We would like to thank the Whelan lab for generously providing the VSV-S-eGFP virus used in this protocol (described in Case et al. 2020). We also thank Drs. Bill Cameron and Juthaporn Cowan (and team) for collecting the patient blood samples (REB protocol ID 20200371-01H). The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the generous support from the Ottawa Hospital Foundation and a grant from the Canadian Institutes of Health Research (#448323) and a Fast Grant from the Thistledown foundation for COVID-19 Science to C.S.I. T.R.J. is funded by an Ontario Graduate Scholarship and cluster Mitacs fellowship. JP is funded by a cluster Mitacs fellowship. T.A. is funded by a CIHR Banting Fellowship. We would also like to thank all the individuals who participated and donated their blood samples for this study.

1. Plating cells (Day 1) for the production and quantification of SARS-CoV-2 pseudovirus
<ol>
	<li>Preparation for tissue culture
	<ol>
		<li>Warm 1x Dulbecco&#39;s Phosphate-Buffered Saline (DPBS); Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (optional); and 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA) to 37 &#176;C in a water bath for approximately 15 min.</li>
		<li>Disinfect a tissue culture hood with 70% ethanol, and place tissue culture dishes, Pasteur pipettes, and serological pipettes in the tissue culture hood as needed. Remove PBS, DMEM, and trypsin from the water bath, and disinfect with 70% ethanol prior to placing in the tissue culture hood.</li>
	</ol>
	</li>
	<li>Plating and maintaining cells
	<ol>
		<li>Grow Vero E6 cells in DMEM (containing 10% FBS) in T75 tissue culture flasks or 15 cm tissue culture dishes in a 37 &#176;C incubator with 5% CO2. Passage the cells as needed when cells are 80-90% confluent.</li>
		<li>Wash the cells by adding 10 mL of 1x PBS to each dish and gently rocking 4-5 times; aspirate the PBS. Add 3 mL of trypsin-EDTA to each dish, rock the plate to ensure the entire surface is covered. Incubate the cells at 37 &#176;C with 5% CO2 for approximately 5 min, or until the cells have detached.</li>
		<li>Add 7 mL of DMEM (containing 10% FBS) to deactivate the trypsin, and resuspend the cells by pipetting up and down several times. Spin down the cells to remove the trypsin using a benchtop centrifuge at 500 &#215; g for 5 min, aspirate the media without disturbing the cell pellet, and resuspend the cells in 10 mL of DMEM (containing 10% FBS). If cells are required for future experiments, place 1 mL in a new dish containing 15 mL of fresh DMEM (containing 10% FBS).</li>
		<li>Count the cells using an automated cell counter or hemocytometer, and add approximately 1 &#215; 107 cells to each 15 cm plate, and incubate at 37 &#176;C with 5% CO2 until they are 100% confluent (1-2 days).</li>
	</ol>
	</li>
</ol>
2. VSV-S-EGFP pseudovirus preparation
<ol>
	<li>Infection
	<ol>
		<li>Infect cells at multiplicity of infection (MOI) 0.01 with VSV-S-eGFP stock virus in 12 mL of serum-free DMEM for 1 h at 37 &#176;C with 5% CO2, occasionally rocking the plates. Replace the inoculum with fresh DMEM (containing 2% FBS and 20 mM HEPES, pH 7.7), and move to an incubator set to 34 &#176;C with 5% CO2. 
		NOTE: A temperature of 34 &#176;C is required for the propagation of VSV-S-eGFP in cell culture.</li>
		<li>Collect cell supernatants upon observation of extensive cytopathic effect (CPE) and cell detachment, approximately 48 h post-infection. Use a fluorescent microscope to visualize the extensive expression of eGFP by infected cells starting at 24 h post-infection.</li>
	</ol>
	</li>
	<li>Collection
	<ol>
		<li>Centrifuge the supernatants using a benchtop centrifuge for 5 min at 1,000 &#215; g at 4 &#176;C to remove the cell debris. Aliquot the supernatant to avoid multiple freeze-thaw cycles, and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Titering the VSV-S-eGFP pseudovirus
