity of infection (MOI). Typically, a high MOI (between 5 and 10) is used for infection timecourses.</li>
<li>Add the desired amount of sucrose purified virus or trypsinized crude virus to 37&deg;C viral growth media and mix well. You should use enough media to just cover the bottom of the flask. For example, 10 mL of media for a T-175 flask.</li>
<li>Remove media from the cells and rinse with room temperature PBS.</li>
<li>Add the virus/viral growth media to each flask.
<ul style="list-style-type:lower-alpha;">
<li>Swirl gently, tilt plates and incubate at 37&deg;C in a 5% CO2 incubator for 1 hour.</li>
<li>Tilt and swirl plates every 15 minutes to spread virus uniformly and keep cells moist.</li>
<li>For a 0hr/Mock time point, add the viral growth media only, with no virus added.</li>
</ul>
</li>
<li>After the 1 hour incubation, remove the media containing the virus, and rinse three times with room temperature PBS.</li>
<li>Add the maximum amount of viral growth medium to each flask. For example, 30 mL of media for a T-175 flask. Start counting this as your 0 hr time point.</li>
</ol>
Part 3: Harvesting cells
<ol>
<li>Check the cells under a microscope and note any cytopathic effect (CPE).</li>
<li>Remove the media and rinse cells with 30 mL of room temperature PBS.</li>
<li>Add 15 mL of trypsin to the cells and incubate for 2-5min at 37&deg;C.</li>
<li>Check under microscope for cell detachment and tap flask gently to dislodge cells.</li>
<li>Transfer cells with a sterile serological pipette into a 50 mL conical tube.</li>
<li>Wash the flask with an equal volume of cell growth media, and add the media to the trypsinized cells in the conical tube.</li>
<li>Centrifuge the cells at 300 x g for 5 minutes at room temperature.</li>
<li>Remove the trypsin/media from the cell pellet.</li>
<li>Resuspend the cell pellet in either TRIzol reagent or the lysis buffer from your desired RNA isolation kit.</li>
<li>If you are using TRIzol, divide the sample into 1 mL aliquots in 1.5mL labeled Eppendorf tubes and freeze at -80&deg;C.</li>
<li>Repeat for all time points, harvesting one flask per time point.</li>
</ol>
Part 4: RNA extraction of samples in TRIzol
<ol>
<li>At this point, your samples should be resuspended in TRIzol reagent and ready to be processed.</li>
<li>Add 200&micro;L of the BCP Phase Separation Reagent for every 1 mL of TRIzol in each tube. You may need to transfer the sample to a larger tube if you are starting out with a large amount of TRIzol.</li>
<li>Vortex or shake vigorously and incubate at room temperature for 2-3 minutes.</li>
<li>Centrifuge the sample at 12,000 x g for 15 minutes.</li>
<li>Transfer the aqueous phase to a fresh tube. The aqueous phase is the clear layer on the top. You should be recovering approximately 600&micro;L for every 1mL of TRIzol you started out with.</li>
<li>Do a second phenol/chloroform extraction, this time using 500&micro;L of chloroform per 1 ml TRIzol, instead of BCP.</li>
<li>Vortex or shake vigorously and incubate at room temperature for 2-3 minutes.</li>
<li>Centrifuge the sample at 12,000 x g for 15 minutes.</li>
<li>Transfer the aqueous phase to a fresh tube. The aqueous phase is the clear layer on the top.</li>
<li>Add 20&micro;g of linear acrylamide to each sample. The linear acrylamide acts as a carrier and helps to precipitate the RNA.</li>
<li>Add 500&micro;L of isopropanol per 1 ml of TRIzol you started out with to each sample and mix well.</li>
<li>Incubate samples at room temperature for 10 minutes.</li>
<li>Centrifuge in a microcentrifuge at maximum speed for 10-15 minutes.</li>
<li>The RNA pellet will be visible at the bottom of the tube. Very carefully, remove the supernatant from the tube, either with a vacuum aspirator, or manually by pipettor. Do not disturb the pellet.</li>
<li>Wash the pellet with 1 mL of 70% ethanol.
<ul style="list-style-type:lower-alpha;">
<li>Add 1 mL of 70% ethanol to the pellet.</li>
<li>Centrifuge at top speed 14,000g for 5-7 minutes.</li>
<li>Very carefully, remove the ethanol from the tube, either with a vacuum aspirator, or manually by pipettor. Check that the pellet is visible at the bottom of the tube.</li>
<li>Discard the supernatant.</li>
</ul>
</li>
<li>Repeat the wash step. After the supernatant is discarded, mark where pellet is on the tube.</li>
<li>Air dry pellet for no longer than 5 minutes. Do not overdry, or the RNA will be difficult to reconstitute!</li>
<li>If you wish to follow the optional DNase treatment to remove any remaining DNA from the sample, resuspend the pellet in 17&micro;L of nuclease-free water. Otherwise, resuspend the pellet in 20&micro;L of nuclease-free water and continue to step 22.</li>
<li>(Optional DNase treatment) Using the Qiagen RNase-free DNase set, add 2&micro;L Buffer RDD and 1&micro;L reconstituted RNase-free DNase I to the resuspended RNA. Mix gently.</li>
<li>(Optional DNase treatment) Incubate at 37&deg;C for 30 minutes.</li>
<li>(Optional DNase treatment) Add 2&micro;L of 2.5 mM EDTA and incubate at 65&deg;C for 5 minutes to inactivate the DNase. Do not exceed the inactivation time or temperature as longer times/higher temperatures can cause degradation of the RNA.</li>
<li>Check the RNA concentration by spectrophotometer.</li>
<li>Store the RNA sample at -80&deg;C.</li>
<li>(Optional QC) Check the RNA quality using an Agilent BioAnalyzer, or by running the sample on a denaturing gel and checking the O.D. readings. (260nm and 260/280 ratio)</li>
</ol>

