Name: Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses
Uploaded: 2023-11-21T12:40:00
Description: Watch this Scientific Journal Video about Studying Host-Virus Interactions with Pseudotyped Viruses at JoVE.com

We acknowledge the contribution of the health-care workers volunteers. This project was supported by the Department of Excellence 2023/2027, MUR, Italy. AR and DZ were supported by PRIN2022 (EU fundings; NextGenerationEU)

The present protocol has been approved by and follows the guidelines of the Ethical Committee of the University of Verona (approval protocol number 1538). Informed written consent was obtained from the human subjects participating in the study. Whole blood samples were collected from healthcare worker (HCW) volunteers who were in the process of receiving anti-SARS-CoV-2 vaccines. These samples were collected in plastic tubes containing anticoagulants for the subsequent isolation of serum15.
<p...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

<ol>
	<li>Ozaki, D. A., 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=International+technology+transfer+of+a+GCLP-compliant+HIV-1+neutralizing+antibody+assay+for+human+clinical+trials.">International technology transfer of a GCLP-compliant HIV-1 neutralizing antibody assay for human clinical trials.</a> Plos One. 7 (1), e30963 (2012).</li><li>Pouget, M., 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=Generation+of+liposomes+to+study+the+effect+of+Mycobacterium+tuberculosis+lipids+on+HIV-1+cis-+and+trans-infections.">Generation of liposomes to study the effect of Mycobacterium tuberculosis lipids on HIV-1 cis- and trans-infections.</a> International Journal of Molecular Sciences. 22 (4), 1945 (2021).</li><li>McKay, L. G. A., 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=The+HCV+envelope+glycoprotein+down-modulates+NF-&#954;B+signalling+and+associates+with+stimulation+of+the+host+endoplasmic+reticulum+stress+pathway.">The HCV envelope glycoprotein down-modulates NF-&#954;B signalling and associates with stimulation of the host endoplasmic reticulum stress pathway.</a> Frontiers in Immunology. 13, 831695 (2022).</li><li>Xiang, Q., Li, L., Wu, J., Tian, M., Fu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+pseudovirus+system+in+the+development+of+vaccine,+antiviral-drugs,+and+neutralizing+antibodies.">Application of pseudovirus system in the development of vaccine, antiviral-drugs, and neutralizing antibodies.</a> Microbiological Research. 258, 126993 (2022).</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, e1963 (2018).</li><li>D'Apice, L., 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=Comparative+analysis+of+the+neutralizing+activity+against+SARS-CoV-2+Wuhan-Hu-1+strain+and+variants+of+concern:+Performance+evaluation+of+a+pseudovirus-based+neutralization+assay.">Comparative analysis of the neutralizing activity against SARS-CoV-2 Wuhan-Hu-1 strain and variants of concern: Performance evaluation of a pseudovirus-based neutralization assay.</a> Frontiers in Immunology. 13, 981693 (2022).</li><li>Falzarano, D., Groseth, A., Hoenen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+reporter-expressing+mononegaviruses:+current+challenges+and+perspectives.">Development and application of reporter-expressing mononegaviruses: current challenges and perspectives.</a> Antiviral Research. 103, 78-87 (2014).</li><li>Gutierrez-Guerrero, A., Cosset, F. -. L., Verhoeyen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vector+pseudotypes:+Precious+tools+to+improve+gene+modification+of+hematopoietic+cells+for+research+and+gene+therapy.">Lentiviral vector pseudotypes: Precious tools to improve gene modification of hematopoietic cells for research and gene therapy.</a> Viruses. 12, 1016 (2020).</li><li>Zufferey, R., Nagy, D., Mandel, R. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+applications+of+SARS-CoV-2+pseudoviruses:+A+mini+review.">Construction and applications of SARS-CoV-2 pseudoviruses: A mini review.</a> International Journal of Biological Sciences. 17 (6), 1574-1580 (2021).</li><li>Zipeto, D., 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+human+immunodeficiency+virus+neutralizing+antibodies+using+fusion+complexes.">Induction of human immunodeficiency virus neutralizing antibodies using fusion complexes.</a> Microbes and Infection. 