This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.

All swine used in the collection of the following data were protected under The Ohio State University Institutional Animal Care and Use Committee (animal use protocol number 2009A0134-R1).
1. Preparation of Viral Transport Medium and Sample Collection Vials
<ol>
	<li>Add 37 g of Brain Heart Infusion to 900 ml of purified water and mix thoroughly with a stir bar and magnetic stirrer while heating to 70 &#176;C to completely dissolve the powder.</li>
	<li>Autoclave the broth at 121 &#176;C for 15 min. Cool on the bench top until the broth reaches room temperature. Refrigerate at 4 &#176;C overnight if needed.</li>
	<li>Dissolve, by mixing with a magnetic stir bar, 6.02 g of Penicillin G sodium salt and 10 g of streptomycin sulfate into 50 ml of purified water at room temperature. Add additional purified water to bring the total volume to 100 ml. Filter the solution through a 0.22 &#181;m polyethersulfone membrane into a sterile bottle.</li>
	<li>Aseptically add the filtered penicillin and streptomycin solution into the cooled Brain Heart Infusion Broth and mix thoroughly (stir bar or swirl flask by hand) to make the viral transport medium.</li>
	<li>Aseptically test the pH of the viral transport medium; if needed, adjust the pH to 7.4 &#177; 0.2 using HCl or NaOH.</li>
	<li>Perform quality assurance testing on the viral transport medium by testing for influenza A virus matrix protein by rRT-PCR, as described by Zhang and Harmon.22</li>
	<li>Dispense 5 ml of the viral transport medium into sterile high-density polyethylene bottles with 8 ml capacity. Label the vials for use in the field.</li>
	<li>Store the ready-to-use vials in standard cryo-boxes at -20 &#176;C until sample collection.</li>
</ol>
2. Collection of Nasal Wipes from Swine
<ol>
	<li>Thaw the appropriate number of vials immediately prior to sample collection; one vial will be needed per pig sampled. Thawing at room temperature takes approximately 30-45 min. Keep the thawed vials cool on ice packs until use.</li>
	<li>Don appropriate personal protective equipment as dictated by the situation (e.g. coveralls, boot covers, respirator, ear plugs, etc.).</li>
	<li>Enter the animal area. If needed, confine the pig to a small area but do no restrain. Do not rouse resting animals.</li>
	<li>Put on a pair of disposable exam gloves. Take care not to contaminate the gloves by touching pigs, people, or inanimate objects.</li>
	<li>Use a gloved hand to remove a sterile 5.08 cm &#215; 5.08 cm (2 in. &#215; 2 in.) cotton gauze pad from its wrapper. Sterile gauze pads should be used whenever possible. Individually wrapped gauze pads are more convenient to use than those wrapped with two gauze pads per package.</li>
	<li>Hold the cotton gauze pad with the fingertips of one hand exposing as much of the pad as possible for sampling.</li>
	<li>Wipe the gauze pad across the pig&#8217;s snout, taking extra care to enter the external nares if possible. Collect visible nasal secretions (approximately 1 ml) with the same gauze pad.</li>
	<li>Using the same hand, fold the gauze pad upon itself to facilitate placement in the vial containing viral transport medium.</li>
	<li>Using the other hand, remove the cap from the vial and place the folded gauze pad into the vial. Recap the vial and shake the vial to ensure the mixing of the gauze pad and viral transport medium.</li>
	<li>Remove the gloves and place in an appropriate receptacle. Change gloves between each pig.</li>
	<li>Verify the vial identifier. Record pig identification number, age, sex, clinical signs, and other notes deemed necessary by investigator.</li>
	<li>Chill the samples immediately after collection. Consider making aliquots of the sample before freezing to prevent multiple freeze-thaw cycles. As soon as possible after collection, place the samples on dry ice for transportation to the laboratory. Store the vials at -80 &#176;C until testing is initiated.</li>
</ol>
3. Detection of Influenza A Virus Nucleic Acid&#160;
<ol>
	<li>Thaw the samples in a 37 &#176;C dry bead bath for 5 min and then finish thawing the samples on the bench top at room temperature for 20-30 min. The simultaneous addition of several vials to the beads will cause the bath to heat; caution should be exercised as to not overheat the vials.</li>
	<li>Remove 100 &#181;l of viral transport medium for RNA extraction.
	<ol>
		<li>Use a high-throughput (96 well plate format) magnetic bead&#160;platform for extracting RNA from a large number of samples.31</li>
	</ol>
	</li>
	<li>Use real-time reverse transcriptase PCR assays for the rapid detection influenza A virus.31</li>
</ol>
4. Isolation of Influenza A Virus from Nasal Wipes
<ol>
	<li>Thaw the samples as described above in Section 3.1</li>
	<li>Treat thawed samples with gentamicin sulfate (1,000 &#956;g/ml), amphotericin B (22.5 &#956;g/ml), and kanamycin sulfate (325 &#956;g/ml).</li>
	<li>Inoculate into Madin-Darby Canine Kidney (MDCK) epithelial cells as described previously.32</li>
</ol>