<ol><li>KH, R. ubins, LE, H. ensley, GW, B. ell, Wang, C., EJ, L. efkowitz, PO, B. rown, DA, R. elman <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection%3A+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> PLoS ONE. 3 (7), e2628-e2628 (2008).</li>
<li>Assarsson, E., Greenbaum, J. A., Sundström, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci%22%5BJournal%5D)">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> Proc. Natl. Acad. Sci. 105 (6), 2140-2145 (2008).</li>
<li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Poxvirus+transcriptome+analysis%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E62-E62 (2008).</li>
<li>Assarsson, E., Greenbaum, J. A., Sundström, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A.+Reply+to+Satheshkumar+and+Moss%3A+Poxvirus+transcriptome+analysis%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E63-E64 (2008).</li>
<li>Guerra, S., López-Fernández, L. A., Pascual-Montano, A., Muñoz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> J Virol. 77 (11), 6493-6506 (2003).</li>
<li>Guerra, S., López-Fernández, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> J Virol. 78 (11), 5820-5824 (2004).</li>
<li>Guerra, S., López-Fernández, L. A., Pascual-Montano, A., Nájera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host+response+to+the+attenuated+poxvirus+vector+NYVAC%3A+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> J Virol. 80 (2), 985-998 (2006).</li>
<li>Guerra, S., Nájera, J. L., González, J. M., López-Fernández, L. A., Climent, N., JM, G. atell, Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> 81 (16), 8707-8721 (2007).</li>
</ol>

Critical Steps
Part 1 &amp; 2
There are several critical steps to setting up a synchronous vaccinia infection, the first being careful sonication (or trypzinizing) of the virus, in order to disaggregate virus particles. Vaccinia is highly prone to aggregating, and disruption of virus particles is important for ensuring even infection of cells. In order to achieve a synchronous infection, a high MOI (greater than 2) should be used to ensure each cell is infected. A m...

