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.remotexs.ntu.edu.sg/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><tbody><tr><td>Plastic rayon dry swab</td><td>Copan, Italy</td><td>167KS01</td><td></td></tr><tr><td>FTA card</td><td>Whatman, GE Healthcare</td><td></td><td>several options</td></tr><tr><td>Harris micro punch 2mm with mat</td><td>Whatman, GE Healthcare</td><td>1154409</td><td></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></td></tr><tr><td>RNA rapid extraction solution</td><td>Ambion</td><td>AM9775</td><td></td></tr><tr><td>MagMAX-96 viral isolation kit</td><td>Ambion</td><td>AM1836</td><td></td></tr><tr><td>One-Step Access RT-PCR system</td><td>Promega Corp.</td><td>A1250</td><td>or comparable</td></tr><tr><td>Agarose MP</td><td>Roche Group</td><td>1388991001</td><td>multi purpose agarose</td></tr><tr><td>5x loading buffer</td><td>Bio-Rad</td><td></td><td>delivered with DNA ladder</td></tr><tr><td>EZ load 100 bp ladder</td><td>Bio-Rad</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></td><td>or comparable</td></tr><tr><td>Zymoclean Gel DNA recovery kit</td><td>Zymo Research Corp.</td><td>D4001</td><td>or comparable</td></tr><tr><td>ABI big dye 3.1 chemistry</td><td>Applied Biosystems</td><td></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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

19.2K 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

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, ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

Environment

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

Medicine

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

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

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