This work was supported by the research grants from the Innovation Capacity Building Project of Jiangsu province (BM2020019), Shenzhen Scientific and Technological Project (No. JSGG20200225150702770), Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29030103), Guangdong Scientific and Technological Project (No. 2020B1111340076) and the Shenzhen Bay Laboratory Open Research Program (No. SZBL202002271003).

All pseudovirus-related experimental operations were performed under ABSL2 condition in the Institut Pasteur of Shanghai Chinese Academy of Sciences (IPS, CAS). Animal experiments were performed based on Institutional Animal Care and Use Committee-approved animal protocols of IPS, CAS.
1. Pseudovirus packaging with Calcium-phosphate transfection
<ol>
	<li>Make single-cell suspensions of HEK293FT cells (9 x 105&#160;per mL) in complete DMEM medium (Table 1). Add 10 mL of the single-cell suspensions to a T75 flask, incubate the cells in a 37 &#176;C, 5% carbon dioxide (CO2) incubator for 20 h before the transfection. 
	NOTE: HEK293FT low passage (&#60;20 passages) is recommended. Ensure that the cell monolayer is 80-90% confluent.</li>
	<li>Replace the medium with 10 mL of fresh complete medium containing chloroquine (100 &#181;M) 2 h before transfection.</li>
	<li>Mix the reagents and plasmid DNA as shown in Table 2 and Figure 1: ddH2O, pCMV/R-HA, pCMV/R-NA, pHR-Luc, pCMV &#916; 8.9, 2.5 M CaCl2, and 2x HEPES buffer. Pipette up and down 5 times gently, incubate the mixture at room temperature (RT) for 20 min.</li>
	<li>Transfer the mixture into the medium above the cells, and rock the flask gently. Incubate the cells in the cell incubator for 15 h.</li>
	<li>Replace the medium with 15 mL of fresh complete DMEM medium. Incubate the cells in a 37 &#176;C, 5% CO2 incubator for 65 h. 
	NOTE: The color of the medium will be light orange or slightly yellow, and at least 80% of the cell should show the cytopathic effect (CPE) under the inverted light microscope.</li>
	<li>Harvest the supernatant, and centrifuge it at 2095 x g for 20 min at 4 &#176;C. Collect the supernatant, aliquot it, and store it at -80 &#176;C.</li>
</ol>
2. Detection of the HA, NA and HIV-1 p24 protein expression of influenza pseudovirus
<ol>
	<li>Detect the HA protein expression by Hemagglutinin (HA) assay.
	<ol>
		<li>Add 50 &#181;L of PBS into columns 2-12, rows A, B, and C of a 96-well round-bottom plate. 
		NOTE: Row A, B, and C belong to 3 independent replication tests, and column 12 is used as the negative control.</li>
		<li>Add 100 &#181;L of pseudovirus into the wells of column 1. Transfer 50 &#181;L from column 1 into Column 2, mix well by pipetting up and down 5 times, perform the two-fold dilutions until the last column 11, and discard the extra 50 &#181;L from column 11.</li>
		<li>Add 50 &#181;L of 0.5% erythrocyte into each well, pipette it up and down gently twice. Incubate the plate for 30-60 min at RT or until the erythrocytes of the negative control form the regular spots at the bottom of the well.</li>
		<li>Observe and record HA titers of the pseudovirus. 
		NOTE: HA unit of the pseudoviruses is the highest dilution factor of the virus that can cause 100% hemagglutination of the red blood cells.</li>
	</ol>
	</li>
	<li>Detect HA and NA protein expression by the western-blot assay. 
	NOTE: All the western-blot steps are performed according to the manual of Molecular cloning (4th edition).</li>
	<li>Detect the HIV-1 p24 protein expression by the HIV-1 p24 ELISA kit (Table of Materials).
	<ol>
		<li>Add 50 &#181;L of the lysis buffer into 450 &#181;L of pseudovirus sample. Mix it thoroughly.</li>
		<li>Add 300 &#181;L of the wash buffer to each well of the microplate. Strike the plate inverted to remove the wash buffer completely. Repeat the wash 5 times.</li>
		<li>Add 200 &#181;L of the standard and samples to each well separately. Incubate them at 37 &#176;C overnight or for 2 h.</li>
		<li>Aspirate the contents of the plate and wash the plate as described in step 2.3.2. 
		NOTE: Leave one well of the assay plate empty to use as a substrate blank.</li>
