We thank SIGA Technologies (Corvallis, OR) for providing ST-246. D.K.R. was supported by an NIH training grant in immunology to Boston University (5T32AI 7309). This work was supported in part by P41 086180, NIH RO1AI1096159-01, and RO3 &#160;(to J.H.C.).

1. Plate Cells
<ol>
	<li>Dissociate HeLa cells from plate and dilute in growth media (DMEM, 2 mM L-glut, 10% FBS) to approximately 2.0 x 105 cells/ml. Dispense 100 &#956;l/well in a black-walled, clear flat-bottom 96-well plate (20,000 cells/well).</li>
	<li>Incubate cells for 24 hr until confluent in a 37 &#176;C incubator + 5% CO2.</li>
</ol>
2. Infect Cells
<ol>
	<li>Dilute virus and infect cells.
	<ol>
		<li>Thaw TrpV (Triple virus; Early Venus, Intermediate mCherry, Late TagBFP) and PLV (Promoter-less Venus) fluorescent virus and disaggregate using sonication for 5 minutes. Alternatively, crude virus stocks can be mixed 1:1 with 0.25 mg/ml trypsin in infection media and incubated at 37 &#176;C for 30 min.</li>
		<li>Dilute crude virus stocks in 37 &#176;C infection media (DMEM, 2 mM L-glut, 2% FBS). For high MOI infections (10 PFU/cell), dilute virus stock to 1.0 x 107 PFU/ml assuming 5 x 104 cells/well and an inoculum volume of 50 &#956;l. Plan on infecting 3 replicate wells for each treatment with TrpV and another 3 replicate wells for the same treatment with PLV to account for background fluorescence specific to each treatment.</li>
		<li>Infect wells by adding 50 &#956;l/well diluted virus in infection media. This is defined as time = 0 hr post infection (0 hpi).</li>
	</ol>
	</li>
	<li>Dilute desired treatment compounds into infection media. For all treatments a single 2x master mix dilution should be made and applied to replicate wells (e.g. 350 &#956;l total volume for six 96-wells with 50 &#956;l each).
	<ol>
		<li>Dilute experimental compounds into infection media.</li>
		<li>Dilute vehicle-control solvent (e.g. PBS, DMSO) into infection media. Note: Similar final concentrations of vehicle-control solvents should be used as applied for experimental compounds (e.g. if your IBT stock is made with DMSO and used at a final concentrations of 1 &#956;l/ml, then use DMSO alone at 1 &#956;l/ml as the vehicle control).</li>
		<li>Dilute control compounds into infection media. Recommended control compounds include: 3.6 &#956;M (1 &#956;g/ml) 1-&#946;-D-Arabinofuranosylcytosine (AraC), 50 &#956;M (11.7 &#956;g/ml) isatin &#946; -thiosemicarbazone (IBT), 60 &#956;M (50 &#956;g/ml) Rifampicin, or 5 &#956;M (1.9 &#956;g/ml) ST-246 8,15,16. See Representative Results for expected effects. Note: make this dilution at twice (2x) the desired final concentration since this is added at a 1:1 ratio to the volume already in the well.</li>
	</ol>
	</li>
	<li>Immediately after addition of virus, add 50 &#956;l infection media containing desired treatments and controls as diluted in step 2.2. Note: To assay for inhibition before early stage expression, compound(s) can either be added directly to the diluted virus inoculum (step 2.1.2) or to host cells before infection (before step 2.1.3).</li>
	<li>Incubate 6-24 hr in a 37 &#176;C incubator + 5% CO2.</li>
</ol>
3. Fix Cells
<ol>
	<li>Fix cells by adding 100 &#956;l 8% paraformaldehyde to infection media already in each well. Incubate at room temperature for 15 min protected from light. Note: It is recommended to add 2x PFA (8%) directly to infection media to prevent aerosolization of infectious vaccinia which can occur if high-titer media is inverted directly into waste dish prior to fixation.</li>
	<li>Remove fixative by inverting plate into the waste dish.</li>
	<li>Add 100 &#956;l of room temperature PBS.</li>
	<li>Seal plate with optically clear adhesive film. Note: Plates can be stored at 4 &#176;C if not read immediately. Be sure to return sealed plates to room temperature before reading to prevent condensation from distorting spectrophotometer measurements.</li>
</ol>
4. Quantify Virus Growth
<ol>
