This work was partially supported by the Spanish Government "Ministerio de Educaci&oacute;n y Ciencia" (project AGL2008-05275-C01/ALI), by the European Union Research Framework 7 funded projects VIROBATHE (Contract No. 513648), VIROCLIME (Contract No. 243923) and by the Catalan Agency of Water, Ag&egrave;ncia Catalana de l'Aigua (ACA), Departament de Control i Millora dels Ecosistemes Aqu&agrave;tics. During the developed study Marta Rusi&ntilde;ol was a fellow of the Catalan Government "AGAUR" (FI-DGR).

1. Concentration of the viral particles present in water samples
<ol>
	<li>Collection and conditioning of water samples
		<ol>
		<li>Collect a minimum of 2 replicates of 10 L per sample in plastic containers with flat bottom and one extra sample as a process control. This last sample will be spiked with a known amount of viral particles and used as a control. 
		Note: It is recommended to have special separate material (bottles, tube, etc.) for spiked samples.</li>
		<li>Check the calibration of the conductimeter and recalibrate if necessary. Prepare a negative control by using tap water previously adjusted to the adequate conductivity (see below 1.6) in one extra plastic 10 L container.</li>
		<li>For spiked samples: Add the standard volume of the control virus (approximately 105 genome copies per 10 L of water) to the sample. Mix by stirring avoiding splashing and aerosols. Positive controls could consist in an uncommon strain of adenovirus such as HAdV-35 or a bacteriophage such as MS2.</li>
		<li>If the sample presents high quantity of suspended material (sand or other materials), let it sediment for 15 minutes. Transfer the water into a new container.</li>
		<li>Adjust the pH of the water sample to 3.5 (&plusmn; 0.1) by the addition of 1 N HCl. This step is important for the concentration of the viruses, so make sure the pH has been properly adjusted. Mix the water thoroughly by vigorous stirring while adding the HCl. (Note: If the pH is lower than 3.5 add 1 M NaOH).</li>
		<li>Adjust the conductivity. If the sample has a conductivity of 1500 &mu;S/cm or higher, this step is not needed. If the conductivity is lower than 1500 &mu;S/cm the formation of flocculated material (flocs) is not guaranteed so adjust the conductivity to 1500 &mu;S/cm by the addition of artificial sea salts (Sigma). Mix vigorously by stirring while adding the sea salts.</li>
		<li>Record the pH of the samples before and after conditioning as well as the volume of HCl used. The conductivity should also be recorded after adjusting the pH. Always disinfect the pH-meter and conductimeter electrodes with a fresh HCl solution, dechlorinate with 10% sodium tiosulphate solution and finally rinse with distilled water.</li>
		</ol>
	</li>
	<li>Preparation of pre-flocculated 1 % skimmed milk (PSM)
		<ol>
			<li>Check the calibration of the pH-meter and the conductimeter and recalibrate if necessary. </li>
			<li>Prepare pre-flocculated skimmed milk solution (1 % PSM, w/v) by dissolving 10 g skimmed milk powder (Difco) in 1 L artificial seawater (dissolve 33.3 g of artificial sea salts in 1 L of dechlorinated tap water and autoclave) and carefully adjusting the pH to 3.5 with 1N HCl. The flocs should be visible. Prepare the solution just before to be used or store at 4&deg;C for 24 h. For dechlorination use 1 ml of 10% tiosulphate solution per 100 ml of water.</li>
		</ol>
	</li>
	<li>Flocculation of viral particles present in water samples
		<ol>
		<li>Add 100 ml of 1 % PSM to the 10-L water sample.</li>
		<li>Stir the samples for 8-10 h to allow the viruses to adsorb to the flocs. Use a timer to switch-off the stirring after 8-10 h.</li>
		<li>Stop the stirring and let the flocs sediment by gravity for 8-10 h.</li>
		</ol>
	</li>
	<li>Collecting and re-dissolving the flocs. Centrifugation
		<ol>
		<li>Remove the supernatant using a peristaltic pump and a plastic pipette connected to a plastic tube. For spiked samples the supernatant should be collected into a bottle and disinfected according to internal procedures. In all cases TAKE CARE not to collect the pellet. </li>
		<li>Collect the sediment with the flocs (approximately 500 ml) into a centrifuge bottle. </li>
		<li>Balance the pots by the addition of PSM pH 3.5. </li>
		<li>Centrifuge the pots in a high-speed centrifuge at 8,000 x g for 30 min at 4&deg;C. As soon as the centrifuge stops, carefully remove the centrifuge pots from the centrifuge. </li>
		<li>Very gently pour off and discard the supernatant. Follow appropriate measures for infectious material.</li>
		<li>Add 7 ml of phosphate buffer to dissolve the pellet in each centrifuge bottle.</li>
		<li>Once the flocs have been dissolved, measure and add phosphate buffer to reach a total volume of 10 ml.</li>
		<li>Homogenize the viral concentrate by vortexing and distribute the 10 ml into clean microtubes which should be frozen at -80&deg;C until needed in further analysis.</li>
		</ol>
	</li>
</ol>
2. Nucleic acid extraction
<ol>
