We wish to thank all members of the Whittaker and Daniel labs for helpful comments. Research funding was provided by the NIH grants R21 AI111085 and R01 AI135270. T.T. acknowledges support from the Presidential Life Science Fellowship in Cornell University and National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1650441. L.N. acknowledges support from a Samuel C. Fleming Family Graduate Fellowship and National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1650441. This work is also supported by National Science Foundation 1504846 (to S.D. and G.R.W.).

1. Cell Seeding for Pseudotyped Particle Production
NOTE: Perform this step in the biosafety cabinet.
<ol>
	<li>By standard cell culture techniques, obtain an 80&#8211;90% confluent 75 cm2 flask of HEK-293T/17 cells passaged in complete Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM-C) containing 10% (vol/vol) fetal bovine serum (FBS), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 IU/mL penicillin, and 100 &#181;g/mL streptomycin. Prepare DMEM-T medium for transfections (its composition is the same as DMEM-C but without antibiotics).</li>
	<li>Wash cells with 10 mL of pre-warmed (37 &#176;C) Dulbecco&#39;s Phosphate Buffered Saline (DPBS) twice. 
	NOTE: Handle HEK293T/17 cells with care as they easily detach.</li>
	<li>Aspirate the supernatant and detach cells with 1 mL of 0.25% trypsin solution pre-warmed at 37 &#176;C. Place the flask of cells at 37 &#176;C, 5% CO2 incubator for 3&#8722;5 min or until cells start detaching. 
	NOTE: Avoid incubating cells with trypsin for more than 5 min as this typically leads to cell clumping.</li>
	<li>Deactivate trypsin by adding 4 mL of DMEM-C medium and count cells using a cell counting slide and light microscope. 
	NOTE: To avoid having to count too many cells, an additional dilution step may be required beforehand. Remember to factor in this dilution when calculating the actual cell density of trypsinized cells.</li>
	<li>Dilute cells to 5 x 105 cells/mL with DMEM-C.</li>
	<li>Seed 6-well tissue culture plate with 2 mL of cell solution per well and gently move the plate back and forth and side to side to evenly distribute cells, avoiding circular motion. 
	NOTE: This is a key step. Evenly distributed cells will ensure that cells do not clump at the center of wells. In turn, this will ensure good transfection efficiencies and pseudotyped particle production.</li>
	<li>Incubate the plate overnight (16&#8211;18 h) in a 37 &#176;C, 5% CO2 cell culture incubator.</li>
</ol>
2. Three-plasmid Co-transfection
NOTE: Perform this step in the biosafety cabinet.
<ol>
	<li>Observe cells under an inverted light microscope to check for the cell morphology and density. 
	NOTE: Ideally, cell density should be in the 40&#8211;60% confluency range. It is critical that cells are neither too confluent (80&#8722;90% confluent) nor too sparsely distributed (20&#8722;30% confluent) in each well. A cell density of 40&#8722;60% confluency will ensure good pseudotyped particles production.</li>
	<li>Plasmids mix
	<ol>
		<li>Calculate the plasmid mix for each envelope glycoprotein following the quantities for one well of a 6-well plate shown in Table 1. Multiply quantities if transfecting several wells and include an extra well to avoid running out of mix. 
		NOTE: Along with the SARS-CoV S and MERS-CoV S encoding plasmids, include empty vector control for the generation of negative control particles which lack viral envelope glycoproteins (&#916;env particles) along with a positive control glycoprotein such as vesicular stomatitis virus (VSV) G glycoprotein that is known to robustly infect a very wide range of cells (VSV-Gpp). Plasmids are available upon request.</li>
		<li>Mix calculated volumes of plasmids and reduced serum cell culture medium (see Table of Materials) in a microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Lipid-based transfection reagent mix (see Table of Materials)