		<li>Add 200 &#181;L of the p24 detector antibodies to each well. Incubate them at 37 &#176;C for 1 h. Aspirate and wash the plate as described in step 2.3.2.</li>
		<li>Add 100 &#181;L of the streptavidin-Peroxidase working solution to each well, and incubate them at 37 &#176;C for 30 min. Aspirate and wash the plate as described in step 2.3.2.</li>
		<li>Add 100 &#181;L of the substate working solution to each well, including the substrate blank well, and incubate them at 37 &#176;C for 30 min. 
		NOTE: Blue color will develop in the wells.</li>
		<li>Add 100 &#181;L of the stop buffer to each well. 
		NOTE: The blue color will change to yellow immediately.</li>
		<li>Read the optical density at 450 nm within 15 min. Record the p24 titers of the pseudovirus</li>
	</ol>
	</li>
</ol>
3. Pseudovirus titration
<ol>
	<li>Make single-cell suspensions of MDCK cells (5 x 104 per mL) in complete DMEM medium, add 1250, 2500, 5000, 10000, 20000, and 40000 cells to different wells of a 96-well flat-bottom plate. Incubate the cells in a 37 &#176;C, 5% CO2 incubator for 20 h.</li>
	<li>Take out the pseudovirus from the -80 &#176;C refrigerator, thaw it on the ice, and vortex the pseudovirus.</li>
	<li>Add 6 HAU (6x HA units) pseudoviruses to each well of 96-well round-bottom plate and replenish each well with complete DMEM up to 120 &#181;L.</li>
	<li>Pipette up and down the mixture 5 times to mix thoroughly. Incubate the mixture plate in a 37 &#176;C, 5 % CO2 incubator for 1 hour.</li>
	<li>Transfer 100 &#181;L of the mixture to the cells in the 96-well plate. Put the cell culture plate back into the incubator for 48 h, 60 h, and 72 h after virus infection.</li>
	<li>Take the plate out of the incubator and perform the luciferase assay (Table of Materials).</li>
	<li>Remove the medium from the wells of the plate carefully. Rinse the cells with 200 &#181;L of PBS and remove the PBS as much as possible. 
	NOTE: Flip the plate and discard the supernatant by reverse slapping it on absorbent paper. If the testing cell line could not firmly attach to the bottom, remove the medium aspiration carefully.</li>
	<li>Add 50 &#181;L of the lysis buffer to each well, and rock the culture plate several times. Store the plate at -80 &#176;C overnight. 
	NOTE: Mix 1 volume of 5x lysis buffer with 4 volumes of water to make the 1x lysis buffer. Equilibrate the 1x lysis buffer to RT before use. Rock the plate to ensure that the cells are covered with the lysis buffer completely. The single freeze-thawprocess can help the cell be lysed completely.</li>
	<li>Take the plate out, equilibrate at RT for 2 h. 
	NOTE: The cell should be lysed completely.</li>
	<li>Rock the plate several times gently, and transfer all the cell lysates to opaque 96-well plates.</li>
	<li>Add 50 &#181;L of the substrate to each well, rock the plate several times gently, and mix substrate and lysis buffer thoroughly.</li>
	<li>Measure the light produced within 5 min using a luminometer. Record the luciferase titers of the pseudovirus. 
	&#8203;NOTE: When a proper substrate is added, light is emitted as a by-product via a chemical reaction in which luciferin is converted to oxyluciferin by the luciferase enzyme. The amount of light produced is proportional to the amount of the luciferase enzyme.</li>
</ol>
4. Immune sera preparation
NOTE: The immune serum will be used for cell-related experiments, and the experimental operation should be carried out under aseptic conditions.
<ol>
	<li>Prepare 12 eight-week-old female BALB/c mice. Divide equally the mice into two groups. 
	NOTE: One group was DDV group and the other was the negative control group.</li>
	<li>DDV group immunization: Prime twice intramuscularly with 100 &#181;g of a codon-optimized DNA plasmid encoding A/Thailand/(KAN-1)/2004 (TH) HA protein at week 0 and week 3 and boost once intraperitoneally with 512 HAU TH virus-like particles (VLP) at week 6.</li>
	<li>Control group immunization: Prime twice intramuscularly with 100 &#181;g empty vector plasmid DNA at week 0 and week 3 and boost once intraperitoneally with HIV-1 gag VLP at week 6.</li>
	<li>Collect the mouse blood 2 weeks after the last immunization. 
