Name: detection of sars-cov-2 neutralizing antibodies using high-throughput fluorescent imaging of pseudovirus infection
Uploaded: 2021-06-05T14:00:00
Description: Watch this Scientific Journal Video about Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection at JoVE.com

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 cultur...

<ol>
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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 ...

This method provides a way of answering one of the more important questions concerning the SARS-coronavirus-2 vaccines under development. Is the vaccine able to elicit a neutralizing antibody response? The main advantage of this technique is that it can be done in a containment level two facility.It is also relatively fast and inexpensive, making it well-suited to analyzing many samples at once. Demonstrating the neutralizing acid procedure, we have Ricardo Marius, a senior technician in the lab and Taylor Jamieson, an MD PhD student. Begin by culturing Vero E6 cells in DMEM supplemented with 10%FBS at 37 degrees Celsius and 5%carbon dioxide.To passage the cells, first wash them by adding 10 milliliters of PBS and gently rocking the dish four to five times. After aspirating the PBS, add three milliliters of trypsin EDTA and rock the dish to ensure that the entire surface is covered before incubating it at 37 degrees Celsius. Once the cells have detached, deactivate the trypsin by adding seven milliliters of DMEM supplemented with 10%FBS, then resuspend the cells by pipetting up and down several times.Remove the trypsin by centrifugation and aspirate the supernatant without disturbing the cell pellet before resuspending the cells in 10 milliliters of fresh DMEM. After counting, add approximately one time 10 to the seventh cells to each 15 centimeter plate and incubate the culture for one to two days until the cells are 100%confluent. To prepare the VSV spike EGFP pseudovirus for titering, infect the cells at 0.01 multiplicity of infection with the stock virus diluted in 12 milliliters of serum-free DMEM.After adding the virus, incubate the cells for one hour at 37 degrees Celsius and 5%carbon dioxide with occasional rocking. At the end of the incubation, replace the inoculum with fresh DMEM supplemented with 2%FBS and 20 millimolar heaps, then incubate the cells at 34 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, use a fluorescent microscope to visualize the EGFP expression of the infected cells.Once an extensive cytopathic effect and cell detachment are observed, collect the culture supernatant and remove the debris by centrifugation. To avoid multiple freeze-thaw cycles, aliquot the supernatant before storage at minus 80 degrees Celsius. To determine the viral titer, plate Vero E6 cells in six well plates at a seeding density of six times 10 to the fifth cells per well and incubate overnight.The next day, set up a tenfold serial dilution series of the viral stock by adding 900 microliters of medium to seven micro-centrifuge tubes and adding 100 microliters of viral stock to the first tube. After brief vortexing, transfer 100 microliters of diluted virus to each subsequent tube, vortexing between each tube. Then replace the supernatant in each well of the Vero E6 culture plate with 500 microliters of the 10 to the minus second to 10 to the minus seventh viral dilutions.After a 45 minute incubation in the cell culture incubator with gently rocking every 15 minutes, replace the inoculum with an overlay solution and incubate the cells at 34 degrees Celsius with 5%carbon dioxide for 48 hours. At the end of the incubation, to visualize the plaques by crystal violet staining, wash the cells one time with PBS before adding two milliliters of 0.1%crystal violet to each well. Place the plate on a rocker for approximately 20 minutes at room temperature before gently washing each well two times with PBS.After the last wash, allow the plates to air dry for at least one hour before using the formula as indicated to calculate the titer of the virus in each well in plaque forming units per milliliter. To plate cells for a neutralization assay, use a multi-channel pipette to add 100 microliters of Vero E6 cells to each well of a 96 well plate at a density of two times 10 to the fifth cells per milliliter, and incubate the plate for 24 hours at 37 degrees Celsius and 5%carbon dioxide. The following day, heat inactivate the patient serum samples to be tested in a 56 degrees Celsius water bath for 30 minutes.Then set up a tenfold dilution series of the patient's serum samples in an empty 96 well tissue culture plate by adding eight microliters of the serum to 72 microliters of serum-free DMEM with antibiotics in each well of row A.Then add 40 microliters of serum-free DMEM to each well of rows B to G and 80 microliters to each well of row H.Using a 12 well multichannel pipette, mix the samples in row A, then transfer 40 microliters of the sample from each well of row A to each well of row B.After mixing, repeat the dilution for each subsequent row of wells until row F.Next, add 40 microliters of VSV spike EGFP at 0.05 multiplicity of infection to each well in rows A to G, and mix by pipetting four to five times before incubating the plate for one hour at 37 degrees Celsius and 5%carbon dioxide. At the end of the incubation, carefully aspirate the supernatant from each well of the Vero E6 culture plate and transfer 60 microliters of antibody virus mixture from each well of the sample dilution plate to the corresponding well of the cell culture plate. When all the samples have been transferred, place the cell culture plate at 37 degrees Celsius and 5%carbon dioxide for one hour with rocking every 20 minutes.After the incubation is complete, add 140 microliters of carboxymethylcellulose overlay solution to each well of the cell culture plate and transfer the plate to a 34 degrees Celsius and 5%carbon dioxide incubator. After 24 hours, image the plates on an automated fluorescent imager at 488 nanometer wavelength, and use the automated counting feature of the imager to quantify the individual EGFP foci. In this representative example, a commercially available neutralizing antibody against the SARS-coronavirus-2 spike receptor binding domain was used as a positive control, alongside IgG as a negative control.The percent inhibition was calculated based on the number of EGFP foci detected via fluorescent imaging. Pseudovirus neutralization by convalescent patient samples collected approximately three months post SARS-coronavirus-2 infection showed that hospitalized patients demonstrated an increased neutralizing capacity compared to those who did not require hospitalization. This procedure can also be used to evaluate other COVID-19 prevention and therapeutic agents that aim to block viral infection.

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Research

JoVE Journal

Immunology and Infection

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

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 ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

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 ...