	<ol>
		<li>Calculate the volumes for the transfection reagent mix from the quantities shown in Table 2 for one well (1:3 transfection ratio, multiply quantities as needed). Include extra wells to avoid running out of transfection reagent mix.</li>
		<li>Mix calculated volumes of lipid-based transfection reagent (3 &#181;L per well) and reduced serum cell culture medium (47 &#181;L per well) in a microcentrifuge tube, making sure to add the transfection reagent into the reduced serum cell culture medium and not the other way around.</li>
	</ol>
	</li>
	<li>Incubate both mixes (for one well: 50 &#181;L of plasmids mix and 50 &#181;L of lipid-based transfection reagent mix) separately for 5 min at room temperature.</li>
	<li>Add the contents of the transfection reagent mix to the plasmids mix at a 1:1 ratio (for 1 well: 50 &#181;L of each mix)</li>
	<li>Perform thorough up-down pipetting of the resulting mix.</li>
	<li>Incubate the mix for at least 20 min at room temperature.</li>
	<li>Aspirate the spent medium of cells.</li>
	<li>Add gently 1 mL of pre-warmed (37 &#176;C) reduced serum cell culture medium per well.</li>
	<li>Add dropwise 100 &#181;L of transfection mix to each well. 
	NOTE: Exercise care when adding the transfection mix to wells of HEK-293T as they detach easily.</li>
	<li>Incubate cells in a 37 &#176;C, 5% CO2 cell culture incubator for 4&#8211;6 h.</li>
	<li>Add 1 mL per well of pre-warmed (37 &#176;C) DMEM-T medium, which does not contain antibiotics 
	NOTE: This is a key step. Transfection reagents increase cell permeability and increase sensitivity to antibiotics. To ensure good transfection efficiency and pseudotyped particle production, it is important to avoid using cell culture medium containing antibiotics</li>
	<li>Incubate cells in a 37 &#176;C, 5% CO2 cell culture incubator for 48 h.</li>
</ol>
3. Pseudotyped Particles Collection
NOTE: Perform this step in the biosafety cabinet.
<ol>
	<li>Observe cells under inverted light microscope to check for cell morphology and general condition. Also check the color of the medium which should be light pink/slightly orange. 
	NOTE: This is an important step. If there is too much cell death associated with the transfection or the medium color turned orange/yellow (acidic pH), this will typically be associated with lower yields in infectious pseudotyped particles</li>
	<li>Transfer supernatants of transfected cells to 50 mL conical centrifuge tubes.</li>
	<li>Centrifuge tubes at 290 x g for 7 min to remove cell debris.</li>
	<li>Filter clarified supernatants through a sterile 0.45 &#181;m pore-sized filter.</li>
	<li>Make small volume aliquots (e.g., 500 &#181;L or 1 mL) of pseudotyped virus solution in cryovials.</li>
	<li>Store at -80 &#176;C. 
	NOTE: The protocol can be paused here. Pseudotyped particles are stable at -80 &#176;C for many months but once thawed, avoid re-freezing them as they will lose infectivity</li>
</ol>
4. Pseudotyped Particle Infection of Susceptible Cells
NOTE: Perform this step in the biosafety cabinet.
<ol>
	<li>Cell seeding of susceptible cells in 24-well plate
	<ol>
		<li>Obtain by standard cell culture techniques 80&#8722;90% confluent 75 cm2 flask of susceptible cells: Vero-E6 cells for SARS-CoV pseudotyped particles (SARS-Spp) and Huh-7 cells for MERS-CoV S pseudotyped particles (MERS-Spp). 
		NOTE: To confirm whether the pseudotyped particles that have been produced are infectious, it is important to carefully choose an appropriate susceptible cell line for pseudovirion infectivity assays. Using poorly permissive cells will lead to low infectivity.</li>
		<li>Wash cells twice with 10 mL of pre-warmed (37 &#176;C) DPBS.</li>
		<li>Aspirate the supernatant and detach cells with 1 mL of 0.25% trypsin solution pre-warmed at 37 &#176;C. Place the flask of cells at 37 &#176;C, 5% CO2 incubator for 3&#8722;5 min or until cells start detaching. 