	<li>Measure endpoint fluorescence at 515:530 for Venus, 587:610 for mCherry, and 415:457 for TagBFP (Excitation:Emission wavelength in nm). Four measurements should be averaged per well using optimized gain settings for each channel. Note: The appropriate emission wavelength may be different depending on the specific model and filter characteristics of your plate reader. This can be determined empirically by performing an emission wavelength scan to determine the point where there is the greatest difference between TrpV and PLV for each channel.</li>
	<li>Normalize the raw data to facilitate comparison between experiments.
	<ol>
		<li>For each channel, determine the mean of replicate TrpV and PLV wells.</li>
		<li>Subtract the mean PLV-infected background measurements from the mean TrpV-infected experimental measurements for each treatment.</li>
		<li>Normalize the data by dividing each background subtracted value by the vehicle-only value well.</li>
		<li>Once an experiment has been repeated multiple times, a one-way analysis of variance (one-way ANOVA) test and multiple comparison post-test can be performed to determine if any of the treatments reflects a statistically significant difference from the vehicle-only treatment for each channel.</li>
	</ol>
	</li>
</ol>
5. Alternative Protocol:&#160;Kinetic Plate Assays
<ol>
	<li>Perform all steps through addition of treatments (step 2.3).</li>
	<li>Seal plate with adhesive film and place in plate reader chamber equilibrated to 37 &#176;C. Note: Special care must be taken to keep plates consistently at 37 &#176;C to prevent undue cell stress and condensation. For kinetic assays, it is particularly important to use a buffered medium (sodium bicarbonate and HEPES) to maintain proper pH without addition of 5% CO2.</li>
	<li>Set plate reader gain manually to prevent saturation of later time points. Note: Accurate gain optimization can be obtained by performing an endpoint assays under similar conditions.</li>
	<li>Acquire hourly readings for 8-24 hpi and normalize using similar procedure as in endpoint assay (step 4.2).</li>
	<li>Individual time points can be analyzed for statistical significance using similar methods as the previously described endpoint assay.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+transcription.">Vaccinia virus transcription.</a> Journal of General Virology. 84 (9), 2293-2303 (2003).</li><li>Condit, R. C., Niles, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+viral+transcription+elongation+and+termination+during+vaccinia+virus+infection.">Regulation of viral transcription elongation and termination during vaccinia virus infection.</a> Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1577 (2), 325-336 (2002).</li><li>D&#237;az-Guerra, M., Rivas, C., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+expression+of+the+2-5A+synthetase/RNase+L+system+results+in+inhibition+of+vaccinia+virus+replication.">Inducible expression of the 2-5A synthetase/RNase L system results in inhibition of vaccinia virus replication.</a> Virology. 227 (1), 220-228 (1997).</li><li>Cohrs, R. J., Condit, R. C., Pacha, R. F., Thompson, C. L., Sharma, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+ppp(A2&#8217;p)nA-dependent+RNase+by+a+temperature-sensitive+mutant+of+vaccinia+virus.">Modulation of ppp(A2&#8217;p)nA-dependent RNase by a temperature-sensitive mutant of vaccinia virus.</a> Journal of Virology. 63 (2), 948-951 (1989).</li><li>S&#225;nchez-Puig, J. M., S&#225;nchez, L., Roy, G., Blasco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+different+leukocyte+cell+types+to+Vaccinia+virus+infection.">Susceptibility of different leukocyte cell types to Vaccinia virus infection.</a> Virology Journal. 1 (1), (2004).</li><li>Bengali, Z., Satheshkumar, P. S., Yang, Z., Weisberg, A. 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The authors have nothing to disclose and no patents are pending for viruses or screening methods.

