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 border="0" cellpadding="0" cellspacing="0" style="width:1213px;" width="1212">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:419px;">Human embryonic kidney (HEK) HEK-293T/17 cells&#160;</td>
			<td style="width:287px;">ATCC</td>
			<td style="width:155px;">CRL-11268</td>
			<td style="width:353px;">Clone 17 cells are highly competent for transfection.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">African green monkey kidney epithelial Vero-E6 cells</td>
			<td>ATCC</td>
			<td>CRL-1586</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human hepatic Huh-7 cells&#160;</td>
			<td>Japan National Institutes of Biomedical Innovation, Health and Nutrition</td>
			<td>JCRB0403</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inverted light microscope with 10 &#215; objective&#160;</td>
			<td>Nikon</td>
			<td>TS100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#8217;s modification of Eagle&#39;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 height="40">
			<td height="40" style="height:40px;">Heat-inactivated fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>1614071</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1 M N-2-hydroxyethylpiperazine-N&#39;-2-ethanesulphonic acid (HEPES)&#160;</td>
			<td>Corning Mediatech</td>
			<td>25-060-Cl</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100 &#215; penicillin-streptomycin (PS) solution</td>
			<td>Corning Mediatech</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#8217;s phosphate buffered saline (DPBS) with Ca2+ and Mg2+&#160;</td>
			<td>Corning Mediatech</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.25% trypsin, 2.21 mM ethylenediaminetetraacetic acid (EDTA) 1 &#215; solution&#160;</td>
			<td>Corning Mediatech</td>
			<td>25-053-Cl</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:419px;">Cell counting slides with grids&#160;</td>
			<td style="width:287px;">Kova</td>
			<td style="width:155px;">87144</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Opti-minimal essential medium (Opti-MEM)</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamine 2000 transfection reagent</td>
			<td>Thermo Fisher Scientific, Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.45 &#181;m pore-size sterile filter&#160;</td>
			<td>Pall</td>
			<td>4184</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10 mL syringes&#160;</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5 &#215; luciferase assay lysis buffer&#160;</td>
			<td>Promega</td>
			<td>E1531</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Luciferin, substrate for luciferase assay</td>
			<td>Promega</td>
			<td>E1501</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile water&#160;</td>
			<td>VWR</td>
			<td>E476-1L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GloMax 20/20 luminometer&#160;</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

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JoVE Journal

Immunology and Infection

58.7K Views.  Cornell University. 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.

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.

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 ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

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 ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

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 ...

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived from all sections of the GI tract (gastric, jejunum, duodenum, ileum, colon, rectum) and, upon differentiation, contain most of the cell types that are naturally found in each individual section. For example, intestinal organoids contain nutrient-absorbing enterocytes, secretory cells (Goblet, Paneth, and enteroendocrine), stem cells, as well as all lineage-specific differentiation intermediates (e.g.,&#160;early or immature cell types). The greatest advantage in using gastrointestinal tract-derived organoids to study infectious diseases is the possibility of precisely identifying which cell type is targeted by the enteric pathogen and to address whether the different sections of the gastrointestinal tract and their specific cell types similarly respond to pathogen challenges. Over the past years, gastrointestinal models, as well as organoids from other tissues, have been employed to study viral tropism and the mechanisms of pathogenesis. However, utilizing all the advantages of using organoids when employing highly pathogenic viruses represents a technical challenge and requires strict biosafety considerations. Additionally, as organoids are often grown in three dimensions, the basolateral side of the cells is facing the outside of the organoid while their apical side is facing the inside (lumen) of the organoids. This organization poses a challenge for enteric pathogens as many enteric infections initiate from the apical/luminal side of the cells following ingestion. The following manuscript will provide a comprehensive protocol to prepare human intestinal organoids for infection with enteric pathogens by considering the infection side (apical vs. basolateral) to perform single-cell RNA sequencing to characterize cell-type-specific host/pathogen interactions. This method details the preparation of the organoids as well as the considerations needed to perform this work under biosafety level 3 (BSL-3) containment conditions.

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 BSL-3 Conditions

This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology.

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived ...

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis. Although anti-inflammatory therapies may help control inflammation, they have no efficacy on fibrosis and stenosis in CD. The pathogenesis of CD is not well understood. Current studies focus mainly on delineating dysregulated gut immune response mechanisms. While CD-associated transmural inflammation, intestinal fibrosis, and luminal stenosis all represent mechanical stress to the gut wall, the role of mechanical stress in CD is not well defined. To determine if mechanical stress plays an independent pathogenic role in CD, a protocol of TNBS-induced CD-like colitis model in rodents has been developed. This TNBS-induced transmural inflammation and fibrosis model resembles pathological hallmarks of CD in the colon. It is induced by intracolonic instillation of TNBS into the distal colon of adult Sprague-Dawley rats. In this model, transmural inflammation leads to stenosis at the TNBS instillation site (Site I). Mechanical distention is observed in the portion proximal to the instillation site (Site P), representing mechanical stress but not visible inflammation. Colonic portion distal to inflammation (Site D) presents neither inflammation nor mechanical stress. Distinctive changes of gene expression, immune response, fibrosis, and smooth muscle growth at different sites (P, I, and D) were observed, highlighting a profound impact of mechanical stress. Therefore, this model of CD-like colitis will help us better understand CD&#39;s pathogenic mechanisms, particularly the role of mechanical stress and mechanical stress-induced gene expression in immune dysregulation, intestinal fibrosis, and tissue remodeling in CD.

The present protocol describes the development of a Crohn's-like colitis model in rodents. Transmural inflammation leads to stenosis at the TNBS instillation site, and mechanical enlargement is observed in the segment proximal to the stenosis. These changes allow studying mechanical stress in colitis.