	NOTE: Here, the mice were anesthetized with pentobarbital sodium (65 mg/kg) at the time of blood collection. After blood collection from the submandibular vein, the mice were euthanized immediately with CO2.</li>
	<li>Keep the blood at RT for 2 h, then 4 &#176;C overnight. Centrifuge at 900 x g for 10 min at 4 &#176;C and collect the supernatant. Inactivate the sera by placing it at 56 &#176;C for 30 min.</li>
	<li>Store the immune sera in a -80 &#176;C refrigerator for use. 
	NOTE: Refer to Supplementary Figure 1 for the flowchart that describes the major procedures of mouse immunization.</li>
</ol>
5. Pseudovirus neutralization (PN) assay
<ol>
	<li>Make single-cell suspensions of MDCK cells (5 x 104 per mL) in complete DMEM medium, and add 100 &#181;L of the cell suspension to each well of a 96-well flat-bottom plate. Incubate the cells in a 37 &#176;C, 5% CO2 incubator for 20 h.</li>
	<li>Take out the pseudovirus and sera from the -80 &#176;C refrigerator, thaw it on the ice, and vortex thoroughly.</li>
	<li>Dilute the sera in the complete medium 20 times, and add 100 &#181;L of complete DMEM medium to the wells of a 96-well round-bottom plate from columns 2-10.</li>
	<li>Add 200 &#181;L of the diluted sera into the wells of column 2, transfer 100 &#181;L from column 2 to column 3, dilute the sera as 1:20, 1:40, 1:80, etc., to 1:10240 successively from column 2 to column 10, and discard the 100 &#181;L from column 10.</li>
	<li>Add 100 &#181;L of DMEM complete medium to the wells of column 11 as the negative control.</li>
	<li>Add 100 &#181;L of pseudoviruses to each well, pipette up and down the mixture 5 times thoroughly, and incubate the mixture plate in a 37 &#176;C, 5% CO2 for 1 h.</li>
	<li>Transfer 100 &#181;L of the mixture from the 96-well round-bottom plate to the corresponding well of the 96-well cell culture plate. Put the plate back in the incubator (37 &#176;C, 5% CO2) for 60 h.</li>
	<li>Take the plate out of the incubator. Perform the luciferase assay as described in steps 3.7-3.12.</li>
</ol>
6. Pseudovirus attachment assay
<ol>
	<li>Make single-cell suspensions of MDCK cells (4 x 104 per mL) in complete DMEM medium, and add 100 &#181;L cell suspensions to each well in a 96-well flat-bottom plate. Incubate the cells at 37 &#176;C, 5% CO2 for 20 h.</li>
	<li>Incubate the pseudovirus with sera in DMEM containing 1% BSA at 4 &#176;C overnight. 
	NOTE: The volume of the mixture is 100 &#181;L.</li>
	<li>Block the MDCK cells with 100 &#181;L per well of DMEM containing 1% BSA at 4 &#176;C for 1 h.</li>
	<li>Inoculate the pseudovirus and serum mixture (100 &#181;L) onto the MDCK monolayer and incubate it at 4 &#176;C for 2 h.</li>
	<li>Wash the cell plate 4 times with PBS containing 1% BSA to remove any unbound virus.</li>
	<li>Lyse the cells with 100 &#181;L of luciferase lysis buffer at RT for 30 min.</li>
	<li>Quantify the amount of bound virus on the cells with an HIV-1 p24 ELISA kit as described above (section 2.3).</li>
</ol>
7. Assessment of viral entry
<ol>
	<li>Make single-cell suspensions of MDCK cells (5 x 104 per mL) in complete DMEM medium, and add 100 &#181;L of the cell suspension to each well of a 96-well flat-bottom plate. Incubate the cells at 37 &#176;C, 5% CO2 for 20 h before the assay.</li>
	<li>Inoculate the pseudovirus (100 &#181;L) onto an MDCK monolayer in a 96-well plate at 4 &#176;C for 6 h.</li>
	<li>Wash the cell plate 4 times with PBS containing 1% BSA to remove unbound pseudoviruses.</li>
	<li>Add 100 &#181;L of the serially diluted immune sera or sera from sham vaccination mice in DMEM containing 1% BSA to the monolayer, and incubate cell at 4 &#176;C for 2 h.</li>
	<li>Remove the cell supernatant, and wash the cell culture plate 4 times with PBS containing 1% BSA.</li>
	<li>Add fresh DMEM to the cells and incubate for 60 h in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Measure the relative luciferase activity (RLA) of the cells as described in steps 3.7-3.12. 
	NOTE: Viral entry, as indicated by RLA, was expressed as a percentage of the reading obtained in the absence of sera, which was set as 100%.</li>
</ol>

