We thank Bert Dibbits for technical assistance. The Animal Breeding and Genomics Group, Wageningen University, The Netherlands, generously hosted us in their laboratory. The personnel of the Ottenby Bird Observatory, Sweden, is thanked for trapping and sampling the mallards, in particular Magnus Hellstr&ouml;m, Marcus Danielsson, Christopher Magnusson and Stina Andersson. We thank Sanne Svensson, Jonatan Qvist and Per-Axel Gj&ouml;res for filming at Ottenby, and Mano Camon for filming in the lab. Daniel Bengtson provided beautiful duck photographs for the video portion of this publication. Further free material from the CDC Public Health Image Library (PHIL; <a href="http://phil.cdc.gov/phil/">http://phil.cdc.gov/phil/</a>) was used: The electron micrograph (no. 280; by Dr. Erskine Palmer) and illustration (no. 11823; by Douglas Jordan) of the influenza virus. Financial support was given by the KNJV (Royal Netherlands Hunters Association), the Dutch Ministry of Agriculture, the Faunafonds and the Stichting de Eik Trusts (both in The Netherlands), the Swedish Research Council (grant no. 2007-20774) and the EC-founded Newflubird project. RNA isolation chemicals were a generous gift of Ambion, Inc, the RNA company. This is contribution No. 245 from the Ottenby Bird Observatory.