<ol>
	<li>Ma, W., Kahn, R. E., Richt, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pig+as+a+mixing+vessel+for+influenza+viruses:+Human+and+veterinary+implications.">The pig as a mixing vessel for influenza viruses: Human and veterinary implications.</a> J. Mol. Genet. Med. 3 (1), 158-166 (2008).</li><li>Nelson, M. I., Vincent, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+zoonosis+of+influenza+to+swine:+new+perspectives+on+the+human-animal+interface.">Reverse zoonosis of influenza to swine: new perspectives on the human-animal interface.</a> Trends Microbiol. 23, (2015).</li><li>Zhou, N. 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M., Spackman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+23,+RNA+extraction+from+swine+samples+and+detection+of+influenza+A+virus+in+swine+by+real-time+RT-PCR.">Ch. 23, RNA extraction from swine samples and detection of influenza A virus in swine by real-time RT-PCR.</a> Animal Influenza Virus. , 277-293 (2014).</li><li>Zhang, J., Gauger, P. C., Spackman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+22,+Isolation+of+swine+influenza+virus+in+cell+cultures+and+embryonated+chicken+eggs.">Ch. 22, Isolation of swine influenza virus in cell cultures and embryonated chicken eggs.</a> Animal Influenza Virus. , 265-276 (2014).</li><li>Bowman, A. 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The authors have nothing to disclose.

Collecting samples from pigs using polyester-tipped nasal swabs has proven useful in conducting IAV surveillance; however, the use of the nasal swab procedure hinders surveillance efforts due to the required use of a snare for restraint. Nasal wipes represent a refinement of current swine sampling techniques to minimize stress on people and pigs during sample collection. While the method has been developed for and validated in exhibition swine settings, it can be easily applied to other situations where ante-mortem detec...

Influenza A viruses (IAV) cause respiratory disease in many species, including domestic birds, swine, and humans. Due to reassortment of the segmented IAV genome rapid viral evolution can occur and new IAV strains frequently emerge. Swine are a species that can serve as a mixing vessel for reassortment of IAVs from multiple host species.1 There are currently three major subtypes of IAV commonly circulating among North American swine (H1N1, H1N2, H3N2), but multiple IAV introductions from humans have led to extensive IAV diversity within those subtypes.2 Rapid evolution of IAVs infecting swine has been evident since 1998 when a triple reassortant IAV containing gene segments from human, avian and swine viruses3 became widespread among pigs in the United States.4 The internal gene segments from that triple reassortant IAV remain highly prevalent among IAVs currently infecting swine.5
Worldwide, IAV is a significant cause of respiratory disease in swine in which typical clinical signs include fever, anorexia, lethargy, coughing, labored breathing, sneezing, nasal discharge and poor weight gain. IAV can be particularly costly to sow farms where reproductive failure due to IAV induced fever and weak-born piglets have been documented.6,7 Within the United States, IAV is commonly detected in commercial swine herds and the extensive antigenic and genomic diversity and continuing evolution among IAV infecting swine has hampered control of this virus.8-11
Public health concerns about the emergence of a pandemic IAV strain resulting from reassortment in swine were realized in 2009 when a swine-lineage IAV containing gene segments from the triple-reassortant North American swine lineage and the Eurasian avian-like swine lineage caused a worldwide pandemic in humans.12 The pandemic virus (A (H1N1)pdm09) has since reassorted with endemic swine IAV strains13,14 and some of these newly reassorted strains have been transmitted back to humans.15 The frequency of reassortment events and emergence of new IAV strains with pandemic potential makes active surveillance of circulating IAV viruses in swine imperative, especially at the swine-human interface.
The swine-human interface is important for bi-directional interspecies transmission of IAV. Human-to-swine transmission occurring in commercial swine production is responsible for a large amount of IAV diversity currently present in the swine population. Agricultural fairs are the largest settings for the comingling of people and swine in the United States and are known sites for the zoonotic transmission of IAV.15-21 In 2012, during the outbreak of a variant H3N2 IAV, 93% of cases reported attendance at an agricultural fair in the days prior to onset of disease.15 Genomic analysis of viral isolates from exhibition pigs compared with human isolates confirmed zoonotic transmission.21 Exhibition swine infected with IAV often do not show clinical signs of illness,21-23 indicating the need for direct diagnostic tests.
Sampling of visibly ill pigs alone will not successfully identify IAV prevalence in swine and cannot be relied on to identify new strains of IAV emerging among swine. Active surveillance is absolutely necessary for detection of emerging strains of IAV in swine and to assess their threat for both swine and public health. Most IAV surveillance activities are voluntary and therefore minimally disruptive methods are needed. The three major ante-mortem sample collection procedures for IAV infecting swine are: nasal swabs, oral fluids, and nasal wipes. Current recommendations for sampling individual swine to detect IAV list the insertion of synthetic-fiber tipped swabs into the nostrils as the preferred method to gather nasal secretions and epithelial cells.24,25 Because pigs may try to avoid this procedure, a team of trained personnel must restrain pigs either manually or with a snare depending on the size of the animal.26 The restraint process is laborious for the personnel, and stressful for the pigs. Additionally, exhibition swine are often involved in multiple competitions at a fair so the perception of added stress on a competition animal can make owners resistant to the surveillance efforts.
With probabilities of IAV detection ranging from 80-100% in IAV infected herds, oral fluids have become a popular alternative to nasal swabs for the molecular detection of IAV in populations of swine.27,28 Additionally, oral fluids may provide a wider window of IAV detection than nasal swabs following initial infection. However, IAV isolation from oral fluids has been problematic with only 50% of virus isolation attempts resulting in IAV recovery.29
Using nasal wipes in lieu of nasal swabs during IAV surveillance in swine overcomes the limitations described above. Nasal wipes do not require the use of a restraining snare and can be performed without stressing the animals or witnesses of the procedure. Minimal technical training is needed to collect nasal wipes, which allows non-veterinary professionals, including swine owners, to collect the surveillance samples. Nasal wipes were previously compared to nasal swabs for the detection and isolation of influenza A virus30 and the detailed protocol for this non-invasive method of sampling is described below.