8 (6), 1424-1433 (2006).</li><li>WHO Coronavirus (COVID-19) Dashboard. Available from: <a target="_blank" href="https://covid19.who.int">https://covid19.who.int</a> (2022)</li><li>Zhou, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumonia+outbreak+associated+with+a+new+coronavirus+of+probable+bat+origin.">A pneumonia outbreak associated with a new coronavirus of probable bat origin.</a> Nature. 579 (7798), 270-273 (2020).</li><li>Chen, X., Huang, H., Ju, J., Sun, R., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+vaccination+on+the+COVID-19+pandemic+in+U.S.+states.">Impact of vaccination on the COVID-19 pandemic in U.S. states.</a> Scientific Reports. 12 (1), 1554 (2022).</li><li>Stefani, C., Fantoni, T., Bissoli, M., Thomas, J., Ruggiero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+and+SARS-CoV-2+Co-Infection:+From+Population+Study+Evidence+to+In+Vitro+Studies.">HIV and SARS-CoV-2 Co-Infection: From Population Study Evidence to In Vitro Studies.</a> Life. 12 (12), 2089 (2022).</li><li>Watson, O. 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=Global+impact+of+the+first+year+of+COVID-19+vaccination:+a+mathematical+modelling+study.">Global impact of the first year of COVID-19 vaccination: a mathematical modelling study.</a> The Lancet Infectious Diseases. 22 (9), 1293-1302 (2022).</li><li>Cantoni, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+antibody+neutralisation+activity+against+SARS-CoV-2+variants+and+seasonal+human+coronaviruses+NL63,+HKU1,+and+229E+induced+by+three+different+COVID-19+vaccine+olatforms.">Analysis of antibody neutralisation activity against SARS-CoV-2 variants and seasonal human coronaviruses NL63, HKU1, and 229E induced by three different COVID-19 vaccine olatforms.</a> Vaccines. 11 (1), 58 (2023).</li><li>Siracusano, 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=Different+decay+of+antibody+response+and+VOC+sensitivity+in+na&#239;ve+and+previously+infected+subjects+at+15+weeks+following+vaccination+with+BNT162b2.">Different decay of antibody response and VOC sensitivity in na&#239;ve and previously infected subjects at 15 weeks following vaccination with BNT162b2.</a> Journal of Translational Medicine. 20 (1), 22 (2022).</li><li>Ruggiero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+vaccination+elicits+unconventional+IgM+specific+responses+in+na&#239;ve+and+previously+COVID-19-infected+individuals.">SARS-CoV-2 vaccination elicits unconventional IgM specific responses in na&#239;ve and previously COVID-19-infected individuals.</a> eBioMedicine. 77, (2022).</li><li>Piubelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subjects+who+developed+SARS-CoV-2+specific+IgM+after+vaccination+show+a+longer+humoral+immunity+and+a+lower+frequency+of+infection.">Subjects who developed SARS-CoV-2 specific IgM after vaccination show a longer humoral immunity and a lower frequency of infection.</a> eBioMedicine. 89, 104471 (2023).</li><li>Zhang, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectivity+of+pseudotyped+SARS-CoV-2+variants+of+concern+in+different+human+cell+types+and+inhibitory+effects+of+recombinant+spike+protein+and+entry-related+cellular+factors.">Infectivity of pseudotyped SARS-CoV-2 variants of concern in different human cell types and inhibitory effects of recombinant spike protein and entry-related cellular factors.</a> Journal of Medical Virology. 95 (1), e28437 (2023).</li><li>da Costa, K. A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+A+(N1-N9)+and+Influenza+B+(B/Victoria+and+B/Yamagata)+neuraminidase+pseudotypes+as+tools+for+pandemic+preparedness+and+improved+influenza+vaccine+design.">Influenza A (N1-N9) and Influenza B (B/Victoria and B/Yamagata) neuraminidase pseudotypes as tools for pandemic preparedness and improved influenza vaccine design.</a> Vaccines. 10 (9), 1520 (2022).</li><li>Condor Capcha, J. 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Although using a wildtype virus simulates the actual infection, lentiviral PVs are a safer option to study the mechanisms associated with viral entry and infection without the strict safety requirements necessary to work with pathogenic viruses4,20,21. PVs are composed of a replication-defective viral core surrounded by the surface envelope glycoprotein of a pathogenic virus which is the objective of the study.
<p class="jov...