		NOTE: Avoid incubating cells with trypsin for more than 5 minutes as this typically leads to cell clumping. Huh-7 cells are especially sensitive to this effect.</li>
		<li>Deactivate trypsin by adding DMEM-C medium and count cells using a cell counting slide and light microscope.</li>
		<li>Dilute cells to 5 x 105 cells/mL with DMEM-C.</li>
		<li>Seed wells of a 24-well plate with 0.5 mL of cell solution per well and gently move the plate back and forth and side to side to evenly distribute cells, avoiding circular motion 
		NOTE: This is a key step. Evenly distributed cells will ensure that cells do not clump at the center of wells. In turn, this will ensure good infectivity assays. For each pseudotyped particle (SARS-Spp, MERS-Spp) and condition, prepare three wells for three experimental replicates. Include wells for the non-infected (N.I.), empty vector &#916;env particles and positive control particles such as VSV-Gpp</li>
		<li>Incubate the plate overnight (16&#8722;18 h) in a 37 &#176;C, 5% CO2 cell culture incubator</li>
	</ol>
	</li>
	<li>Pseudotyped particle infection
	<ol>
		<li>Observe cells under light microscope and visually confirm that there is a confluent carpet of cells.</li>
		<li>Bring cryovials of pseudotyped virus to thaw on ice.</li>
		<li>Wash cells three times with 0.5 mL pre-warmed (37 &#176;C) DPBS 
		NOTE: This is a key step. Cells that are not properly rinsed prior to infection typically lead to poor infectivity readouts</li>
		<li>Aspirate the supernatants of cells.</li>
		<li>Inoculate cells with 200 &#181;L of thawed pseudotyped particle solution.</li>
		<li>Incubate cells in a 37 &#176;C, 5% CO2 cell culture incubator for 1&#8722;2 h.</li>
		<li>Add 300 &#181;L of pre-warmed (37 &#176;C) DMEM-C medium.</li>
		<li>Incubate cells in a 37 &#176;C, 5% CO2 cell culture incubator for 72 h.</li>
	</ol>
	</li>
</ol>
5. Infectivity Quantification by Luciferase Assay Readout
NOTE: Perform initial steps in the biosafety cabinet.
<ol>
	<li>Thaw luciferin substrate (stored at -80 &#176;C) and 5x luciferase assay lysis buffer (stored at -20 &#176;C) until they reach room temperature.</li>
	<li>Dilute luciferase assay lysis buffer to 1x with sterile water.</li>
	<li>Aspirate supernatants of cells infected with pseudotyped particles.</li>
	<li>Add 100 &#181;L of 1x luciferase assay lysis buffer to each well.</li>
	<li>Place the plate on a rocker and incubate for 15 min with rocking at room temperature (from this point onwards the plate can be handled outside of a biosafety cabinet).</li>
	<li>Prepare microcentrifuge tubes for each well by adding 20 &#181;L of luciferin substrate in each tube.</li>
	<li>Turn on the luminometer.</li>
	<li>Perform luciferase activity measurement one well at a time by transferring 10 &#181;L of lysate to one tube containing 20 &#181;L of luciferin substrate.</li>
	<li>Flick the tube gently to mix contents, but avoid displacing the liquid on walls of tube.</li>
	<li>Place the tube in device and close lid.</li>
	<li>Measure the luminescence value of the tube by using the luminometer.</li>
	<li>Record the relative light unit&#39;s measurement.</li>
	<li>Repeat steps 5.8&#8211;5.12 until all wells are analyzed. 
	NOTE: With the appropriate equipment such as a plate reading luminometer, this process can be performed automatically. The assay will need to be scaled to the plate format (e.g., 96-well plate format).</li>
</ol>
6. Data Analysis
<ol>
	<li>Calculation and plotting of relative luciferase units&#39; averages and standard deviations
	<ol>
		<li>Use a graph plotting software to calculate luciferase assay measurement averages and standard deviations of experimental and biological replicates.</li>
		<li>Plot data as bar chart with standard deviations. 
		NOTE: When performing statistical analyses on data, make sure to include at least three biological replicates in data sets.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