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			<td height="21" style="height:21px;width:216px;">BBL Brain Heart Infusion</td>
			<td style="width:360px;">Becton, Dickinson and Company</td>
			<td style="width:119px;">211059</td>
			<td style="width:356px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin G Sodium Salt</td>
			<td style="width:360px;">MP Biomedicals, LLC</td>
			<td style="width:119px;">021194537</td>
			<td>1500 u/mg</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin Sulfate</td>
			<td style="width:360px;">AMRESCO LLC</td>
			<td style="width:119px;">0382</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Gentamicin Solution</td>
			<td style="width:360px;">Mediatech, Inc.</td>
			<td style="width:119px;">30-005-CR</td>
			<td>50 mg/ml</td>
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			<td height="21" style="height:21px;width:216px;">Amphotericin B Solution</td>
			<td style="width:360px;">Fisher</td>
			<td style="width:119px;">S 24 25</td>
			<td>250 ug/ml</td>
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			<td height="21" style="height:21px;width:216px;">Kanamycin Sulfate</td>
			<td style="width:360px;">Teknova</td>
			<td style="width:119px;">K2105</td>
			<td>5000 ug/ml</td>
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			<td height="21" style="height:21px;width:216px;">TPP Rapid Filtermax System</td>
			<td style="width:360px;">TPP Techno Plastic Products AG&#160;</td>
			<td style="width:119px;">99150</td>
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			<td height="21" style="height:21px;width:216px;">Nalgen Diagnostic Bottles</td>
			<td style="width:360px;">Thermo Scientific</td>
			<td style="width:119px;">342002-9025</td>
			<td>HDPE with white PP closure</td>
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			<td height="26" style="height:26px;width:216px;">Dermacea Gauze Sponge, 8 ply</td>
			<td style="width:360px;">Covidien</td>
			<td style="width:119px;">441211</td>
			<td>5.08 cm &#215; 5.08 cm (2 in. &#215; 2 in)&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Nitrile Exam Gloves</td>
			<td style="width:360px;">Saftey Choice</td>
			<td style="width:119px;">19-170-010 (A-D)</td>
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</table>

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.

Successful use of this method yields rRT-PCR results that, accompanied with the use of an internal control during RNA extraction and rRT-PCR, show samples did not contain PCR inhibitors from any environmental debris picked up during sampling. After sample inoculation, virus isolation wells should be free of visible environmental debris from the sample. There is reasonable agreement between rRT-PCR results and virus isolation results with the understanding that PCR often yields a higher IAV positive rate than virus isolat...

Watch this Scientific Journal Video about Nasal Wipes for Influenza A Virus Detection and Isolation from Swine at JoVE.com

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

nasal-wipes-for-influenza-a-virus-detection-and-isolation-from-swine

Research

JoVE Journal

Immunology and Infection

9.3K Views.  The Ohio State University. The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. 

Video: Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

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, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

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 ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

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 ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...