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>
	<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. D. <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+mammalian+cells+with+calcium+phosphate-DNA+coprecipitates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+phosphate+transfection.">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/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...

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. ...

This protocol describes the procedures and the critical steps of H5 high pathogen avian influenza pseudo virus preparations and pseudo virus neutralization assays. Also, it discusses the troubleshooting limitation and modifications of these assays. Highly pathogenic pathogens must be carried out in the level three bio safety laboratory, whereas pseudo viruses can be safely handled in a level two bio safety laboratory.To begin, make a single cell suspension of HEK293 FT cells in a complete DMEM medium. Add 10 milliliters of the single cell suspension to a T75 flask, followed by incubation at 37 degrees Celsius. Two hours before transfection, replace the old medium with 10 milliliters of fresh complete medium with chloroquine.Mix the reagents and plasmid DNA as described in the manuscript then transfer this mixture into the medium above the cells and rock gently. After incubation, replace the medium with 15 milliliters of fresh complete DMEM medium. After 65 hours, harvest the supernatant by centrifugation.Collect the supernatant and aliquot it. To detect hemagglutinin protein expression, add 50 microliters of PBS into columns two through 12 of a 96 well round bottom plate. Add 100 microliters of pseudo virus into the wells of column one.Then transfer 50 microliters of pseudo virus from column one into the wells of column two. Perform this twofold dilution until column 11 followed by gentle mixing. Then discard the extra 50 microliters from column 11.Add 50 microliters of 0.5%erythrocyte into each well followed by gentle mixing. After 30 to 60 minutes of incubation at room temperature, observe and record hemagglutinin titers of the pseudo virus. For pseudo virus titration, make a single cell suspension of five times 10 to the fourth cells per milliliter of MDCK cells in a complete DMEM medium.Add 1, 250, 2, 500, 5, 000, 10, 000, 20, 000, and 40, 000 cells to different wells of a flat bottom 96 well plate. After incubation, thaw and vortex the pseudo virus. Add six HAU of pseudo viruses to each well of a 96 well round bottom plate and replenish each well with complete DMEM up to 120 microliters.Transfer 100 microliters of the mixture to the cells in the 96 well plate. Incubate the culture plate for 48, 60, and 72 hours for viral infection and perform a luciferase assay. To perform the luciferase assay, wash the cells by removing the medium carefully and rinsing it with 200 microliters of PBS.Remove as much of the PBS as possible, then add 50 milliliters of the lysis buffer to each well. On the next day, equilibrate the plates at room temperature for two hours, then rock the culture plates several times and transfer all the cell lysates to opaque 96 well plates. Add 50 microliters of the substrate to each plate and mix it well.Record the luciferase titers of the pseudo virus by using the luminometer. Add 100 microliters of the single cell suspension of MDCK cells to each well of a flat bottom 96 well plate and incubate it for 20 hours at 37 degrees Celsius. After the incubation, thaw and vortex the pseudo virus and the sera stored at minus 80 degrees Celsius.Dilute the sera 20 times in a complete medium. Add 100 microliters of DMEM medium to columns three through 10. Add 200 microliters of the diluted sera into the wells of the second column and perform serial dilution.Then add 100 microliters of DMEM medium to the 11th column as the negative control. Add 100 microliters of pseudo viruses to each well and mix it. Transfer 100 microliters of the mixture from the 96 well round bottom plate to the corresponding well of the 96 well cell culture plate.After 60 hours of incubation at 37 degrees Celsius, perform the luciferase assay. The hemagglutinin assay showed that 643 HA units per milliliter of influenza pseudo viruses are present in influenza pseudo virus stocks. The ratios of HA and Gag P24 were within a normal range.The western blot assay showed the presence of four protein types, including HA0, HA1, HA2, and NA proteins, indicating the similarity of influenza pseudo viruses to that of wild type viruses. Pseudo virus titration results indicate that the suitable conditions for the highest relative luciferase activity include the feeding of 5, 000 cells in a well of a 96 well plate and detecting relative luciferase activity readings after 60 hours of virus infection. The immune sera from the DDV group exhibited high neutralization titers against pseudo virus A Thailand 1 Can 104 strain, whereas sera from the control group did not exhibit any neutralization activity.Neutralizing activity of the immune sera was potent compared to the control sera, indicating that a lot of antibodies were directed to the HA head in the immune sera. The significant differences between the immune sera and the controls during the assessment of viral entry indicate that antibodies are directed to the HA stem region in the immune sera. When performing this procedure, remember to keep the pH of the transfection solution and the culture medium stable.

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2.0K 조회수.  Nanjing Advanced Academy of Life and Health. 이 프로토콜은 H5 고병원체 조류 인플루엔자 유사 바이러스 제제 및 유사 바이러스 중화 분석의 절차와 중요한 단계를 설명합니다. 또한 이러한 분석의 문제 해결 제한 및 수정에 대해 설명합니다. 고병원성 병원체는 3급 생물안전 실험실에서 수행해야 하는 반면, 유사 바이러스는 2단계 생물안전 실험실에서 안전하게 처리할 수 있습니다.시작하려면 완전한 DMEM 배지에?...

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