This protocol describes a method to efficiently produce pseudotyped particles bearing the S protein of risk group 3 coronaviruses, SARS-CoV and MERS-CoV, in a BSL-2 setting. These particles, which incorporate a luciferase reporter gene, enable us to easily quantify coronavirus S-mediated entry events by a relatively simple luciferase assay48,49,50,51. In infectivity assays using permissive cells, we confirm that the luciferase activity measured is dependent on the concentration of particles. In addition, ACE2 and DPP4 receptor transfection allows for more efficient entry in poorly permissive cells lines, such as HEK-293T cells. The method is highly adaptable to other viral envelope glycoproteins and has been used extensively48,49,50,51,52,53,55,56,57,58,59, often to complement other assays like biochemical analyses or native virus infections.
The protocol we describe here is based on the retrovirus MLV that incorporates a luciferase reporter. However, it is important to emphasize that there is a very wide array of other pseudotyping systems that have been successfully developed for packaging coronavirus S12,13,25,26,30,31,32 and other viral envelope glycoproteins10,11,14,16,17,23,24,29,33,38,40,42,44,46. Some of these other systems are based on the commonly used MLV retroviral core7,8,9,10,11,12,13,14,15,16,17,18,19,20, or based on the widely used lentiviral HIV-1 pseudotyping system using different strategies23,24,25,26,27,28,29,30,31,32,33,34,35, or with the rhabdovirus vesicular stomatitis virus (VSV) as core, which allows to incorporate a wide variety of envelope glycoproteins, and again with various strategies employed37,38,39,40,41,42,43,44,45,46,47. In addition, other reporters such as fluorescent proteins like GFP11,13 and RFP36, or enzymes other than luciferase like &#946;-galactosidase16,17 and secreted alkaline phosphatase (SEAP)42 have been successfully employed for measurements. Furthermore, in the assay presented in this protocol, a transient transfection was used to express the MLV and CoV S genes and proteins. However, there are other strategies for expression, such as generation of stable cell lines for production of pseudotyped viruses7,14. As each of these systems have their advantages and disadvantages, it is important to consider the following important parameters when deciding which pseudotyping system best suits an investigator&#39;s needs: pseudovirion core (MLV, HIV-1, VSV or others), how selective a particular pseudotyping core is in incorporating a specific viral envelope glycoprotein, reporter for assay readout (GFP, luciferase, SEAP or other), and the transfection strategy (number of plasmids involved in co-transfection, transient transfection or generation of stable cell lines).
There are a number of critical steps in the method that are important to emphasize. Cell density, particularly of the HEK-293T/17 producer cell line is a critical factor in ensuring successful transfection. A cell density in the range of 40&#8211;60% confluency was found to be optimal. Higher densities typically result in low transfection efficiencies and low particle production. Also, it is important to keep in mind that HEK-293T/17 cells are less adherent than other cell lines. Care should be exercised when handling them to avoid detaching them unnecessarily. One option is to treat cell culture plastic surfaces with poly-D-lysine to enhance adherence. Furthermore, higher cell passage often results in poor transfection rates. After adding the transfection reagent to HEK-293T/17 cells, it is also important to remember that cell permeability increases. This is why at this point it is best to avoid using medium containing antibiotics as they may increase cytotoxicity. Before collecting pseudotyped particles, check the color of transfected HEK-293T/17 cell supernatants. Typically, after 48 h of transfection, the cell culture medium color takes an orange-pink tinge. Yellow-colored medium usually translates to poor pseudotyped particles yields and is often a result of issues with cell seeding density or high passage number.
In this protocol, pseudotyped particle production is performed in a 6-well plate format. To increase volume of produced particles, several wells of a 6-well plate can be transfected with the same plasmids mix and the supernatants can be pooled together. The pool can then be clarified, filtered and aliquoted. Alternatively, to scale production up, other kinds of vessels (e.g., 25, or 75 cm2 flasks) can be used. In this case, transfection conditions should be scaled up accordingly. In this protocol, the infectivity assay is performed using a 24-well plate format and a luminometer that only allows measurements one tube at a time. For high throughput screenings, other formats are also possible, such as 96-well plate format and a plate reader luminometer. Volumes and reagents for the luciferase assay need to be adapted accordingly. Storage of pseudotyped particles in cryovials at -80 &#176;C maintains their stability for several months without noticeable decrease in infectivity. It is not recommended to subject them to freeze-thaw cycles as this will decrease their infectivity over time. Thus, it is best to store them in small aliquots such as 0.5&#8211;1 mL and thaw them before an infection.
The method presented here has several limitations. An important one is the fact that pseudotyped particles recapitulate only viral entry events. To analyze other steps in the infectious life cycle, other assays are required. Furthermore, as MLV particles bud at the plasma membrane, it is important to bear in mind that the envelope glycoprotein being studied needs to also traffic to the plasma membrane for incorporation into pseudovirions during production. As such, it is important to know where in the cell a particular viral envelope glycoprotein is expressed in transfection conditions, such as by visualizing subcellular localization with an immunofluorescence assay, and/or by checking for retention signals within the protein. Also, while the protocol describes steps to produce and test infectivity, it does not detail how to measure incorporation of viral envelope glycoproteins into pseudotyped particles. One method is to perform western blot assays on concentrated solutions of particles, as previously described50,51 for MERS-CoV S incorporation. In these assays, the S envelope glycoprotein of MERS-CoV is probed along with the capsid (p30) protein of MLV, which allow us to normalize incorporation of the S protein into particles. Other examples of such assays analyzing viral envelope glycoprotein incorporation into pseudovirions have been performed for SARS-CoV S incorporation in an HIV-1 lentiviral pseudovirion system32 , Ebola glycoprotein (GP) in another MLV pseudotyped particle system17, and influenza hemagglutinin (HA) and neuraminidase (NA) in VSV pseudovirions38. A recent development in characterizing pseudotyped particle production is the use of innovative imaging devices such as Nanosight: it enables us to directly visualize, quantify, and size viral particles50. The device provides detailed information on overall particle production; however, it is important to keep in mind that it does not provide information on envelope glycoprotein incorporation. A future direction for the application of these versatile pseudovirion particles is to analyze individual viral fusion events using single particle tracking, microfluidics and total internal reflection fluorescence microscopy60,61,62. Such approaches were successfully applied to influenza virus and feline coronavirus particles as well as influenza HA- and NA-pseudotyped VSV-based pseudovirions63. The deployment of such techniques applied to coronavirus S-pseudotyped MLV-based particles is currently being developed.

