Authors would like to acknowledge Dr. Frank Scholle for his helpful comments in the manuscript, Chloe Mariant for her help with the microscopy images and Teresa M. Tiedge for her helpful English revision.

1. Titration protocol
NOTE: Use a cytopathic virus infecting adherent cells. For this demonstration, Influenza A Virus (IAV) of swine origin (A/California/07/2009/(H1N1)) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 2, strain NC 1-7-4 were used.
<ol>
	<li>Titrate these viruses in 96 well plates for 7 days in a biosafety cabinet located in a Biosafety Level 2 (BSL-2) laboratory.
	<ol>
		<li>To perform these titrations, seed 96 well plates with the required cell line. For PRRSV, use MA-104 cell line and for IAV use MDCK cell line. For the cell culture, use DMEM medium supplemented with 10% FBS, L-Glutamine and Penicillin-Streptomycin and grow the cells to confluency.</li>
		<li>Before infection, wash the cells using 200 &#181;L of PBS.</li>
		<li>Dilute the virus stocks using 10 fold dilutions series by mixing 900 &#181;L of media and 100 &#181;L of virus. Make sure to properly vortex the tube to ensure proper mixing of the medium and virus and to avoid dilution errors.</li>
		<li>Mark the layout of the plate on the lid. Wash the wells with 1x phosphate saline buffer (PBS). Add 50 &#181;L of the inoculum in the corresponding wells following previously described titration methods2,3.</li>
		<li>Incubate at 37 &#176;C in a 5% CO2 incubator for 7 days.</li>
	</ol>
	</li>
</ol>
2. Cytopathic effect (CPE) assessment&#160;via microscopy
<ol>
	<li>After the 7-day incubation, wash all wells with 200 &#181;L of 1x PBS twice.</li>
	<li>Assess all wells of the plate under a light microscope to visually detect CPE. In the case of both PRRSV and IAV, their CPE consists of cell death and subsequent detachment from the plate, leading to monolayer disruption. However, other viruses may present different types of CPE.</li>
</ol>
3. Staining protocol
NOTE: Cytopathic effect (CPE) was assessed via crystal violet staining.
<ol>
	<li>After the 7-day incubation, wash all the wells using 200 &#181;L of 1x PBS twice.</li>
	<li>Fix the cells by adding 50 &#181;L/well of 4% paraformaldehyde (PFA) in 1x PBS and incubate for 15 min at room temperature (RT).</li>
	<li>After incubation, wash the cells twice with 200 &#181;L of 1x PBS. Then, add 50 &#181;L/well of crystal violet diluted to 4% in water and incubate for 5 min at RT. 
	NOTE: Crystal violet chemical stains the cells that remain attached to the plate at the time of fixation, leaving the sections of the well where the cells detached as unstained.</li>
	<li>Finally, aspirate crystal violet from the wells and optionally leave the plates to air dry for 2-5 min at RT or wash the plate with 200 &#181;L of water to remove excess stain prior to visualization.</li>
	<li>Use previously described methods to mathematically calculate the titer. Here, Karber formula and Muench formula were applied for PRRSV and IAV, respectively2,3. Details of these equations are presented in the representative result section.</li>
</ol>
4. Immunocytochemical (ICC) labeling
NOTE: Immunocytochemistry labeling for both viruses was performed following previously described methods9,10,11.
<ol>
	<li>After the 7-day incubation of the titration, fix the cells using PFA as described in step 3.2.</li>
	<li>Wash the plates with a solution composed by 100 mL of 1 M Tris hydrochloride, 1 g of Saponin, and 8.5 g of NaCl in 900 mL of H2O. Then, complete two additional washes with a mixture of 1x PBS and 5% Fetal Bovine Serum (FBS) and incubate with that Tris-Saponin-NaCl solution at Room temperature (RT) for 20 min.</li>
	<li>Incubate all the wells with the primary antibody for 2 h at RT. 
	NOTE: The volume of primary antibodies for each virus was 100 &#181;L and was prepared using a 1:300 dilution of 2% FBS in 1x PBS.</li>
	<li>Wash the cells twice with the Tris, Saponin, and NaCl solution prepared in step 4.2.</li>
	<li>Incubate all the wells with the secondary antibody for 1 h at RT. 
	NOTE: The volume of secondary antibody was 100 &#181;L and was prepared using a 1:250 dilution of 2% FBS in 1x PBS.</li>
	<li>Wash the cells with the Tris, Saponin, and NaCl dilution as described in step 4.2.</li>
	<li>Aspirate the previous dilution and incubate the plates with 200 &#181;L/well of aminoethyl carbazole (AEC) solution at a final dilution of 1:50 in water following the manufacturer&#39;s directions for 30 min at RT.</li>
	<li>After incubation, discard the AEC solution and add 100 &#181;L/well of 1x PBS for imaging via microscopy.</li>
	<li>Mathematically derive the titer as described in step 3.5.</li>
</ol>