	<li>Perform a nucleic acid extraction with the QIAamp Viral RNA Mini Kit following manufacturer instructions. This kit enables the use of an automated platform (such as Qiacube, Qiagen).</li>
</ol>
3. Quantitative PCR of human adenoviruses (HAdV), JC polyomaviruses (JCPyV), porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV)
<ol>
	<li>Quantification of genome copies in the samples
		<ol>
		<li>Prepare the qPCR mix in a clean separate area by using TaqMan Environmental PCR Master Mix 2x (Applied Biosystems) . The reaction takes place in a 96-well optical reaction plate covered with optical adhesive covers. Concentrations of the Master Mix, primers and probes are described in Table 1.</li>
		<li>Once the mix has been prepared, aliquot 15&mu;l into each well including the controls (see 3.2). The total volume for one reaction after addition of target will be 25&mu;l (15&mu;l mix + 10&mu;l sample or standard).</li>
		<li>Add the nucleic acid extractions from the samples (10&mu;l) in a separate area. Run direct and a ten-fold dilution in purified water of each sample in duplicate. Cover the wells containing the samples with part of an adhesive cover, keep the other part of the cover for the following step.</li>
		<li>Add dilutions of the DNA standard suspensions (10&mu;l) from 100 to 106 GC/10&mu;l by triplicate and using a micropipette exclusively used for the standard DNA. It is advisable to add standards in an area equipped with UV radiation for destroying plasmid DNA and to clean the micropipette after each use. Cover the wells containing the standards with the pre-cut adhesive cover. 
		Note: To prepare standard suspensions to be used in the quantification of genome copies, the target DNA region should be cloned into a plasmid and linearized. In the following address you will find a detailed procedure on how to create standard curves with plasmid DNA templates to be used in qPCR: 
		<a href="http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf">http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf</a></li>
		<li>Perform the qPCR into an adequate system selecting the appropriate parameters (considering the use of adhesive cover and the total volume in each well, etc). Following activation of the AmpliTaq Gold for 10 min at 95&deg;C, 40 cycles of amplification are performed as follows: 15 s at 95&deg;C and 1 min at 60&deg;C for HAdV, JCPyV and BPyV, and 15 s at 95&deg;C, 20s at 55&deg;C and 20s at 60&deg;C for PAdV.</li>
		<li>Once the reactions are completed, store data and results as described in the user&#x2019;s manual of the equipment used. The amount of DNA will be defined as the median of the data obtained after correcting the dilution factor when needed.</li>
		</ol>
	</li>
	<li>Controls
		<ol>
		<li>Use positive and negative controls. The assay must include more than one non-template control (NTC) to prove mix does not produce fluorescence. It is advisable to run the positive process control in order to evaluate potential enzymatic inhibition due to inhibitors present in the studied samples.</li>
		<li>Record results of qPCR assays of two different dilutions of the standard DNA and from the process control. Use results to prepare control charts for quality control (QC) programs related to the sensitivity and efficiency of the assays.</li>
		</ol>
	</li>
	<li>Confirmation of results
		<ol>
		<li>Positive results may be further confirmed using nested-PCR and nucleotide sequencing of the amplicons, producing additional data on the nucleotide sequences of the strains detected1,5,6,7,9,12. </li>
		</ol>
	</li>
</ol> 
Following the procedure described, human and animal viruses have been detected and quantified in bathing waters, seawater and river water2,3. As a representative example, ground water samples from areas presenting high levels of nitrates were evaluated to define the sources of the contamination. Ten-liter water samples were collected from 4 different wells in rural areas of a Northeast region in Spain. Five replicates were collected in each well being one replicate seeded with human adenovirus 2 used as process control. Samples were processed according to the protocol represented in Figure 1. The four replicates analyzed in one of the four sites studied showed positive results for PAdV (mean value 7.74x102 GC/L) which would be related to the presence of pig slurries in the areas surrounding the sampling site and would support fecal porcine contamination as the source of nitrates in groundwater (Table 2).
4. Representative Results:
<img src="/files/ftp_upload/2820/2820fig1.jpg" alt="Figure 1" /> 
Figure 1. Procedure for the detection and quantification of viruses in water.
<img src="/files/ftp_upload/2820/2820table1.jpg" alt="Table 1" /> 
Table 1. Concentration of primers and probes for qPCR assays.
<img src="/files/ftp_upload/2820/2820table3.jpg" alt="Table 3" /> 
Table 2. Detection and quantification of animal and human adenoviruses and polyomaviruses in ground water samples. 
n number of replicates analyzed 
% percentage of positive replicates 
(-) non detected

