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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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+increase+in+human+monkeypox+incidence+30+years+after+smallpox+vaccination+campaigns+cease+in+the+Democratic+Republic+of+Congo.">Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo.</a> Proceedings of the National Academy of Sciences of the U S A. 107 (37), 16262-167 (2010).</li><li>Sodeik, B., Griffiths, G., Ericsson, M., Moss, B., Doms, R. 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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. 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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 border="0" cellpadding="0" cellspacing="0" width="1249">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:747px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:104px;">11995-065</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fetal Bovine Serum - Optima, Heat Inactivated (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">200 mM L-Glutamine, (L-glut)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3712</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Trypsin, 2X, Sterile, Irradiated</td>
			<td>Worthington Biochemical Corp.</td>
			<td>TRLVMF</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl Sulfoxide, Cell Culture (DMSO)</td>
			<td>American Bioanalytical</td>
			<td>AB03091</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1-&#946;-D-Arabinofuranosylcytosine&#160; (AraC), MW= 280 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>C6645</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">isatin &#946; -thiosemicarbazone (IBT), MW= 234 g/mol</td>
			<td>Fisher Scientific</td>
			<td>NC9075202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Rifampicin, MW= 823 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">ST-246, MW= 376 g/mol</td>
			<td>SIGA Labs, Corvallis, OR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">8% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TempPlate RT optically clear film</td>
			<td>USA Scientific</td>
			<td>2978-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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. ...

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Research

JoVE Journal

Immunology and Infection

11.3K 조회수.  Boston University School of Medicine.  우리는 스펙트럼 구별 리포터 형광의 단계 - 특이 적 발현을 통해 바이러스 감염 및 유전자 발현의 실시간 측정을 가능하게하는 형광 리포터 백시 니아 바이러스의 사용을 설명한다. 우리 세부 정확하게 바이러스 복제가 저분자 저해에 응답하여 영향되는 단계를 식별하기위한 플레이트 기반 방법. 

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

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

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

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

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

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

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

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

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 ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...