1. Duck Trapping and Cloacal Swabbing
<ol>
 <li>Trap dabbling ducks of the genus Anas (or other birds) in a cage by attracting them with lure ducks or food, and put them into individual card board boxes. Transportation time in the box should be kept to a minimum, for instance, by installing a field station within a short distance to the trap. Logistic circumstances may vary in each study. The advice and approval of the relevant animal ethics committee needs to be sought for each new set-up. Alternatively, also hunter shot birds can be sampled.</li>
 <li>Take the duck out of the box by holding its wings tight to its body. Sample a fresh dropping, which will be present in most of the cases, directly from the bottom of the box. Pick them up with a sterile swab and apply the fluid to a Whatman FTA card. If not, perform cloacal swabbing.</li>
 <li>Turn the duck on its back. This permits access to the cloaca and facilitates sampling. The cloaca is a protruding structure situated below the abdomen, close to the base of the tail. An experienced person could hold and sample the bird at the same time, or else a second person can assist.</li>
 <li>Carefully insert the sterile plastic rayon swab (Copan, Italy) approximately 1 cm and make a gentle swirl of the cloaca. Roll the swab with the fluids from the cloaca over the surface of a Whatman FTA card. After sampling has been performed immediate release of the animal is desireable.</li>
 <li>Dry the FTA cards at room temperature for at least 1 hour, then store each sample individually in paper bags (e.g. envelopes). Storage is possible at room temperature, possibly with silica beads in humid climates. The sending and receiving institutions' biosafety officers can permit to send FTA cards by regular mail, since the pathogens become inactive upon contact with the FTA card surface.</li>
</ol>
2. Viral RNA Isolation
<ol>
 <li>Extract the FTA card material including faeces/cloacal fluid. Punch three discs with a Harris 2 mm puncher and place all of those into a well of an RNase free 96well plate. Between each new sample, clean the puncher carefully with alcohol and a Kim precision wipe (Kimtech). Always use positive and negative controls. A positive control can consist of a laboratory strain freshly applied to an FTA card, or a natural sample which is know to yield a positive result. As negative control, punch out discs from an empty FTA card. Additionally, leave another well free for a PCR control later in your experiment (with water as template).</li>
 <li>Add 70&mu;l RNA rapid extraction solution (Ambion) and heat-seal the plate (AbGene Thermo-Sealer). Incubate 5 minutes on a plate shaker at room temperature with the determined speed that does not cause spill-over (this depends on the used plate shaker model; test in advance).</li>
 <li>Carry viral RNA isolation according to the manufacturer's protocols with the MagMAX-96 viral RNA isolation kit (Ambion). In brief: Add 130&mu;l prepared lysis/binding solution (from the kit) to each well. Transfer 50&mu;l extracted FTA card material to each well. Shake plate for 1 minute.</li>
 <li>Add 20&mu;l prepared bead mix (from the kit) to each sample and mix by pipetting up and down. Shake on determined speed for 5 minutes. RNA molecules will bind to the magnetic beads.</li>
 <li>Move the plate to a magnetic 96-well stand to capture the beads. Depending on the model of magnetic stand used, this can take more than 5 minutes. When the solution has turned completely clear, remove and discard the supernatant. Then remove the plate from the magnetic stand.</li>
 <li>Wash the beads twice with wash solution 1 and twice with wash solution 2 (from the kit). For each of the 4 wash steps add 150&mu;l prepared wash solution to each sample, shake for 1 minute on determined speed, move the plate to the magnetic stand, capture beads for about 5 minutes (or until the solution is completely clear), remove and discard the supernatant. Then remove the plate from the magnetic stand, add the next wash solution, and carry out the washing steps as previously. After the last washing step, remove as much wash solution as possible and air dry the beads at room temperature in the plate shaker for 2 minutes.</li>
 <li>Add 50&mu;l of elution buffer (from the kit) to release RNA from the beads and shake for 4 minutes on the plate shaker. Capture beads as previously described. The supernatant now contains the isolated RNA which is ready for downstream applications.</li>
</ol>
3. RT-PCR of the AIV Matrix Gene
Carry out reverse transcription PCR (RT-PCR) with the One-Step Access RT-PCR system (Promega) in 25 &mu;l reactions (adjusted from Kraus et al.18 and Fouchier et al.22):
<table border="1">
 <tbody>
 <tr>
 <td>Nuclease free water</td>
 <td>1.5&mu;l</td>
 </tr>
 <tr>
 <td>AMV/Tfl5 buffer</td>
 <td>5&mu;l</td>
 </tr>
 <tr>
 <td>dNTPs</td>
 <td>0.5&mu;l</td>
 </tr>
 <tr>
 <td>primer M52C22 (10 &mu;M)</td>
 <td>2.5&mu;l</td>
 </tr>
 <tr>
 <td>primer M253R22 (10 &mu;M)</td>
 <td>2.5&mu;l</td>
 </tr>
 <tr>
 <td>MgSO4 (25 mM)</td>
 <td>7&mu;l</td>
 </tr>
 <tr>
 <td>AMV reverse transcriptase (5u/&mu;l)</td>
 <td>0.5&mu;l</td>
 </tr>
 <tr>
 <td>Tfl polymerase (5u/&mu;l)</td>
 <td>0.5&mu;l</td>
 </tr>
 <tr>
 <td>RNA sample</td>
 <td>5&mu;l</td>
 </tr>
 </tbody>
</table>
PCR conditions in a Biometra T1 thermocycler are: initial reverse transcription of 45 minutes at 45&deg;C, followed by 2 minutes initial denaturation at 94&deg;C and 40 cycles of: 94&deg;C for 1 minute, 56&deg;C for 1 minute, and 68&deg;C for 2 minutes. An additional 7 minutes elongation at 68&deg;C concludes the amplification.