Host cell entry constitutes the initial steps of the viral infectious life cycle. For enveloped viruses, this involves binding to a single host cell receptor or several receptors, followed by fusion of viral and cellular membranes. These essential functions are carried out by viral envelope glycoproteins1,2. The coronavirus envelope glycoprotein is called the spike (S) protein and is a member of the class I viral fusion proteins2,3,4,5,6. Studying viral envelope glycoproteins is critical for understanding many important characteristics of a given virus, such as: lifecycle initiation, its host and cellular tropism, interspecies transmission, viral pathogenesis, as well as host cell entry pathways. Viral pseudotyped particles, also named pseudovirions, are powerful tools that enable us to easily study the function of viral fusion proteins. Pseudotyped particles or pseudovirions are chimeric virions that consist of a surrogate viral core with a heterologous viral envelope protein at their surface. The protocol&#39;s main purpose is to show how to obtain coronavirus spike pseudotyped particles that are based on a murine leukemia virus (MLV) core and contain a luciferase reporter gene. As examples, the method to produce pseudotyped particles with the spike proteins of the highly pathogenic severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses are presented. The protocol describes the transfection procedure involved, how to infect susceptible target cells, and infectivity quantification by luciferase assay.
Since the entry steps of the pseudovirions are governed by the coronavirus S at their surface, they enter cells in a similar fashion to native counterparts. As such, they are excellent surrogates of functional infectivity assays. Pseudotyped particles are usually derived from parental model viruses such as retroviruses (MLV7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22 and the lentivirus human immunodeficiency virus &#8212; HIV23,24,25,26,27,28,29,30,31,32,33,34,35) or rhabdoviruses (vesicular stomatitis virus &#8212; VSV36,39,40,41,42,43,44,45,46,47). When used in pseudotyping, the parental viruses&#39; genomes are modified to remove essential genes, rendering them defective for accomplishing a complete replication cycle. This feature allows them to be used in intermediate biosafety level facilities (BSL-2) and is an important advantage over using highly pathogenic native viruses that require higher biosafety facilities (BSL-3, BSL-4 which are not as readily available) when conducting virus entry studies. Here, the S proteins of risk group 3 pathogens SARS-CoV and MERS-CoV are used as examples of viral envelope proteins being incorporated into MLV pseudotyped particles, generating SARS-CoV S and MERS-CoV S pseudovirions (SARS-Spp and MERS-Spp, respectively). These pseudovirions have been successfully used in studies focusing on entry events of these viruses48,49,50,51. Another advantage is that the technique described here is not limited to pseudotyping coronavirus S proteins: it is very flexible and can be used to incorporate representatives of all three classes of viral fusion proteins. Examples include influenza hemagglutinin (HA, class I)52, Ebola virus glycoprotein (GP class I), E1 protein of the alphavirus Semliki Forest virus (SFV, class II), and VSV glycoprotein (G, class III)53. In addition, more than one kind of viral glycoprotein can be co-incorporated into a pseudotyped particle, as in the case of influenza HA- and NA- pseudotyped particles51.
Based on the work performed by Bartosch et al.20, this protocol describes the generation of MLV pseudotyped particles with a three-plasmid co-transfection strategy using the widely available and highly transfection-competent HEK-293T cell line54. The first plasmid encodes the MLV core genes gag and pol but lacks the MLV envelope env gene. The second plasmid is a transfer vector that encodes a firefly luciferase reporter gene, an MLV &#936;-RNA packaging signal, along with 5&#39;- and 3&#39;-flanking MLV long terminal repeat (LTR) regions. The third plasmid encodes the fusion protein of interest, in this case either the SARS-CoV S or MERS-CoV S protein. Upon co-transfection of the three plasmids using a transfection reagent, viral RNA and proteins get expressed within transfected cells allowing generation of pseudotyped particles. Since MLV is used as pseudovirion backbone, this occurs at the plasma membrane: the RNAs containing the luciferase gene reporter and packaging signal get encapsulated into nascent particles that also incorporate plasma membrane-expressed coronavirus spike proteins. The particles that bud out from cells contain the coronavirus S protein at their surface and are harvested for use in infectivity assays. Because pseudotyped particles harbor the coronavirus S protein and not the MLV envelope protein, when used for infecting cells, they behave like their native coronavirus counterparts for entry steps. The viral RNA containing the luciferase reporter and flanking LTRs is then released within the cell and the retroviral polymerase activities enable its reverse transcription into DNA and integration into the host cell genome. Quantification of the infectivity of viral pseudotyped particles in infected cells is then performed with a simple luciferase activity assay. Because the DNA sequence that gets integrated into the host cell genome only contains the luciferase gene and none of the MLV or coronavirus protein-encoding genes, they are inherently safer to use than replication-competent native viruses.
In addition to being safer surrogates and highly adaptable to allow incorporation of various kinds of envelope glycoproteins, the pseudotyped particles described here are also highly versatile and can be used to study many aspects of virus entry. This includes but is not limited to: testing host cell susceptibility to virus infection, analyzing the cellular entry pathways an enveloped virus uses, studying the effects of pharmacological inhibitors and drug screenings, conducting neutralization antibody assays, characterizing host cell entry of enveloped viruses that cannot be cultured, and generating viral vectors for gene delivery, stable cellular expression of genes of interest, or gene silencing.