Pseudotyped viruses (PVs) are molecular tools used in microbiology to study host-virus and pathogen-pathogen interactions1,2,3,4. PVs consist of an inner part, the viral core that protects the viral genome, and an outer part, the envelope glycoproteins on the surface of the virus that defines the tropism5. A pseudovirus is replication-incompetent in the target cell because it does not contain all the genetic information to generate new viral particles. This combination of peculiar features makes PVs a safe alternative to a wildtype virus. Wildtype viruses, on the other hand, are highly pathogenic and cannot be used in BSL 2 laboratories for analysis6.
The infectivity of PVs can be monitored by a reporter gene, usually coding for a fluorescent protein (GFP, RFP, YFP) or an enzyme that produces chemiluminescent products (luciferase). This is contained in one of the plasmids used for PV production and incorporated in the genome of the pseudovirus7.
Several types of PV cores currently exist, including lentiviral-derived particles based on the HIV-1 genome. The great advantage of HIV-1-based PVs over other platforms is their intrinsic integration process in the target cell genome8. Although HIV-1 is a highly contagious virus and is the causative agent of AIDS, these lentiviral vectors are safe to use because of the extensive optimization steps over the years. Optimal safety conditions were achieved with the introduction of 2nd-generation lentiviral vectors, in which viral genes were depleted without influencing transduction capabilities9. The 3rd and 4th generations contributed to the increased safety of lentiviral vector handling with the further splitting of the viral genome into separate plasmids10, 11. The latest generations of PVs are generally employed to produce lentiviral vectors for gene therapy.
PVs can be used to study interactions between viruses and host cells, during both the production and the infection phases. PVs are especially employed in pseudovirus neutralization assays (PVNA). PVNAs are widely validated to assess the neutralization potential of serum or plasma by targeting the viral glycoprotein on the PV&#39;s envelope12,13. Neutralization activity, expressed as the inhibitory concentration 50 (IC50), is defined as the dilution of serum/plasma that blocks 50% of viral particle entry14. In this protocol, we described the set-up of a PVNA to test the antibody activity against Severe Acute Respiratory Syndrome - Coronavirus 2 (SARS-CoV-2) in sera collected before and after receiving a booster vaccine dose.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;m filter</td>
			<td>SARSTEDT</td>
			<td>83 1826</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>SARSTEDT</td>
			<td>83 3920</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>SARSTEDT</td>
			<td>8,33,924</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>Merck</td>
			<td>10403892</td>
			<td></td>
		</tr>
		<tr>
			<td>Black Opaque 96-well Microplate</td>
			<td>Perkin Elmer</td>
			<td>60005270</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium&#160;</td>
			<td>SIGMA-ALDRICH</td>
			<td>D6546 - 500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (PBS 1x)</td>
			<td>AUROGENE</td>
			<td>AU-L0615-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>AUROGENE</td>
			<td>AU-S1810-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism version 7</td>
			<td>graphpad dotmatics</td>
			<td>NA</td>
			<td>In the manuscript, we replace the commercial name with &#39;data analysis program&#39;</td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T/ACE2 cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>HEK293T has been transduced to overexpress ACE2 with a lentiviral vector.</td>
		</tr>
		<tr>
			<td>L-glutamine&#160;</td>
			<td>AUROGENE</td>
			<td>AU-X0550-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer - Victor3</td>
			<td>Perkin Elmer</td>
			<td>HH35000500</td>
			<td>In the manuscript, we replace the commercial name with&#160; &#39;luminometer&#39;&#160;</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11058021</td>
			<td>In the manuscript, we replace the commercial name with &#39;reduced serum medium&#39;&#160;</td>
		</tr>
		<tr>
			<td>p8.91 packaging plasmid</td>
			<td>Di Genova et al., 2021</td>
			<td></td>
			<td>A kind gift from Prof. Nigel Temperton (ref 16.)</td>
		</tr>
		<tr>
			<td>pCSFLW reporter plasmid</td>
			<td>Di Genova et al., 2021</td>
			<td></td>
			<td>A kind gift from Prof. Nigel Temperton (ref 16.)</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>AUROGENE</td>
			<td>AU-L0022-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, branched (PEI) (25 kDa)</td>
			<td>SIGMA-ALDRICH</td>
			<td>408727</td>
			<td></td>
		</tr>
		<tr>
			<td>RRL.sin.cPPT.SFFV/Ace2.IRES-puro.WPRE (MT126)</td>
			<td>Addgene</td>
			<td>145839</td>
			<td>This plasmid was used to generate HEK293Tcells/ACE2</td>
		</tr>
		<tr>
			<td>SARS-CoV-2 Spike expressing plasmid</td>
			<td>Addgene</td>
			<td>pGBW-m4137382</td>
			<td></td>
		</tr>
		<tr>
			<td>steadylite plus Reporter Gene Assay System</td>
			<td>Perkin Elmer</td>
			<td>6066759</td>
			<td>In the manuscript, we replaced the commercial name with &#39;luciferase reading reagent&#39;</td>
		</tr>
		<tr>
			<td>Trypsin EDTA 1x</td>
			<td>AUROGENE</td>
			<td>AU-L0949-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

pseudotyped viruses as a molecular tool to monitor humoral immune responses against sars-cov-2 via neutralization assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