4. Screening for AI-positive Samples and Purification of Targeted Fragments from Gel
<ol>
 <li>Load 2&mu;l of the PCR product mixed with 2&mu;l 5x loading dye (BioRad) and 6&mu;l water on a 1% agarose gel (Roche) stained with 1% ethidium bromide (2.5&mu;l per 100 ml gel) for a pre-screening.</li>
 <li>Run the gel for 1 hour at 120V, along with a DNA size standard (BioRad EZ load 100 bp ladder), visualise with a gel documentation system. See an example in Figure 1.</li>
 <li>Select samples with amplified fragments in the expected size range (between 200 bp and 300 bp (target fragment is 244 bp). Load the whole PCR reaction volume (of which &tilde;23&mu;l are left) of these candidates with 6&mu;l 5x loading dye on a 2% ethidium bromide stained agarose gel and run for 2 hours.</li>
 <li>Place the gel on a UV-transilluminator (Bioblock Scientific) and inspected visually. See an example in Figure 2. Excise bands of the correct size from gel with a scalpel and placed into individual 1.5 ml reaction tubes. Purify fragments from gel, for instance with the Zymoclean Gel DNA Recovery Kit (Zymo Research).</li>
</ol>
5. Sequencing and Identification of PCR Products
<ol>
 <li>Carry out Sanger sequencing of the target fragments, for instance on an ABI 3730 capillary sequencer with ABI Big Dye 3.1 chemistry (Applied Biosystems). Prepare sequencing reactions in 10&mu;l volumes containing 10-20 ng gel-purified template cDNA, 1.75&mu;l 5x dilution buffer, 0.5&mu;l Big Dye V3.1 premix, 1 &mu;l forward primer (M52C20, 10 mM), and ddH2O. Cycling conditions are: 1 min initial denaturation, followed by 25 cycles of: 10 s at 96&deg;C, 5 s at 45&deg;C and 4 min at 60&deg;C. Purify and prepare the sequencing reaction according to your internal protocols.</li>
 <li>Identify resulting cDNA sequences against nucleotide databases such as GenBank23, e.g. by web based tools such as BLAST at the National Centre for Biotechnology Information (NCBI, <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a>).</li>
</ol>
6. Representative Results:
Mallards (Anas platyrhynchos) were sampled at Ottenby Bird Observatory in December 2007. From each mallard a sample on FTA card was taken as described in this protocol. After shipping, the FTA cards were kept in a freezer at -20&deg;C for two years. The same FTA card sample of the laboratory isolate tested in Kraus et al.18 was included as positive control, as well as nine tenfold serial dilutions of it. Two negative controls were i) extraction from an empty FTA card, to test if there was carry-over from the puncher, and ii) RT-PCR reaction in which nuclease free water was used as template, to test if contamination occurred during, or in preparation of the PCR reaction.
84 samples were analysed. A gel picture of the PCR products from these 84 samples can be found in Figure 1. From natural samples a multitude of unspecific bands can be observed due to the presence of various microbial contaminations in the faeces. However, the target fragment of the primer pair is 244 bp long. The whole PCR-reaction volume of a subset of the samples which produced fragments in approximately the correct size range (between 200 bp and 300 bp) was loaded on gel (Figure 1). An illustration of which of the bands were cut from the gel can be found in Supplementary Figure 1. In addition to the positive control, two of these samples (69899 and 69912) were positive by the new protocol. A BLAST search against the NCBI nucleotide database revealed their identity as AI matrix gene (Figure 3), while all the other bands resembled bacterial sequences most closely, or did not yield a readable sequence at all.
<img alt="Figure 1" src="/files/ftp_upload/2832/2832fig1.jpg" /> 
Figure 1. Gel picture of a preliminary screening of the PCR products. 2 &mu;l PCR product of positive control, serial dilutions of the positive control, two negative controls and 84 cloacal samples were loaded. 48 samples are shown in the top gel panel, and 48 samples in the bottom panel. Red arrows indicate gel lanes used for 100 bp DNA size standard. For illustration, red circles show bands in the expected size range. Blue circles indicate an unspecific amplicon (top left) or a primer dimer artefact below 100 bp in size (bottom right).
<img alt="Figure 2" src="/files/ftp_upload/2832/2832fig2.jpg" /> 
Figure 2. Selection of samples with fragments in the correct size range. Samples with bands between 200 bp and 300 bp (target fragment 244 bp) were chosen. The green arrow indicates the positive control, the two red arrows indicate samples which were confirmed to be AIV positive by comparing their cDNA sequences to the NCBI nucleotide database.
<img alt="Figure 3" src="/files/ftp_upload/2832/2832fig3.jpg" /> 
Figure 3. Screen capture of a representative BLAST search at NCBI. One of the cDNA sequences obtained from the excised fragments was queried against the nucleotide database at the NCBI website. The sample is correctly identified as AI Matrix gene fragment. <a href="https://www.jove.com/files/ftp_upload/2832/2832fig3large.jpg">To view a full sized version of this image, click here.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/2832/2832supfig1.jpg" /> 
Supplementary Figure S1. Bands of selected samples excised from gel. This picture was taken from the gel depicted in Figure 1 after candidate bands between 200 bp and 300 bp were cut out.