<table><tbody><tr><td>TRIzol Reagent</td><td>Reagent</td><td>Invitrogen</td><td>15596-026</td><td>Similar reagents, such as TriPure from Roche, will also work.</td></tr><tr><td>BCP Phase Separation Reagent</td><td>Reagent</td><td>Molecular Research Center</td><td>BP151</td><td></td></tr><tr><td>RNase-Free DNase Set</td><td>Reagent</td><td>Qiagen</td><td>79254</td><td>DNase treatment is an optional step.</td></tr></tbody></table>

vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Time course infections of hela cells using vaccinia virus begin by setting up the viral infections, infecting the cells, and then harvesting the RNA from the infected cells at several time points post infection. This RNA can later be amplified for hybridization to microarrays for analysis of gene expression. Hi, I am Judy Ann from the laboratory of Kate Irvins at the Whitehead Institute for Biomedical Research.Today we will show you a procedure for a time course infection with the vaccinia virus in mammalian cells and the subsequent RNA extraction from the infected cells at different time points post-infection. This technique is a beginning step in a larger protocol aimed to extract and amplify RNA in order to assess virus's gene expression during infection, which will complete in two other JoVE videos. So let's get started To set up for the infection, grow hela cells and flasks and wait until the cells are approximately 80%confluent.Next, be sure to prepare enough viral growth medium for the experiment. Using regular DMEM with 2%FBS and no added antibiotics, the infection can be performed with either recruited stock of vaccinia virus with a known titer or sucrose purified virus with a known titer. If you're concerned about host gene expression, if you are using sucrose purified virus, skip to the next session.If you are using a crude vaccinia virus, stock sonicate and aliquot of the virus just before infection, and a cup sonicate with a bath of mostly ice and a little water sonicate around 30 W for 20 seconds. Vortex the tube and repeat the sonication three times adding more ice if needed to keep the cup packed with ice and chilled, be sure to vortex in between each sonication step. If you do not possess a cup ator, mix an equal volume of the crude virus stock in point 25 milligrams per milliliter.Trypsin vortex vigorously incubate the virus stock trips and mix in a 37 degrees Celsius water bath for 30 minutes. Vortex at five to 10 minute intervals to infect cells. First, calculate the amount of viral particles needed to infect the monolayer at the desired multiplicity of infection.Or MOI. Typically a high MOI between five and 10 is used for infection time courses. Next, add the desired amount of sucrose purified virus or tryp crude virus to 37 degrees Celsius viral growth media and mix well.You should use enough media to just cover the bottom of the flask. 10 milliliters of media would be enough for a T 1 75 flask. Remove the media from the cells and rinse with room temperature.PBS.Add the virus or viral growth media to each flask for a zero hour mock time point. Add the viral growth media only with no virus added swirl gently and tilt the plates incubated 37 degrees Celsius in a 5%CO2 incubator for one hour. Be sure to tilt and swirl the plates every 15 minutes to spread the virus uniformly and keep the cells moist.After incubation. Remove the media containing the virus and rinse three times with room temperature.PBS. Add the maximum amount of viral growth medium to each flask.For example, 30 milliliters of media for a T 1 75 flask. You can start counting this as your zero hour time point at the desired time point.Post-infection. Check the cells under a microscope and note any cytopathic effects or CPE.Remove the media and rinse the cells with 30 milliliters of room temperature PBS. Next, add 15 milliliters of trypsin to the cells and incubate for two to five minutes. At 37 degrees Celsius, check under a microscope for cell detachment and tap the flask gently to dislodge cells.Transfer the cells with a sterile serological pipette into a 50 milliliter conical tube. Wash the flask with an equal volume of cell growth media and add the media to the trypsin cells in the conical tube. Centrifuge the cells at 500 Gs for five minutes at room temperature.After centrifugation, remove the tryin media from the cell pellet resuspend, the cell pellet, and either triol reagent or the lysis buffer from your desired RNA isolation kit. If you are using triol, divide the sample into one milliliter quats and 1.5 milliliter labeled eend dwarf tubes and freeze negative 80 degrees Celsius. Repeat for all time points.Harvesting one flask per time point. At this point, your sample should be resuspended and triol reagent and ready to be processed. Add 200 microliters of the BCP phase separation reagent.For every one milliliter of triol in each tube, BCP or one bromo three chloro propane is used in place of phenol. To reduce genomic DNA contamination you may need to transfer the sample to a larger tube. If you are starting out with a large amount of triol vortex or shake vigorously and incubate room temperature for two to three minutes after incubation, centrifuge the sample at 12, 000 GS for 15 minutes after centrifugation.An acqui phase should be visible as a clear layer on top. Transfer the acquiesce phase to a fresh tube. You should be recovering approximately 600 microliters for every milliliter of triazole you started out with.Do a second phenol chloroform extraction. This time using 500 microliters of chloroform per one milliliter triazole instead of BCP. The second chloroform extraction is used to remove any traces of organic solvent from