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

Pseudotyped viruses are molecular tools that can be used to study host-virus interactions and to test neutralizing activity of serum samples. In the work we present today, a general methodology to produce pseudotyped viruses, based on free plasmid, will be presented. For neutralization assays, Y-type viruses are generally used, but they are highly pathogenic and cannot be handled in a BSL-2 laboratories, rendering this system purely applicable on large scale.The technology of pseudo viruses can be a safer alternative compared to live virus to allow the study of pathogen to host interaction and to test neutralizing activity of antibodies. Future experiments will investigate the role of specific antibodies in the maintenance of immunity after vaccination using the technology of pseudo viruses.

This protocol describes the production of SARS-CoV-2 PVs and a downstream application of these PVs to analyze the neutralization activity of serum/plasma of subjects receiving anti-COVID-19 vaccination17. Furthermore, this protocol can be applied to produce pseudotypes of each SARS-CoV-2 variant of concern (VOC) to test the evolution of the neutralizing response. Despite this protocol facilitating the study of humoral immune response after COVID-19 vaccination, it can be adapted to easily test the...

Watch this Scientific Journal Video about Studying Host-Virus Interactions with Pseudotyped Viruses at JoVE.com

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 (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

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Research

JoVE Journal

Immunology and Infection

SARS-CoV-2 Pseudotype Virus Production and Serum Neutralization Assay

To begin, in a six-well plate, seed one milliliter of human embryonic kidney 293T cells. Add two milliliters of complete DMEM. The next day, replace the medium with fresh antibiotic-free medium to prepare the cells for transfection.To transfect the adherent cells, first, prepare mix A by adding plasmid DNA to 100 microliters of Opti-MEM. Next, add 17.5 microliters of polyethylenimine to 100 microliters of Opti-MEM. Mix the contents of the mix A and B, then incubate.During incubation, gently flick the tube every three to four minutes to enhance mixing. Add the whole volume of the mixture to the plate with the HEK 293T cells. After 16 to 20 hours of transfection, replace the culture medium with fresh complete DMEM.Incubate the plate at 37 degrees Celsius under 5%carbon dioxide to produce the pseudotype viruses. After 72 hours of transfection, harvest the supernatant into a collection tube. Centrifuge the supernatant at 1, 600 G for seven minutes at room temperature.Then filter the supernatant through a 0.45 micrometer cellulose acetate filter. Distribute 50 microliters of complete DMEM in the wells of a 96-well plate. Leave row A empty.Add 100 microliters of the pseudovirus stock to the wells in row A.Next, pipette 50 microliters of the solution from row A to B.Repeat this process up to row G to obtain serial dilutions. Remove the exhausted medium from a plate with cultured susceptible HEK 293T ACE2 cells, and wash the cells with four milliliters of DPBS. Then detach the cells with one milliliter of trypsin EDTA in DPBS saline.Now add 50 microliters of the susceptible cell suspension into each well containing the pseudotype viruses. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide for 48 hours. Add 100 microliters of the luciferase reagent to each well.After incubation, transfer the contents of each well into a black 96-well plate. Then read the plate in a plate reader. In a 96-well plate, add 50 microliters of fresh complete DMEM to columns one to 10 from rows B to H.Add 95 microliters of fresh complete DMEM to the corresponding wells of row A.Now add 50 microliters of complete DMEM to the wells in column 11 and 100 microliters of DMEM to column 12.Pipette five microliters of heat-inactivated serum samples to row A.With a multi-channel pipette, mix the samples in the first row and transfer 50 microliters of medium-containing serum from row A to B up to row H.Distribute 50 microliters of dilute pseudovirus containing medium to each well from columns one to 11. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide for one hour. Prepare a suspension of susceptible HEK293T ACE2 cells in five milliliters.Add 50 microliters of the cell suspension to each well, then incubate before performing the luciferase assay. Lower serum dilution resulted in decreased viral entry into the cells. This is confirmed by the increased percentage of neutralization.