<ol>
	<li>Serial dilution to determine viral titer
	<ol>
		<li>Plate Vero E6 cells on 6-well plates at a seeding density of 6 &#215; 105 cells per well in DMEM (with 10% FBS), and incubate overnight at 37 &#176;C with 5% CO2. 
		NOTE: Vero cells that overexpress TMPRSS2 and hACE2 can also be used.</li>
		<li>Set up a 10-fold serial dilution series of the VSV-S-eGFP virus in cold serum-free DMEM by placing 900 &#181;L of media in seven microcentrifuge tubes. Label tubes from 1 to 7. Add 100 &#181;L of the viral stock to the first tube and vortex briefly. Transfer 100 &#181;L of diluted virus from tube 1 to tube 2 and vortex briefly; continue until tube 7.</li>
		<li>Aspirate the medium from all wells of the 6-well plate containing Vero E6 cells, and replace with 500 &#181;L of dilutions 10-2 to 10-7. Incubate the plates for 45 min at 37 &#176;C with 5% CO2, gently rocking every 15 min.</li>
		<li>Aspirate the inoculum and replace with overlay containing a 1:1 mixture of 2x DMEM and 6% carboxymethyl cellulose (CMC), final concentration of 3% CMC, supplemented with 10% FBS; incubate at 34 &#176;C with 5% CO2 for 48 h. 
		NOTE: Pre-warm the overlay mixture in a 37 &#176;C water bath for 15 min during the infection step. A 1:1 mixture of 1% agarose with 2x DMEM supplemented with 10% FBS may be used in place of CMC. To overlay with agarose, boil 1% agarose (in dH2O) and mix with cold 2x DMEM. Ensure the mixture is a suitable temperature before adding to the cell monolayer (approximately 37-40 &#176;C). If agarose is used for the overlay, cells must be fixed by incubating in 2 mL of 3:1 methanol:acetic acid for at least one hour prior to staining.&#160;</li>
	</ol>
	</li>
	<li>Staining and calculating viral titer
	<ol>
		<li>Visualize plaques by staining with crystal violet or direct visualization of eGFP under a fluorescent microscope.</li>
		<li>To stain plates, aspirate the overlay, wash once with PBS, then add 2 mL of 0.1% crystal violet (in 80% methanol and dH2O) to each well. Place on a plate rocker for approximately 20 min at room temperature prior to de-staining. Remove the crystal violet, and gently wash each well twice using dH2O or PBS. 
		NOTE: Washing and staining steps should be performed gently by pipetting slowly towards the side of each well to ensure the cell monolayer remains intact throughout the procedure.</li>
		<li>Allow plates to dry for at least 1 h prior to counting plaques. Ensure that plaque counts are obtained from wells containing 20 to 200 plaques. To calculate the titer of the virus in plaque-forming units (PFU) per mL, multiply the dilution factor of the well counted with the volume of infection, and divide this number from the plaques present in the respective well. 
		<img alt="Equation 1" src="/files/ftp_upload/62486/62486eq01.jpg" /></li>
	</ol>
	</li>
</ol>
4. Plating cells (Day 1) for the measurement of neutralization of SARS-CoV-2 pseudovirus by commercially available antibodies and convalescent patient serum
<ol>
	<li>Preparation for tissue culture
	<ol>
		<li>Prepare medium and tissue culture hood as described in section 1. In addition, prepare the necessary number of 96 well plates to accommodate a minimum of 2 replicates per sample (i.e., 1 plate per 6 samples); add additional replicates if sample volume permits.</li>
	</ol>
	</li>
	<li>Plating and maintaining cells
	<ol>
		<li>Grow and maintain Vero E6 cells as described in section 1. Prepare at least 10 mL of cell suspension per 96-well plate at a concentration of 2 &#215; 105 cells per mL (plate 1.5 &#215; 105 cells per mL for assays performed after 48 h, if needed). Add 100 &#181;L of cell suspension to each well of a 96-well plate using a multichannel pipette, and incubate for 24 h at 37 &#176;C with 5% CO2. 
