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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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.

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

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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 ...

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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

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

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

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 ...

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 ...

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 ...

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 ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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