<ol>
	<li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+prime+and+virus-like+particle+boost+from+a+single+H5N1+strain+elicits+broadly+neutralizing+antibody+responses+against+head+region+of+h5+hemagglutinin.">DNA prime and virus-like particle boost from a single H5N1 strain elicits broadly neutralizing antibody responses against head region of h5 hemagglutinin.</a> The Journal of Infectious Diseases. 209 (5), 676-685 (2014).</li><li>Wang, G., Yin, R., Zhou, P., Ding, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+the+immunization+with+the+sequence+close+to+the+consensus+sequence+and+two+DNA+prime+plus+one+VLP+boost+generate+H5+hemagglutinin+specific+broad+neutralizing+antibodies.">Combination of the immunization with the sequence close to the consensus sequence and two DNA prime plus one VLP boost generate H5 hemagglutinin specific broad neutralizing antibodies.</a> Plos One. 12 (5), 0176854 (2017).</li><li>Li, Q., Liu, Q., Huang, W., Li, X., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+on+the+development+of+pseudoviruses+for+enveloped+viruses.">Current status on the development of pseudoviruses for enveloped viruses.</a> Reviews in Medical Virology. 28 (1), 1963 (2018).</li><li>Duverg&#233;, A., Negroni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudotyping+lentiviral+vectors:+When+the+clothes+make+the+virus.">Pseudotyping lentiviral vectors: When the clothes make the virus.</a> Viruses. 12 (11), 1311 (2020).</li><li>Carnell, G. W., Ferrara, F., Grehan, K., Thompson, C. P., Temperton, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudotype-based+neutralization+assays+for+influenza:+A+systematic+analysis.">Pseudotype-based neutralization assays for influenza: A systematic analysis.</a> Frontiers in Immunology. 6 (161), (2015).</li><li>Trombetta, C., Perini, D., Mather, S., Temperton, N., Montomoli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+serological+techniques+for+influenza+vaccine+evaluation:+Past,+present+and+future.">Overview of serological techniques for influenza vaccine evaluation: Past, present and future.</a> Vaccines. 2 (4), 707-734 (2014).</li><li>Wei, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+influenza+vaccines:+opportunities+and+challenges.">Next-generation influenza vaccines: opportunities and challenges.</a> Nature Reviews Drug Discovery. 19 (4), 239-252 (2020).</li><li>Krammer, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza.">Influenza.</a> Nature Reviews Disease Primers. 4 (3), (2018).</li><li>Wei, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+broadly+neutralizing+H1N1+influenza+antibodies+by+vaccination.">Induction of broadly neutralizing H1N1 influenza antibodies by vaccination.</a> Science. 27 (329), 1060-1064 (2010).</li><li>Chernov, V. M., Chernova, O. A., Sanchez-Vega, J. T., Kolpakov, A. I., Ilinskaya, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+contamination+of+cell+cultures+vesicular+traffic+in+bacteria+and+control+over+infectious+agents.">Mycoplasma contamination of cell cultures vesicular traffic in bacteria and control over infectious agents.</a> Acta Naturae. 6 (3), 41-51 (2014).</li><li>Michael, R. G., Joseph, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual (Fourth Edition). , (2012).</li><li>Kumar, P., Nagarajan, A., Uchil, P. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+DNA+into+eukaryotic+cells.">Transfection of DNA into eukaryotic cells.</a> Current Protocols in Molecular Biology. 9, 11-19 (1996).</li><li>Kachkin, D. V., Khorolskaya, J. I., Ivanova, J. S., Rubel, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+isolation+of+plasmid+DNA+for+transfection+of+mammalian+cell+cultures.">An efficient method for isolation of plasmid DNA for transfection of mammalian cell cultures.</a> Methods and Protocols. 3 (4), 69 (2020).</li><li>. Luciferase assay system Available from: <a target="_blank" href="https://www.promega.com.cn/products/luciferase-assays/reporter-assays/luciferase-assay-system">https://www.promega.com.cn/products/luciferase-assays/reporter-assays/luciferase-assay-system</a> (2021)</li><li>. Corning 96-Well Solid Black or White Polystyrene Microplates Available from: <a target="_blank" href="https://www.fishersci.com/shop/products/costar-96-well-black-white-solid-plates">https://www.fishersci.com/shop/products/costar-96-well-black-white-solid-plates</a> (2021)</li></ol>