		NOTE: Mix the cell suspension well, and rock each plate gently in all directions to ensure even distribution of cells.</li>
	</ol>
	</li>
</ol>
5. Antibody or serum dilutions and infections (Day 2)
NOTE: This protocol can be applied to measure the neutralization of VSV-S-eGFP by both commercially available antibodies and patient serum, as well as serum collected from animals for pre-clinical vaccine development studies. *Take note of the additional steps listed when handling patient/animal serum samples.
<ol>
	<li>Serial dilution series
	<ol>
		<li>Prior to diluting serum samples, heat-inactivate each sample in a 56 &#176;C water bath for 30 min to inactivate complement. Ensure necessary safety precautions are taken when handling patient samples; only open sample containers when in the tissue culture hood, and ensure appropriate containment level 2 personal protective equipment is worn.</li>
		<li>Set up the dilution series in an empty 96-well plate (Plate 1).</li>
		<li>Begin all dilutions in Row A of the 96-well plate in 80 &#181;L. *For patient samples, begin the dilution series at 1 in 10 by placing 8 &#181;L of serum in 72 &#181;L of serum-free DMEM (with 1% penicillin/streptomycin). 
		NOTE: The concentration of neutralizing antibody used will depend on the activity predicted by the manufacturer. For the SARS-CoV-2 Spike neutralizing antibody used here (see the Table of Materials), start with 5 &#181;g/mL to observe at least 50% neutralization.</li>
		<li>Add 40 &#181;L of of serum-free DMEM (with 1% penicillin/streptomycin) to rows B to G, and 80 &#181;L to row H. Do not add virus to row H, as it serves as a cell-only control.</li>
		<li>Using a 12-well multichannel pipette, mix and transfer 40 &#181;L of diluted serum or antibody from row A to row B. Mix and repeat until row F, discard 40 &#181;L from row F. Do not add serum to row G as it represents the virus-only control row.</li>
	</ol>
	</li>
	<li>Infection and overlay
	<ol>
		<li>Prepare an appropriate volume of diluted VSV-S-eGFP to treat each well at MOI 0.05 (or 2000 pfu). (When completing this calculation, keep in mind that only 60 &#181;L of the total 80 &#181;L volume will be moved onto the cells; therefore, be sure to multiply the volume of the virus by 1.33 to maintain 2000 pfu). 
		NOTE: As VSV-S-eGFP is temperature-sensitive, ensure viral stocks remain on ice whenever not in use, and avoid multiple freeze-thaw cycles as titers will differ after repeated freezing.</li>
		<li>Add 40 &#181;L of diluted virus to each well in rows A to G of Plate 1 and mix by pipetting up and down 4-5 times. Incubate the plate for 1 h at 37 &#176;C with 5% CO2.</li>
		<li>Remove the plate containing cells from the incubator (Plate 2), and carefully aspirate the media from all wells. Transfer 60 &#181;L of antibody/virus mixture from Plate 1 to Plate 2, and incubate for 1 h at 37 &#176;C with 5% CO2, rocking the plate every 20 min.</li>
		<li>Top up each well with 140 &#181;L of overlay containing CMC in DMEM for a final concentration of 3% CMC (with 10% FBS and 1% penicillin/streptomycin). Place Plate 2 in an incubator set to 34 &#176;C with 5% CO2 for approximately 24 h before imaging. 
		&#8203;NOTE: Pre-warm the overlay mixture in a 37 &#176;C water bath for 15 min during the infection step.</li>
	</ol>
	</li>
</ol>
6. Imaging and quantification (Day 3)
<ol>
	<li>Image plates using an automated fluorescent imager (using a fluorescein isothiocyanate (FITC) filter or an alternative filter with an excitation wavelength of 488 nm). Quantify viral infection by creating a protocol that automatically identifies and counts individual eGFP foci. If an automatic counting feature is not available, use ImageJ software to quantify the number of eGFP foci.</li>
</ol>