<table><tbody><tr><td>Human embryonic kidney (HEK) HEK-293T/17 cells&amp;nbsp;</td><td>ATCC</td><td>CRL-11268</td><td>Clone 17 cells are highly competent for transfection.</td></tr><tr><td>African green monkey kidney epithelial Vero-E6 cells</td><td>ATCC</td><td>CRL-1586</td><td></td></tr><tr><td>Human hepatic Huh-7 cells&amp;nbsp;</td><td>Japan National Institutes of Biomedical Innovation, Health and Nutrition</td><td>JCRB0403</td><td></td></tr><tr><td>Inverted light microscope with 10 &amp;times; objective&amp;nbsp;</td><td>Nikon</td><td>TS100</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modification of Eagle&#039;s medium (DMEM) with 4.5 g/L glucose, L-glutamine without sodium pyruvate</td><td>Corning Mediatech</td><td>10-017-CV</td><td></td></tr><tr><td>Heat-inactivated fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific, Gibco</td><td>1614071</td><td></td></tr><tr><td>1 M N-2-hydroxyethylpiperazine-N&#039;-2-ethanesulphonic acid (HEPES)&amp;nbsp;</td><td>Corning Mediatech</td><td>25-060-Cl</td><td></td></tr><tr><td>100 &amp;times; penicillin-streptomycin (PS) solution</td><td>Corning Mediatech</td><td>30-002-Cl</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS) with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Corning Mediatech</td><td>21-030-CV</td><td></td></tr><tr><td>0.25% trypsin, 2.21 mM ethylenediaminetetraacetic acid (EDTA) 1 &amp;times; solution&amp;nbsp;</td><td>Corning Mediatech</td><td>25-053-Cl</td><td></td></tr><tr><td>Cell counting slides with grids&amp;nbsp;</td><td>Kova</td><td>87144</td><td></td></tr><tr><td>Opti-minimal essential medium (Opti-MEM)</td><td>Thermo Fisher Scientific, Gibco</td><td>31985-070</td><td></td></tr><tr><td>Lipofectamine 2000 transfection reagent</td><td>Thermo Fisher Scientific, Invitrogen</td><td>11668-027</td><td></td></tr><tr><td>0.45 &amp;micro;m pore-size sterile filter&amp;nbsp;</td><td>Pall</td><td>4184</td><td></td></tr><tr><td>10 mL syringes&amp;nbsp;</td><td>BD</td><td>309604</td><td></td></tr><tr><td>5 &amp;times; luciferase assay lysis buffer&amp;nbsp;</td><td>Promega</td><td>E1531</td><td></td></tr><tr><td>Luciferin, substrate for luciferase assay</td><td>Promega</td><td>E1501</td><td></td></tr><tr><td>Sterile water&amp;nbsp;</td><td>VWR</td><td>E476-1L</td><td></td></tr><tr><td>GloMax 20/20 luminometer&amp;nbsp;</td><td>Promega</td><td>2030-100</td><td></td></tr></tbody></table>