&#160;Here, we have described the practical usage of a multi-stage vaccinia reporter virus (TrpV), which provides reproducible, real-time feedback measures of virus replication. By using well-defined poxvirus inhibitors, we were able to show that TrpV responds in a manner consistent with the understood mechanism of action for each inhibitor. While the TrpV virus provides the most comprehensive information about virus stage progression, two-stage (IREV, LREV) and single-stage (EV, IV, LV) viruses can also be used similarl...

Traditionally, virus expression is studied using molecular biology techniques (e.g. northern blotting, western blotting, microarray hybridization, etc.) 1. While these methods are capable of providing detailed information with respect to categorizing expression changes of individual mRNAs or proteins, they are typically not amenable to real-time and high-throughput processes. Alternative approaches using fluorescence-based reporters have been applied previously when working with poxviruses; however, their development and usage has been motivated by varied aims. Several such methods were designed for selection of recombinant viruses 2,3. These techniques express EGFP upon proper incorporation and expression of exogenous DNA from recombinant vaccinia clones. Similarly, several vaccinia strains stably expressing soluble EGFP or GFP-tagged proteins expressed under a native vaccinia promoter have been widely used. These are typically used to quantify virus entry and replication during antibody-based neutralization assays, chemical inhibition, or comparison of antiviral effectiveness 4&#8211;6. While these viruses have proven useful, they are limited in their ability to provide detailed information about the point of inhibition due to their usage of a single ambiguous early/late viral promoter. Previous methods have also made use of EGFP protein with suboptimal signal-to-noise characteristics.

Due to the complexity of poxvirus replication and the lack of existing tools available to assay real-time changes in viral gene expression, we have developed a suite of single and multi stage reporter viruses 7,8. As described in previous publications, these viruses express one, two, or three spectrally distinct fluorescent proteins from native vaccinia promoters during early, intermediate, or late stages in infection. These viruses can be used as indicators of virus replication progress using a fluorescence microscope and they are equally well suited for high-throughput plate-and-reader based assays. These viruses are easy to use in place of wild type virus, growing with similar kinetics to reach equivalent titers 7. During the planning and creation of these viruses, much care was taken in selecting fluorophores that have high signal-to-background characteristics with superior folding efficiency to facilitate reliable quantification with rapid feedback to changes in expression (Venus, mCherry and TagBFP). Additionally, combinations of viral promoters were chosen which produce high fidelity, unambiguous stage-specific expression, providing information about the full range of early (C11R promoter), intermediate (G8R promoter) and late (F17R promoter) gene expression, in contrast to ambiguous early/late promoters.

These viruses can be used as a tool for investigating the life cycle of the prototypical poxvirus, vaccinia virus. Much is still not known about the host-virus interaction despite its long history of study. Vaccinia is complex, producing over 200 unique proteins, many of which are immunomodulatory and host antagonistic. Upon infection, vaccinia virus immediately begins early mRNA transcription. This is facilitated by an RNA polymerase and transcription factors that were loaded on the viral genome during virion packaging and held in a paused state until subsequent infection. This early expression mainly produces proteins required for suppression of the host immune system (mRNA decapping, dsRNA sequestration, and decoy receptor proteins as well as inhibitors of apoptosis, stress response, and Toll, IL, and NF-&#954;B signaling) and genome replication. Early expression also produces transcription factors necessary for intermediate expression. Intermediate expression includes the expression of late stage transcription factors. This expression cascade leads to production of structural and enzymatic virus proteins during late stages of infection, which are necessary for complete assembly of the mature vaccinia virion.&#160;&#160;&#160;&#160;&#160;

Our set of fluorescent reporter viruses allows for rapid progress to be made in the understanding of poxvirus biology. One of the most common and time-consuming methods in the field of virology is the growth assay. This typically involves infecting cells, enacting a series of treatments, harvesting virus by lysing infected cells, and quantifying the resulting viral titer by plaque assay. Using the reporter viruses described here allows real-time measurement of virus growth that can be easily assayed and compared between numerous treatments performed in parallel. We foresee this set of reagents will be used in various protocols for identifying alterations in viral gene expression in response to drug treatment, RNAi knockdown, or host range restriction.

This method also enables high-content analysis on a larger scale than previously available, allowing for high-throughput anti-viral drug screens for specifically-defined target stages of interest. While numerous potential treatments to combat poxvirus infection have been identified, the only FDA approved therapies effective for treating poxvirus infection are the acyclic phosphonate nucleoside, Cidofovir, and treatment with vaccinia immune globulin 9,10. Despite the eradication of smallpox in 1977 11, poxviruses remain a significant threat to human health 12. Cessation of widespread vaccination against Variola virus has led to increased susceptibility to other poxviruses 13. For example, regions of Central Africa previously protected by vaccination are experiencing a surge in Monkeypox virus infections 14. Significant concern has also been raised regarding the latent susceptibility to intentional release of Variola virus. Due to the limited therapies currently available, there is an urgent need for development of novel treatments. These reporter viruses allow rapid and high-throughput small molecule inhibitor screening for inhibition of a specific stage of viral replication. Identification of inhibitors that target viral expression stages currently not the target of inhibition will facilitate development of combination therapies with increased potency.