The authors have nothing to disclose.

HEK293FT cells are usually used as packaging cells to produce pseudoviruses. Regular mycoplasma detection is essential during the cell culture. Mycoplasma contamination can drastically decrease the pseudovirus yields and sometimes close to zero. Compared with other contaminations, mycoplasma contaminations do not lead to pH value changes or turbidity of the cell culture medium.Even a high concentration of mycoplasma is not visible with naked eyes or under a microscope. Three popular methods of mycoplasma detection are my...

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing continual flu outbreaks in poultry and wild birds, accounting for enormous socio-economical losses in the global poultry industry. Sometimes, humans also get infected with it, faced with a high fatality rate1,2. However, HPAI virus research is often hindered, given that it cannot be handled outside of biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses are safe enough to practice in biosafety level 2 laboratories.
H5 HPAI pseudoviruses belong to chimeric viruses consisting of surrogate virus cores, lipid envelopes with the surface glycoproteins from influenza viruses, and reporter genes. Pseudovirus cores are usually derived from lentiviral human immunodeficiency virus (HIV), retroviruses such as murine leukemia virus (MLV), and vesicular stomatitis virus (VSV)3. Specifically, the HIV-1 packaging system is widely used to produce influenza pseudovirus, where the primary genes provided are gag and pol. The HIV gag gene expresses core proteins. The pol gene expresses the integrase and reverse transcriptase, both of which are necessary for the expression of the reporter gene in transduced cells. Mimicking the genome of the surrogate virus, the reporter gene is embraced into the pseudovirus core in RNA form. The reporter gene will express the protein in host cells. The gene expression levels of reporter genes can be used to measure pseudovirus infection efficiency3,4. The primary reporter is firefly luciferase to measure the relative luminescence units (RLU) or relative luciferase activity (RLA) in transduced cells. Other reporters such as lacZ, Gaussia, and Renilla luciferase are also used, only to a lesser extent5.
Pseudoviruses are ideal tools to study neutralizing antibodies against H5 HAPI viruses. To measure the neutralizing antibody potency, pseudovirus neutralization (PN) assays6 are used.Hemagglutinin (HA) and neuraminidase (NA) are glycoproteins on the surface of the influenza A virus7,8. The HA is composed of a globular head domain for receptor binding and a stem domain for membrane fusion. The NA protein has the sialidase activity to facilitate virus release7,8. A PN assay can measure neutralizing antibodies directed to HA proteins. Neutralizing antibodies directed to the head and stem region of HA can also be detected by viral attachment and entry assays. Compared with wild-type viruses, pseudovirus neutralization experiments have more sensitive detection values, can be safely handled in a Level 2 biosafety laboratory, and are generally easier to operate in practice.
This protocol presents in detail the procedures and critical steps of H5 HPAI pseudovirus preparations and PN assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays. In this study, the A/Thailand/1(KAN)-1/2004(TH) strain from H5N1 HPAI viruses was used as an example. To obtain the immune sera used in assays, this protocol selected the HA protein originating from the TH strain as the immunogen to immunize mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% Chiken Erythrocyte</td>
			<td>Bio-channel</td>
			<td>BC-RBC-C001</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>96-well cell culture plates (flat-bottom)</td>
			<td>Thermo fisher scientific</td>
			<td>167008</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>96-well cell culture plates (round-bottom)</td>
			<td>Thermo fisher scientific</td>
			<td>163320</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Allegra X-15R</td>
			<td>Beckman coulter</td>
			<td>--</td>
			<td>Equipment/Centrifuge</td>
		</tr>
		<tr>
			<td>BD Insulin Syringes</td>
			<td>BD</td>
			<td>324910</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Calcium Chloride Anhydrous</td>
			<td>AMRESCO</td>
			<td>1B1110-500G</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>chloroquine diphosphate</td>
			<td>Selleck</td>
			<td>S4157</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>12100-046</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>HEK293FT</td>
			<td>Gibco</td>
			<td>R700-07</td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>HEPES FREE ACID</td>
			<td>AMRESCO</td>
			<td>0511-250G</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>HIV-1 p24 Antigen ELISA</td>
			<td>ZeptoMetrix</td>
			<td>801111</td>
			<td>Reagent kit</td>
		</tr>
		<tr>
			<td>Luciferase Assay System Freezer Pack</td>
			<td>Promega</td>
			<td>E4530</td>
			<td>Reagent kit</td>
		</tr>
		<tr>
			<td>MDCK.1</td>
			<td>ATCC</td>
			<td>CRL-2935</td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes 1.5 mL</td>
			<td>Thermo fisher scientific</td>
			<td>509-GRD-Q</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Nunc Conical Centrifuge Tubes 15 mL</td>
			<td>Thermo fisher scientific</td>
			<td>339650</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Nunc Conical Centrifuge Tubes 50 mL</td>
			<td>Thermo fisher scientific</td>
			<td>339652</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Nunc EasYFlask 75 cm2</td>
			<td>Thermo fisher scientific</td>
			<td>156499</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Pipette Tips (10 &#956;L)</td>
			<td>Thermo fisher scientific</td>
			<td>TF102-10-Q</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Pipette Tips (100 &#956;L)</td>
			<td>Thermo fisher scientific</td>
			<td>TF113-100-Q</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Pipette Tips (1000 &#956;L)</td>
			<td>Thermo fisher scientific</td>
			<td>TF112-1000-Q</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Serological pipets (5 mL)</td>
			<td>Thermo fisher scientific</td>
			<td>170355N</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Serological pipets (10 mL)</td>
			<td>Thermo fisher scientific</td>
			<td>170356N</td>
			<td>consumable material</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Varioskan Flash</td>
			<td>Thermo fisher scientific</td>
			<td>--</td>
			<td>Equipment/Microplate reader</td>
		</tr>
		<tr>
			<td>Water Jacket Incubator</td>
			<td>Thermo fisher scientific</td>
			<td>3111</td>
			<td>Equipment/Cell incubator</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium salt</td>
			<td>Sigma</td>
			<td>57-33-0</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