production of pseudotyped particles to study highly pathogenic coronaviruses in a biosafety level 2 setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Representative results of infectivity assays of SARS-CoV S and MERS-CoV S pseudotyped particles are shown in Figure 1. As expected, for both Figure 1A and 1B, the VSV G pseudotyped positive control particles (VSV-Gpp) gave very high average infectivity in the 106 to 107 relative luciferase units (RLU) range respectively. For SARS-CoV S pseudotyped particles (Figure 1A) infection of susceptible Vero-E6 cells, a strong average infectivity was measured at around 9.8 x 104 RLU. This value is almost 3 orders of magnitude higher than the values measured for the non-infected control (1.1 x 102 RLU), or the &#916;env particles (1.5 x 102 RLU), which do not harbor any viral envelope glycoproteins at their surface. Similarly, for MERS-CoV S pseudotyped particles (Figure 1B) infection of Huh-7 cells, a high average infectivity was measured at around 1.0 x 106 RLU. This is almost 4 orders of magnitude higher than the values measured for the non-infected control (0.8 x 102 RLU), or the &#916;env particles (2.0 x 102 RLU). An additional infectivity assay was performed in which the SARS-Spp and MERS-Spp were serially diluted and used to infect Vero-E6 cells (Figure 2A). This assay confirms that the luciferase activity measured is dependent on the concentration of the particles used to infect cells. To confirm the role of angiotensin converting enzyme 2 (ACE2) and dipeptidyl peptidase 4 (DPP4), the receptors of SARS-CoV and MERS-CoV, respectively, in mediating attachment and entry of the pseudotyped particles, we used poorly-permissive HEK-293T cells and transfected them to express either ACE2 or DPP4 (Figure 2B). The transfected cells were then used for an infectivity assay. This analysis demonstrates that upon overexpression of ACE2 and DPP4, there is a ~4-log and ~2-log increase for SARS-Spp and MERS-Spp infectivity, respectively, confirming that receptor usage of pseudovirions is similar to that of native viruses.
The examples shown here demonstrate the importance of including negative (non-infected, &#916;env particles) as well as positive control (VSV-Gpp) conditions when producing pseudotyped particles. Indeed, the positive control VSV-Gpp particles allow us to assess whether a particular batch of pseudotyped particles was successful in yielding functional and infective pseudovirions. Expected results of typical infection by VSV-Gpp particles in most mammalian cell lines are in the 106&#8722;107 RLU range. Problems with HEK-293T/17 producer cells (high passage number, issues with cell densities) or poor transfection efficiencies can impact overall pseudotyped particle production and infectivity. Furthermore, the negative control conditions are also important as they allow us to assess the baselines of RLU measurements in a particular cell line (non-infected condition) and non-specific internalization of particles (&#916;env infection) which is not mediated by a viral envelope protein. Ideally, for a given type of particle pseudotyped with a viral envelope protein of interest, it is recommended to obtain values that are a few orders of magnitude higher than the negative control values, as shown here in Figure 1A,B. However, if for a given cell type a pseudotyped virus infection gives very little infectivity (i.e., close to negative controls such as in Figure 2B in non-transfected N.T. conditions) it does not necessarily mean that the pseudotyped particle production was faulty. It could well be that the particular cell line used is not or poorly permissive to infection. It is recommended to check whether a given cell type is expected to be susceptible to infection by the virus being investigated. Transfecting poorly permissive cells with plasmid(s) expressing the viral receptor(s) can allow more efficient viral entry and infection to occur, as shown in Figure 2B, where upon transfection of ACE2 and DPP4 receptors in target HEK-293T cells, there is a ~4- and ~2-log increase in infectivity, respectively.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Plasmid/reagent</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>pCMV-MLVgagpol MLV gag and pol encoding plasmid</td>
			<td>300 ng</td>
		</tr>
		<tr>
			<td>pTG-Luc transfer vector with luciferase reporter</td>
			<td>400 ng</td>
		</tr>
		<tr>
			<td>pcDNA-SARS-S, pcDNA-MERS-S or empty vector</td>
			<td>300 ng</td>
		</tr>
		<tr>
			<td>Reduced serum cell culture medium</td>
			<td>To 50 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Quantities of plasmids and reduced serum cell culture medium required to transfect one well of a 6-well plate of HEK-293T/17 cells for pseudotyped particle production. From the concentration of plasmid preparations, calculate the required volume to reach the required amount of plasmid DNA. If more than one well is transfected with the same plasmids, multiply volumes by required number of wells to transfect, and include an extra well in calculations to avoid running out of mix in later steps. The total amount of DNA being transfected is 1 &#181;g/well. Plasmids are available upon request to authors.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>Transfection reagent</td>
			<td>3 &#181;L</td>
		</tr>
		<tr>
			<td>Reduced serum cell culture medium</td>
			<td>47 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 2: Quantities of transfection reagent and reduced serum cell culture medium required to transfect one well of a 6-well plate of HEK-293T/17 cells for pseudotyped particle production. Multiply volumes by required number of wells to transfect and include extra wells in calculations to avoid running out of mix in later steps. The transfection reagent: plasmid DNA ratio used is 3:1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59010/59010fig1.jpg" /> 
Figure 1: Coronavirus S-pseudotyped particle infectivity assays in susceptible host cells using a murine leukemia virus (MLV) backbone and luciferase reporter gene. (A) SARS-CoV S pseudotyped particle infectivity assay in Vero-E6 cells. (B) MERS-CoV S pseudotyped particle infectivity assay in Huh-7 cells. For both (A) and (B), plotted data corresponds to the average relative luciferase units from three independent experiments, with error bars corresponding to standard deviation (s.d.). Data plotted in log10 scale on y-axis. N.I.: non-infected control; &#916;env: infection with pseudotyped particles lacking viral envelope glycoproteins and VSV-Gpp: infection with pseudotyped particles bearing positive control VSV G envelope glycoprotein. Other abbreviation used, SARS-Spp: infection with SARS-CoV S pseudotyped particles, MERS-Spp: infection with MERS-CoV S pseudotyped particles. <a href="https://www.jove.com/files/ftp_upload/59010/59010fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59010/59010fig2.jpg" /> 
Figure 2: Concentration-dependence of CoV S-pseudovirion infectivity and role of ACE2 and DPP4 receptors in SARS-Spp and MERS-Spp entry. (A) Concentration-dependence of SARS-Spp and MERS-Spp infectivity measured by luciferase activity assay. Pseudovirions were serially diluted and used to infect Vero-E6 cells. (B) Infectivity assay of SARS-Spp and MERS-Spp in poorly permissive HEK-293T target cells transfected to express ACE2, and DPP4 receptors, respectively. Data plotted in log10 scale on y-axis as in Figure 1 from duplicate experiments, with error bars corresponding to standard deviation (s.d.). N.T.: non-transfected control HEK-293T cells; ACE2: angiotensin converting enzyme 2 (SARS-CoV receptor) and DPP4: dipeptidyl peptidase 4 (MERS-CoV receptor). Other abbreviations used are the same as in Figure 1. <a href="https://www.jove.com/files/ftp_upload/59010/59010fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting at JoVE.com

