Name: preparation of pseudo-typed h5 avian influenza viruses with calcium phosphate transfection method and measurement of antibody neutralizing activity
Uploaded: 2021-11-22T13:30:00
Description: Watch this Scientific Journal Video about Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity at JoVE.co

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<...

<ol>
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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. 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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>. 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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...

Watch this Scientific Journal Video about Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity at JoVE.co

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. ...

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