preparation of pseudo-typed h5 avian influenza viruses with calcium phosphate transfection method and measurement of antibody neutralizing activity

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. Occasionally, humans also fall victim to it, which results in high mortality. Nonetheless, HPAI virus research is often hindered, considering that it must be handled within biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses prove to be the ideal tools to study neutralizing antibodies against H5 HPAI viruses. This protocol describes the procedures and critical steps of H5 HPAI pseudovirus preparations and pseudovirus neutralization assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays.

HA, NA and HIV-1 p24 protein expression of influenza pseudovirus 
To identify the viral packaging efficiency, influenza pseudovirus stocks were first detected by HA assay (Figure 2A). HA units per milliliter of influenza pseudoviruses are 643 (Table 3). Western-blot assay and sandwich ELISA assays were used to test HA, NA, and HIV-1 p24 protein expression. Then the ratios of HA unit and the amount of gag p24 for pseudoviruses were calculated. The result...

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Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity

Here we describe a protocol for pseudovirus packaging and the measurement of antibody neutralizing activity.

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. ...

preparation-pseudo-typed-h5-avian-influenza-viruses-with-calcium

Research

JoVE Journal

Immunology and Infection

2.1K Views.  Nanjing Advanced Academy of Life and Health. Dit protocol beschrijft de procedures en de kritieke stappen van H5 hoogpathogene aviaire influenza pseudo-viruspreparaten en pseudo-virusneutralisatietests. Ook bespreekt het de probleemoplossing, beperking en wijzigingen van deze testen. Hoogpathogene pathogenen moeten worden uitgevoerd in het bioveiligheidslaboratorium van niveau drie, terwijl pseudovirussen veilig kunnen worden behandeld in een bioveiligheidslaboratorium van niveau twee.Maak om te beginnen een enkele celsuspensie v...

Video: Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity

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 ...

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.

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 ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

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, ...

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

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 ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

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 ...

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 ...

Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity

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