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The authors have no competing financial interests related to this publication.

The method described here may be adapted to suit varying lab environments and resources as needed. Importantly, the main limitation of this protocol is the necessity for a containment level 2 space and tissue culture hood. The application of a replicating RNA virus pseudotyped with the SARS-CoV-2 spike, such as VSV-S-eGFP, is a formidable alternative to the SARS-CoV-2 virus, which requires a containment level 3 working area, but may remain a limitation for some groups. All other steps described here are quite flexible an...

In December 2019, a novel coronavirus was identified, which we now know as SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19)1. SARS-CoV-2 is a betacoronavirus belonging to the Coronaviridae family. These enveloped viruses comprise a large positive-sense RNA genome and are responsible for respiratory and intestinal infections in both humans and animals2. As of May 2021&#160;there have been more than 157 million reported cases of COVID-19 globally and more than 3.2&#160;million deaths3. The development of an effective vaccine has become the primary goal of researchers around the globe with at least 77&#160;preclinical vaccines under investigation and 90&#160;currently undergoing clinical trials4.
Coronaviruses encode four structural proteins including the spike protein (S), nucleocapsid (N), envelope protein (E), and the membrane protein (M). Entry of SARS-CoV-2 requires interaction of the receptor-binding domain (RBD) of S with the host receptor, human angiotensin-converting enzyme 2 (hACE2), and subsequent membrane fusion following proteolytic cleavage by host cellular serine protease, transmembrane protease serine 2 (TMPRSS2)5,6,7,8,9,10. Humoral immunodominance of the S protein of SARS-CoV has been previously reported and has now been shown also for SARS-CoV-211,12,13. Indeed, neutralizing antibody responses against S have been detected in convalescent serum from SARS-CoV patients 24 months after infection14, highlighting their critical role in the long-term immune response. The S protein has been identified as a promising vaccine target and has thus become a key component of most vaccines under development15,16.
While the rapid detection of neutralizing antibodies is a critical aspect of vaccine development, it may also shed light on the rate of infection and sero-epidemiologic surveillance in impacted areas17. A replication-competent VSV pseudotyped with the SARS-CoV-2 S glycoprotein, in place of the wild-type VSV glycoprotein, to study SARS-CoV-2 infection in biosafety level 2 settings was kindly donated by Whelan and co-workers18. VSV expressing spike (VSV-S) will be utilized to determine the neutralizing antibody response against SARS-CoV-2 spike protein. As the VSV-S used here also expresses enhanced green fluorescent protein (eGFP), eGFP foci may be detected within 24 h to quantify infection, whereas plaque formation can take 48 to 72 h. Summarized here is a simple and effective protocol to determine the ability of convalescent patient serum to neutralize VSV-S-eGFP infection. This method may also be easily adapted to interrogate other potential therapeutics that aim to disrupt the host-viral interaction of SARS-CoV-2 S protein.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA (Gibco)</td>
			<td>Fisher scientific</td>
			<td>LS25200114</td>
			<td></td>
		</tr>
		<tr>
			<td>ArrayScan VTI HCS</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Automated fluorescent imager</td>
		</tr>
		<tr>
			<td>carboxymethyl cellulose</td>
			<td>Sigma</td>
			<td>C5678</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (Gibco)</td>
			<td>Fisher scientific</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (Powder) (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-800-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Fisher scientific</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher scientific</td>
			<td>BP-310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Isotype Control (mouse)</td>
			<td>Thermo Fisher Scientific</td>
			<td>31903</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse Mab</td>
			<td>SinoBiological</td>
			<td>40592-MM57</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero E6 cells</td>
			<td>ATCC</td>
			<td>&#160;CRL-1586</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of sars-cov-2 neutralizing antibodies using high-throughput fluorescent imaging of pseudovirus infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

This protocol outlines a rapid and effective method for detecting neutralizing antibodies against SARS-CoV-2 S protein via inhibition of VSV-S-eGFP pseudovirus infection (quantifiable by loss of eGFP foci detected). A schematic representation of the protocol is depicted in Figure 1. It is recommended that a commercially available antibody be used as a positive control each time the assay is run to ensure the consistency of the assay. Here, we demonstrate a dilution curve using a commercially...

Watch this Scientific Journal Video about Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection at JoVE.com

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

detection-sars-cov-2-neutralizing-antibodies-using-high-throughput

Research

JoVE Journal

Immunology and Infection

4.2K Views.  Ottawa Hospital Research Institute. The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein. 

Video: Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

Quantifying Vaccine-Induced Neutralizing Antibodies in SARS-CoV-2 Variants

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

Author Spotlight: A Pseudotype Virus System for Assessing Omicron Subvariants and Neutralizing Antibodies in SARS-CoV-2 Research

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

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

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 (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 ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Quick and Early Detection of Rabies Antibodies to Evaluate the Immune Response in a Patient

Detection of Rabies IgG and IgM Antibodies Using the Rabies Indirect Fluorescent Antibody Test

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. This test provides rapid results and can be used to detect rabies antibodies in several different scenarios. The rabies IFA test is especially useful for the quick and early detection of antibodies to evaluate the immune response in a patient who has developed rabies. Although other methods for antemortem rabies diagnosis take precedence, this test may be utilized to demonstrate recent rabies virus exposure through antibody detection. The IFA test does not provide a virus-neutralizing antibody (VNA) titer, but the pre-exposure prophylaxis (PrEP) response can be evaluated through positive or negative antibody presence. This test can be utilized in various situations and can provide results for a number of different targets. In this study, we used several paired serum samples from individuals who received PrEP and demonstrated their rabies antibody presence over time using the IFA test.

Author Spotlight: Rabies-Specific Antibody Isotypes Detection in Sera or Cerebral Spinal Fluid Using an IFA Test 

The aim of this manuscript is to examine the use of the rabies indirect fluorescent antibody test for the detection of rabies-specific IgG and IgM antibodies.

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. ...