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Trapping, handling and sampling of birds were done following national legislation and were approved by the Swedish Board of Agriculture and the research animal ethics committee.

The protocol described here provides a supplementary method to screen faecal or similar samples for the presence of AIV. It was especially designed to make sample collection quick and easy. This makes it possible for less trained persons, such as hunters or wildlife managers, to contribute to AI surveillance. No cool chains need to be applied, although freezing the samples is recommended where possible. A few days of room temperature, for instance during transport to the laboratory, were not a problem for RNA molecules on FTA cards, as long as the cards remained dry. Other non-cooled storage systems that are currently evaluated by the research community, such as alcohol15 or guanidine16, require special shipping arrangements because of their hazardous nature. In contrast, the FTA card method does not require shipping of hazardous materials. However, another interesting storage medium in this respect is RNAlater&trade; that is not hazardous, either17. Sample analysis can be carried out in any standard molecular laboratory. No special equipment other than for usual PCR reactions and capillary sequencing was needed. All steps could be carried out without a biosafety level because already at sample collection the potential pathogenic agents were inactivated by the antibacterial and antiviral activity of the FTA card.
Cross-contamination and subsequent false positive samples were not observed in our trials with wild bird samples. However, when working with RNA and PCR it is always advisable to pay special attention to clean working places and separate rooms for pre- and post-PCR steps. Working in a fume hood decreases the risk of aerosol contamination in the laboratory. Pipetting needs to be carried out with filter tips.
From a previous study on samples taken simultaneously from the same ducks we know that six of the 84 samples were positive by the traditional RealTime RT-PCR method24 and the Ct values from RealTime RT-PCR are known. The two positive samples detected by our method stem from ducks which had Ct values &lt;30 (indicating a high concentration of viral RNA). The other four samples which were positive with the traditional protocol had Ct values &gt;30 (less concentrated) and could not be detected by our protocol.
Only samples with relatively high virus titers were positive in our assay and it is likely that sensitivity of the method was the source of failure to detect all positive samples. Further, the sample size in the current study was very low and the method can be completely developed and assessed if more controlled and rigorous experiments are carried out. However, if these samples would have been collected from a remote area, traditional analysis would not have been possible at all. Additionally, these first results stem from pilot experiments which need further optimisation. A period of two years storage in a regular -20&deg;C freezer after sample collection probably also affected the quality of the viral RNA. This possible RNA degradation is an important issue when dealing with room temperature storage of inactivated viruses. Samples stored in alternative liquids as mentioned above suffer from significant degradation which impacts analysis of longer stretches of the viral genome16. Although not tested in our study, FTA cards have proven to be well suited to preserve intact RNA molecules in other RNA systems that are very similar to Avian Influenza viruses25, 26.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent or instrument</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>Plastic rayon dry swab</td>
			<td>Copan, Italy</td>
			<td>167KS01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FTA card</td>
			<td>Whatman</td>
			<td>&nbsp;</td>
			<td>several options</td>
		</tr>
		<tr>
			<td>Harris micro punch 2mm with mat</td>
			<td>Whatman</td>
			<td>1154409</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase free 96well plate</td>
			<td>Greiner Bio-One</td>
			<td>652290</td>
			<td>or comparable</td>
		</tr>
		<tr>
			<td>Kim precision wipes</td>
			<td>Kimtech</td>
			<td>75512</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNA rapid extraction solution</td>
			<td>Ambion</td>
			<td>AM9775</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MagMAX-96 viral isolation kit</td>
			<td>Ambion</td>
			<td>AM1836</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>One-Step Access RT-PCR system</td>
			<td>Promega</td>
			<td>A1250</td>
			<td>or comparable</td>
		</tr>
		<tr>
			<td>Agarose MP</td>
			<td>Roche</td>
			<td>1388991001</td>
			<td>multi purpose agarose</td>
		</tr>
		<tr>
			<td>5x loading buffer</td>
			<td>BioRad</td>
			<td>&nbsp;</td>
			<td>delivered with DNA ladder</td>
		</tr>
		<tr>
			<td>EZ load 100 bp ladder</td>
			<td>BioRad</td>
			<td>170-8353</td>
			<td>or comparable</td>
		</tr>
		<tr>
			<td>Ethidium bromide 1%</td>
			<td>Fluka</td>
			<td>46067</td>
			<td>or comparable</td>
		</tr>
		<tr>
			<td>1.5ml reaction tubes</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>or comparable</td>
		</tr>
		<tr>
			<td>Zymoclean Gel DNA recovery kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td>or comparable</td>
		</tr>
		<tr>
			<td>ABI big dye 3.1 chemistry</td>
			<td>Applied Biosystems</td>
			<td>&nbsp;</td>
			<td>or comparable</td>
		</tr>
	</tbody>
</table>

avian influenza surveillance with fta  cards: field methods, biosafety, and transportation issues solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

Watch this Scientific Journal Video about Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved at JoVE.com

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

avian-influenza-surveillance-with-fta-cards-field-methods-biosafety

Research

JoVE Journal

Immunology and Infection

19.1K Views.  Wageningen University. A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

Video: Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

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 ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.