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Two patent applications were filed in 2009 to protect the intellectual property of protocols for quantification of PAdV and BPyV.

The procedure described would fulfill the conditions for a fitting method for routine environmental and public health laboratories: reproducible, reliable, straightforward and cost-effective. The protocol is simple; however it must be followed carefully. Low conductivity in the samples without adding the requested concentration of artificial seawater salts would dramatically reduce the recovery of viruses as would be the case if the stirring time for flocculation is significantly reduced (less than 5 hours for example).<...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>High speed centrifuge (8,000xg)</td>
 <td>Berckman Coulter</td>
 <td>Avanti J-20XP</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pH-meter, thermometer and conductimeter</td>
 <td>Afora</td>
 <td>LPPC3003</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Plastic tubes 100-200 cm length</td>
 <td>Deltalab</td>
 <td>350059</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile graduated disposable pipettes</td>
 <td>Labclinics</td>
 <td>PN10E1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile plastic tubes of 1.5 and 10-15 mL (Eppendorf, Falcons, etc.)</td>
 <td>Afora</td>
 <td>KA298/00</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Centrifuge pots (500 mL)</td>
 <td>Fisher Scientific</td>
 <td>SE5753512</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Magnetic stirrers and magnets (one per sample)</td>
 <td>Fisher Scientific</td>
 <td>10510</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass or plastic containers having flat bottoms to allow the use of magnetic stirrers </td>
 <td>Deltalab</td>
 <td>191642</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>A peristaltic pump for removing the supernatant (or a water-jet vacuum pump)</td>
 <td>Watson-Marlow</td>
 <td>323E/D</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Timer to switch-off the stirring after 8-10 hours</td>
 <td>Deltalab</td>
 <td>900400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hydrochloric acid (1N and 0.1N)</td>
 <td>Panreac</td>
 <td>141020.1611</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium hydroxide (1N)</td>
 <td>Panreac</td>
 <td>131687.1211</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Artificial seawater sea salts </td>
 <td>Sigma</td>
 <td>S9883</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Skimmed milk (SM)</td>
 <td>Difco</td>
 <td>232100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate buffer pH 7,5</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1:2 v/v of sterile Na2HPO4 0,2M and NaH2PO4 0,2M at pH 7.5</td>
 </tr>
 <tr>
 <td>Thiosulphate </td>
 <td>Panreac</td>
 <td>121879.1209</td>
 <td>Make a 10% solution in water </td>
 </tr>
 <tr>
 <td>QIAamp Viral RNA Mini Kit </td>
 <td>Qiagen</td>
 <td>52904</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96-well optical reaction plate (500 units)</td>
 <td>Applied Biosystems</td>
 <td>43426659</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Optical adhesive covers (100 units)</td>
 <td>Applied Biosystems</td>
 <td>4311971</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TaqMan Environmental PCR Master Mix 2x</td>
 <td>Applied Biosystems</td>
 <td>4396838</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses at JoVE.com

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

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 ...

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12.1K vistas.  University of Barcelona. El estudio describe un método costo-efectivo para la identificación de la fuente de contaminación fecal / orina o la contaminación por nitratos en el agua utilizando qPCR para la cuantificación específica de los virus de ADN humano / porcino / bovino, adenovirus y poliomavirus, propuesto como herramientas de MST.

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

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 ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

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 ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...