Much can be gleaned about vaccinia cell biology by observing changes in each stage&#8217;s gene expression. Attenuation by chemical or genetic manipulation of an infected host cell is typically expressed in terms of reduced viral titers. However, by comparing changes in each stage of the viral expression cascade, one can obtain a more complete understanding of how virus fitness is impacted by a particular treatment. These data have been shown to correlate well with the traditional virus titer output, but provide more detailed mechanistic information as well as high throughput capabilities 7.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td></td></tr><tr><td>Fetal Bovine Serum - Optima, Heat Inactivated (FBS)</td><td>Atlanta Biologicals</td><td>S12450H</td><td></td></tr><tr><td>200 mM L-Glutamine, (L-glut)</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td><td>Corning</td><td>3603</td><td></td></tr><tr><td>384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td><td>Corning</td><td>3712</td><td></td></tr><tr><td>Trypsin, 2X, Sterile, Irradiated</td><td>Worthington Biochemical Corp.</td><td>TRLVMF</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Dimethyl Sulfoxide, Cell Culture (DMSO)</td><td>American Bioanalytical</td><td>AB03091</td><td></td></tr><tr><td>1-&amp;beta;-D-Arabinofuranosylcytosine&amp;nbsp; (AraC), MW= 280 g/mol</td><td>SigmaAldrich Co</td><td>C6645</td><td></td></tr><tr><td>isatin &amp;beta; -thiosemicarbazone (IBT), MW= 234 g/mol</td><td>Fisher Scientific</td><td>NC9075202</td><td></td></tr><tr><td>Rifampicin, MW= 823 g/mol</td><td>SigmaAldrich Co</td><td>R3501</td><td></td></tr><tr><td>ST-246, MW= 376 g/mol</td><td>SIGA Labs, Corvallis, OR</td><td></td><td></td></tr><tr><td>8% Paraformaldehyde (formaldehyde) aqueous solution</td><td>Electron Microscopy Sciences</td><td>157-8</td><td></td></tr><tr><td>TempPlate RT optically clear film</td><td>USA Scientific</td><td>2978-2700</td><td></td></tr><tr><td>Opti-MEM Reduced Serum Medium</td><td>Gibco</td><td>31985-070</td><td></td></tr></tbody></table>

vaccinia reporter viruses for quantifying viral function at all stages of gene expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

As an example of the typical usage of these viruses, the triple fluorescence-reporter virus was used to compare the point of inhibition of several well-defined poxvirus inhibitors.
HeLa cells were plated in tissue culture treated black-walled clear flat-bottom 96-well plates and incubated overnight. Confluent monolayers were infected at a multiplicity of infection (MOI) of 10 using either triple reporter virus (TrpV; Table 1) or promoter-less Venus (PLV) as detailed on the pla...

Watch this Scientific Journal Video about Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression at JoVE.com

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

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.

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

vaccinia-reporter-viruses-for-quantifying-viral-function-at-all

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

11.3K Views.  Boston University School of Medicine. 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.

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

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.

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.

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

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.

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

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 ...

Biology

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

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.

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

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 3

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

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.

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. ...

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

A Luciferase-Fluorescent Reporter Influenza Virus to Track Viral Infections

Source: Chiem, K., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59890">A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection.</a> J. Vis. Exp. (150), (2019).
This video demonstrates a method to track viral infections in mouse models using a recombinant luciferase and fluorescence protein-expressing bi-reporter Influenza A virus. This method enables both in vivo and ex vivo studies, enabling researchers to gain insights into viral dynamics and the effects of interventions.

Wirus wirusa grypy reporterowej lucyferazy-fluorescencji do śledzenia infekcji wirusowych

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. In ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells

Source: Dhungel, P., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59626">In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells.</a> J. Vis. Exp. (2019)
This video demonstrates an assay to study the translation regulation in poxvirus-infected cells. Uninfected and virus-infected cells were co-transfected with mRNAs encoding for firefly luciferase (Fluc) and Renilla luciferase (Rluc), with the Fluc mRNA containing a 5&#39; Poly(A) leader sequence. Upon adding bioluminescent substrates on lysed cells, measure the signal from both the luciferases to identify the translational advantage of the Fluc mRNA in virus-infected cells, conferred by the leader sequence.

Test reporterowy lucyferazy do badania regulacji translacji w komórkach zakażonych ospą

1. Transfect mRNA to Cells

	Seed HeLa cells in a 24-well plate (to be approx. ~80-90% confluent the next day) and incubate overnight in an incubator at ...

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

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

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

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

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

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

Wirus wirusa grypy reporterowej lucyferazy-fluorescencji do śledzenia infekcji wirusowych

Test reporterowy lucyferazy do badania regulacji translacji w komórkach zakażonych ospą