<ol>
	<li>K&#228;rber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+to+the+collective+treatment+of+pharmacological+series+experiments.">Contribution to the collective treatment of pharmacological series experiments.</a> Naunyn-Schmiedeberg's Archive for Experimental Pathology and Pharmacology. 162 (4), 480-483 (1931).</li><li>Ramakrishnan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+50%+endpoint+titer+using+a+simple+formula.">Determination of 50% endpoint titer using a simple formula.</a> World Journal of Virology. 5 (2), 85-86 (2016).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+per+cent+endpoints.">A simple method of estimating fifty per cent endpoints.</a> American Journal of Epidemiology. 27 (3), 493-497 (1938).</li><li>Spearman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+method+of+right+and+wrong+cases+(constant+stimuli)+without+Gauss's+formulae.">The method of right and wrong cases (constant stimuli) without Gauss's formulae.</a> British Journal of Psychology. 2 (3), 227 (1908).</li><li>Darling, A. J., Boose, J. A., Spaltro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+assay+methods:+accuracy+and+validation.">Virus assay methods: accuracy and validation.</a> Biologicals. 26 (2), 105-110 (1998).</li><li>Kim, J., Chae, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+virus+isolation,+polymerase+chain+reaction,+immunohistochemistry,+and+in+situ+hybridization+for+the+detection+of+porcine+circovirus+2+and+porcine+parvovirus+in+experimentally+and+naturally+coinfected+pigs.">A comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs.</a> Journal of Veterinary Diagnostic Investigation. 16 (1), 45-50 (2004).</li><li>Suchman, E., Blair, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytopathic+effects+of+viruses+protocols.">Cytopathic effects of viruses protocols.</a> American Society of Microbiology. , (2007).</li><li>Karakus, U., Crameri, M., Lanz, C., Y&#225;ng&#252;ez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Influenza Virus. , 59-88 (2018).</li><li>Tingstedt, J. -. E., Nielsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+immune+responses+in+the+lungs+of+pigs+infected+in+utero+with+PRRSV:+an+immunohistochemical+study.">Cellular immune responses in the lungs of pigs infected in utero with PRRSV: an immunohistochemical study.</a> Viral Immunology. 17 (4), 558-564 (2004).</li><li>Guarner, J., 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=Immunohistochemical+and+in+situ+hybridization+studies+of+influenza+A+virus+infection+in+human+lungs.">Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs.</a> American Journal of Clinical Pathology. 114 (2), 227-233 (2000).</li><li>Nicholls, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+highly+pathogenic+influenza+and+pandemic+influenza+virus+in+formalin+fixed+tissues+by+immunohistochemical+methods.">Detection of highly pathogenic influenza and pandemic influenza virus in formalin fixed tissues by immunohistochemical methods.</a> Journal of Virological Methods. 179 (2), 409-413 (2012).</li></ol>

The authors have nothing to disclose.

Viral titrations are routinely used in virology research, with PFU detection and TCID50 assays most commonly used1,2,3,4. Both methods rely on the detection of CPE in infected cells, and even though they can be visually assessed via microscopy, a staining is usually applied to obtain a more objective result or to even reduce incubation times. In the case of TCID50, one of the most comm...