the extraction as even trace amounts can inhibit subsequent amplification steps.Vortex or shake vigorously, then incubate a room temperature for two to three minutes. Centrifuge the sample at 12, 000 GS for 15 minutes after centrifugation. Transfer the acquiesce phase to a fresh tube.Remember, the aqueous phase is the clear layer on top. Next, add 20 micrograms of linear acrylamide to each sample. The linear acrylamide acts as a carrier and helps to precipitate the RNA.Add 500 microliters of isopropanol per one milliliter of triol you started out with to each sample and mix. Well incubate the samples at room temperature for 10 minutes after incubation centrifuge and a micro centrifuge at maximum speed for 10 to 15 minutes. The RNA pellet will be visible at the bottom of the tube after centrifugation.Very carefully, remove the supernatant from the tube either with a vacuum aspirator or manually by pipetter. Do not disturb the pellet. Wash the pellet with one milliliter of 70%ethanol and centrifuge at top speed for five to seven minutes.Very carefully. Remove the ethanol from the tube either with a vacuum aspirator or manually by pipetter. Discard the supernatant.Check that the pellet is visible at the bottom of the tube. Repeat this wash step after the supernatant is discarded. Mark where the pellet is on the tube.Air dry the pellet for no longer than five minutes. Do not over dry or the RNA will be difficult to reconstitute. Next, if DNA's treatment is planned resus, suspend the pellet in 17 microliters of nuclease free water.If D'S treatment is not planned, resus suspend in 20 microliters. For D'S treatment, use the KaiGen RNA free DNA set. Add two microliter buffer, RDD and one microliter reconstituted RNAs free.DNAs one to the resuspended, RNA mixed gently incubated 37 degrees Celsius for 30 minutes. Next, add two microliters of 2.5 millimolar EDTA and incubate at 65 degrees Celsius for five minutes. To inactivate the DNA, do not exceed the inactivation time or temperature as longer times and higher temperatures can cause degradation of the RNA.Now you have completed the DNA's treatment. Check the RNA concentration by spectrophotometer. Traces of phenol or BCP can be detected as a spike at 270 nanometers beyond the standard peak at 260 nanometers.When measuring the absorbance of total RNA after extraction, if such contamination appears, retract the RNA using a filter or column-based RNA extraction method before proceeding with amplification. You can store the RNA sample at negative 80 degrees Celsius. You can check the RNA quality if you desire, by using an Agilent bioanalyzer or by running the sample on a denaturing gel and checking the OD readings on the bioanalyzer.Good RNA quality is indicated by the RNA integrity number or RIN. The RIN is based on a numbering system from one to 10, with one being the most degraded profile and 10 being the most intact. We've just shown you how to perform a time course infection with the vaccinia virus and how to extract the RNA from infected cells at various time points post infection.When doing this procedure, it's important to remember that there are several critical steps in setting up a synchronous vaccinia infection. First careful sonication or trypsin ization of the virus. It's important to disaggregate virus particles since vaccinia is highly prone to aggregating, which would prevent an even infection of cells.Second, in order to achieve asynchronous infection, a high MOI of greater than two should be used to ensure each cell is infected. A mixture of infected and uninfected cells will lead to multiple rounds of infection, heterogeneous mixtures of time points, and asynchronous viral, and host transcriptional responses. Third, the infection should be carried out in minimal amounts of media to allow maximum absorption of the virus onto the cells.In addition, regular shaking of the flask or culture dishes every 10 minutes enables distribution of the virus across the flask and ensures that the cells do not dry out. So that's it. Thanks for watching and good luck with your experiments.Be sure to watch the other videos in this series where we show the procedure for linear amplification of RNA samples and the preparation and cleanup of fluorescently labeled RNA.

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Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression.  Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

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

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach.  This protocol describes how to process  tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (γHV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for γHV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

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

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Viral Tracing of Genetically Defined Neural Circuitry

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

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

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.

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

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.

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

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

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

Learn vaccinia virus infection and RNA extraction from HeLa cells for temporal gene expression analysis. This protocol establishes methods to study viral-host interactions using microarray hybridization techniques.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1

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