Name: Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity (Video) | JoVE Journal
Uploaded: 2021-11-22T13:30:00.000+00:00
Duration: 7 min 15 s
Description: Learn to prepare H5 avian influenza pseudoviruses using calcium phosphate transfection in HEK293 FT cells and measure antibody neutralizing activity. This protocol enables safe vaccine research in BSL-2 laboratories.

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5 minutes

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/pubmed?term=((DNA+prime+and+virus-like+particle+boost+from+a+single+H5N1+strain+elicits+broadly+neutralizing+antibody+responses+against+head+region+of+h5+hemagglutinin%5BTitle%5D)+AND+%22The+Journal+of+Infectious+Diseases%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Plos+One%22%5BJournal%5D)">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/pubmed?term=((Current+status+on+the+development+of+pseudoviruses+for+enveloped+viruses%5BTitle%5D)+AND+%22Reviews+in+Medical+Virology%22%5BJournal%5D)">Current status on the development of pseudoviruses for enveloped viruses.</a> Reviews in Medical Virology. 28 (1), 1963(2018).</li>
<li>Duvergé, A., Negroni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudotyping+lentiviral+vectors%3A+When+the+clothes+make+the+virus%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">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/pubmed?term=((Pseudotype-based+neutralization+assays+for+influenza%3A+A+systematic+analysis%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">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/pubmed?term=((Overview+of+serological+techniques+for+influenza+vaccine+evaluation%3A+Past%2C+present+and+future%5BTitle%5D)+AND+%22Vaccines%22%5BJournal%5D)">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/pubmed?term=((Next-generation+influenza+vaccines%3A+opportunities+and+challenges%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">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/pubmed?term=((Influenza%5BTitle%5D)+AND+%22Nature+Reviews+Disease+Primers%22%5BJournal%5D)">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/pubmed?term=((Induction+of+broadly+neutralizing+H1N1+influenza+antibodies+by+vaccination%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Mycoplasma+contamination+of+cell+cultures+vesicular+traffic+in+bacteria+and+control+over+infectious+agents%5BTitle%5D)+AND+%22Acta+Naturae%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMichael%2C+RG+%22Molecular+Cloning%3A+A+Laboratory+Manual+%28Fourth+Edition%29%22">Molecular Cloning: A Laboratory Manual (Fourth Edition)</a>. , Cold Spring Harbor Laboratory Press. New York. (2012).</li>
<li>Kumar, P., Nagarajan, A., Uchil, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfection+of+mammalian+cells+with+calcium+phosphate-DNA+coprecipitates%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Transfection of mammalian cells with calcium phosphate-DNA coprecipitates.</a> Cold Spring Harbor Protocols. 2019 (10), (2019).</li>
<li>Robert, E. K., Chen, C. A., Okayama, H., John, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+phosphate+transfection%5BTitle%5D)+AND+%22Current+Protocols+in+Molecular+Biology%22%5BJournal%5D)">Calcium phosphate transfection.</a> Current Protocols in Molecular Biology. 9, (2003).</li>
<li>Robert, E. K., Chen, C. A., Okayama, H., John, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfection+of+DNA+into+eukaryotic+cells%5BTitle%5D)+AND+%22Current+Protocols+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((An+efficient+method+for+isolation+of+plasmid+DNA+for+transfection+of+mammalian+cell+cultures%5BTitle%5D)+AND+%22Methods+and+Protocols%22%5BJournal%5D)">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><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&amp;rsquo;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 cm&lt;sup&gt;2&lt;/sup&gt;</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 &amp;mu;L)</td><td>Thermo fisher scientific</td><td>TF102-10-Q</td><td>consumable material</td></tr><tr><td>Pipette Tips (100 &amp;mu;L)</td><td>Thermo fisher scientific</td><td>TF113-100-Q</td><td>consumable material</td></tr><tr><td>Pipette Tips (1000 &amp;mu;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

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 results ...

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. ...

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JoVE Journal

Immunology and Infection

Source: Chang, X., et al. Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity. J. Vis. Exp. (2021)This video demonstrates a luciferase-based pseudovirus titration assay to determine the optimal target cell density for maximum infection. By comparing luminescence across cell densities, the method ensures high assay sensitivity and reproducibility for downstream applications.

Determining Optimal Target Cell Density Using a Luciferase-Based Pseudovirus Infection Assay

Source: Chang, X., et al. Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity. J. Vis. Exp. (2021).This video demonstrates a luciferase-based assay to quantify pseudovirus infection in epithelial cells. The pseudovirus encodes the luciferase gene, which is expressed in infected cells. After washing, and cells are incubated with lysis buffer at an ultra-low temperature, and then thawed to induce lysis.

Assessing Pseudovirus Infection Using Luciferase Reporter Assay

This video demonstrates Microneutralization assays to measure neutralizing antibodies using Madin-Darby Canine Kidney cells overexpressing α2,6-linked sialic acid in human sera. This assay is often used for influenza human...

A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

This video demonstrates a technique for generating escape variants of neutralizing influenza virus monoclonal antibodies. Antibody-neutralized virus suspension is injected into embryonated chicken eggs to propagate escape variants selectively. This results in a virus population predominantly of escape variants, confirmed via a Hemagglutination assay.

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

Optimization of a Quantitative Micro-neutralization Assay

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 A Virus Studies in a Mouse Model of Infection

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.

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

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.

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

The protocol describes a method for culturing avian T cells in vitro by isolating memory T cells from infected ducks, allowing for the generation of highly purified specific duck T cells. Additionally, an intracellular cytokine staining (ICS) method was established to accurately measure IFN-γ secretion in duck T cells.

In Vitro Culture for H5N1-Specific Duck T Cells and Detection of Immune Responses Using Intracellular Cytokine Staining Method

Learn to prepare H5 avian influenza pseudoviruses using calcium phosphate transfection in HEK293 FT cells and measure antibody neutralizing activity. This protocol enables safe vaccine research in BSL-2 laboratories.

Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity (Video) | JoVE Journal

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

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