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

production-pseudotyped-particles-to-study-highly-pathogenic

Research

JoVE Journal

Immunology and Infection

58.9K Views.  Cornell University. Dit protocol stelt onderzoekers in staat om pseudotypevirussen te genereren die veilig kunnen worden gebruikt om virale instapgebeurtenissen van hoogpathogene virussen te bestuderen, zoals sal coronavirus en de meeste coronavirus. Deze veelzijdige techniek is gebaseerd op de eenvoudige setup tijdelijke transfectie. Dit systeem kan ook worden toegepast op deeltjes pseudotyped met fusie-eiwitten van andere virussen te produceren, niet alleen coronavirus S.Voor de beste resultaten is het belangr...

Video: Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

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.

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 ...

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

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. Occasionally, humans also fall victim to it, which results in high mortality. Nonetheless, HPAI virus research is often hindered, considering that it must be handled within biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses prove to be the ideal tools to study neutralizing antibodies against H5 HPAI viruses. This protocol describes the procedures and critical steps of H5 HPAI pseudovirus preparations and pseudovirus neutralization assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays.

Here we describe a protocol for pseudovirus packaging and the measurement of antibody neutralizing activity.

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. ...

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 ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Biology

Multiplex RT-qPCR Assay for Respiratory Virus Detection

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public&#39;s health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses 

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

Samenvatting

Meer video's verkennen