Viral titration via TCID50 assay is a commonly used technique in infectious disease research1. Although variations on the math behind this method have been proposed over time1,2,3,4, the currently applied methods of infection detection rely on visual confirmation through the presence of cytopathic effect (CPE) using microscopy5. To confirm CPE visualization more objectively on TCID50 assays, immunocytochemical (ICC) intracellular staining targeting the proteins of the virus is one of the most-commonly used methods6 as different viruses can produce varying forms of CPE. In our case, the cell morphological changes are similar when infected with both Influenza A virus (IAV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), where infected cells round up and detach from the plate. In the case of PRRSV, it causes a CPE known as &#34;total destruction&#34;, where all cells end up detaching from the well. IAV on the other hand, can present both total destruction and an additional CPE known as &#34;subtotal destruction&#34; where a small number of cells do not detach after infection7. However, this technique is time consuming to perform and requires the use of relatively costly reagents. It is important to note that ICC does not label CPE, but rather the number of cells successfully infected by the virus. This implies that the cells that were successfully infected by the end of the incubation will be seen as positive even if the infection has not caused CPE yet, and thus, a higher percentage of ICC positive cells compared to CPE is expected. For that reason, in this study we describe a complementary method of visual detection of CPE in a TCID50 assay based on crystal violet, a chemical with a positive charge that attaches to cell membranes and is used to stain adherent cells. Crystal violet is often utilized in virology research to measure plaque-forming units, among others8.
In this study, we compare the sensitivity of non-stained microscopy CPE detection with crystal violet staining and immunocytochemical staining based on viral protein recognition, which is known to be more objective due to its high sensitivity. This study shows that both crystal violet and immunocytochemical staining are more accurate than visual microscopy-based CPE detection and can be used to objectively identify infected wells in a TCID50 titration. Given their ability to reach nearly identical level of accuracy on the cytopathic viruses tested in cell lines, crystal violet is presented as a faster and more affordable way to determine viral titrations on a TCID50 assay. The proposed method using crystal violet staining takes a total time of 40 min to perform, with 15 min for paraformaldehyde (PFA) incubation, 5 min for crystal violet incubation and a maximum of 15 min for material preparation, buffer washes, and drying. The immunocytochemistry protocol applied for comparison takes an average time of 4 h 30 min and was performed as previously described9,10. The method proposed aims to help visualize a completed viral titration. Infection and incubation times can be performed with different layout depending on the virus. Here we tested two RNA viruses with cytopathic effect on cell lines.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well cell culture plates</td>
			<td>Genesee</td>
			<td>25-221</td>
			<td>Clear, flat bottom</td>
		</tr>
		<tr>
			<td>AEC solution</td>
			<td>Thermo Fisher</td>
			<td>1122</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Thermo Fisher</td>
			<td>C581-25; C581-100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>BioWest</td>
			<td>S1480</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehide</td>
			<td>Thermo Fisher</td>
			<td>J19943</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Influenza Antibody</td>
			<td>Bioss</td>
			<td>BS-0344R</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary PRRSV Antibody</td>
			<td>Bioss</td>
			<td>BS-10043R</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Thermo Scientific</td>
			<td>AAA1882014</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondaty antibody</td>
			<td>Invitrogen</td>
			<td>31460</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Hydrochloride</td>
			<td>Thermo Scientific</td>
			<td>AM9856</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of crystal violet to improve visual cytopathic effect-based reading for viral titration using tcid50 assays

Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are the two main methods to calculate the titer of a virus stock and are often based on microscopy detection or cell staining for visualization. In the case of TCID50 assay, objective visualization is commonly based on immunocytochemical (ICC) staining of intracellular virus to calculate titers combined with visual CPE detection via microscopy. However, ICC staining is costly and time consuming. In this study, we compared visual CPE observation via microscopy, ICC staining and crystal violet staining to determine the titers of two CPE-forming viruses, Influenza A virus (IAV) of swine origin and Porcine Reproductive and Respiratory Syndrome virus (PRRSV). We show that both crystal violet and ICC staining are more accurate than visual CPE detection, presenting nearly identical levels of precision on both IAV and PRRSV. For this reason, here we present crystal violet staining as a faster and more affordable way to determine viral titrations on a TCID50 assay for CPE-forming viruses titrated in cell lines.

The equations used for mathematically calculating the titer were previously described2,3.
Briefly, for PRRSV, we apply the Karber method:
Titer (TCID50) = 10 T + 1.3 where:
<img alt="Equation 1" src="/files/ftp_upload/63063/63063eq01.jpg" />
In this formula d = negative log of the last dilution with complete positive virus response: five posi...

Watch this Scientific Journal Video about Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays at JoVE.com

Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays

This protocol shows an accurate and objective approach to visualize viral titrations using crystal violet, by comparing it with optical microscopy and immunocytochemical staining.

Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are ...

use-crystal-violet-to-improve-visual-cytopathic-effect-based-reading

Research

JoVE Journal

Immunology and Infection

12.2K Views.  North Carolina State University. This protocol shows an accurate and objective approach to visualize viral titrations using crystal violet, by comparing it with optical microscopy and immunocytochemical staining.

Video: Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

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 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

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

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

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

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

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