Name: production of high-titer infectious influenza pseudotyped particles with envelope glycoproteins from highly pathogenic h5n1 and avian h7n9 viruses
Uploaded: 2020-01-15T12:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses at JoVE.com

This work was supported by grants from Zhejiang Provincial Medicine and Health Science and Technology Plan (Grant Numbers, 2017KY538), Hangzhou Municipal Medicine and Health Science and Technology Plan (Grant Numbers, OO20190070), Hangzhou Medical Science and Technology key Project (Grant Numbers, 2014Z11) and Hangzhou municipal autonomous application project of social development and scientific research (Grant Numbers, 20191203B134).

1. Day 1: Cell Culture and Seeding
<ol>
	<li>Cultivate human embryonic kidney (HEK) 293T/17 cells in 60 mm dishes with Dulbecco&#39;s modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin (DMEM Complete Medium, DCM) in a 37 &#176;C, 5% carbon dioxide (CO2) incubator until about 80% confluent. 
	NOTE: HEK 293T/17 low passage cells are recommended.</li>
	<li>Carefully wash the cells with 5 mL of phosphate buffered saline (PBS) 1x. 
	NOTE: Manual handling of the HEK 293T/17 cells must be very gentle, because they easily detach.</li>
	<li>Remove PBS and dissociate the cells with 1 mL of 0.25% trypsin-ethylene diamine tetraacetic acid (EDTA) solution. Place the dish in a 37 &#176;C, 5% CO2 incubator for no more than 5 min until the cells are dissociated.</li>
	<li>Deactivate trypsin by adding 5 mL of DCM. Disperse the cells into a single-cell suspension by pipetting up and down several times.</li>
	<li>Transfer the cell suspensions to a prechilled 15 mL centrifuge tube. Collect the cells by centrifugation at 250 x g for 5 min at 4 &#176;C.</li>
	<li>Decant as much of the supernatant as possible. Resuspend the cell pellet with 6 mL of DCM medium and count the cells. Dilute the cells to 1 x 106 cells/mL with DCM medium.</li>
	<li>Seed the cells into a 6 well plate with 1 mL of cell suspension per well. Gently pat the plate to evenly distribute the cells. Incubate the plate overnight (14&#8211;16 h) in a 37 &#176;C, 5% CO2 incubator.</li>
</ol>
2. Day 2: Four-plasmid Cotransfection Mediated by Lipofection
<ol>
	<li>Check the cell morphology and density under an inverted light microscope. Ideally, cells should be approximately 85% confluent at transfection. Replace the medium with 1 mL of serum-free DMEM medium per well, and then put the plate back into the incubator. 
	NOTE: Manual handling of the HEK 293T/17 cells must be very gentle, because they easily detach.</li>
	<li>For each well of cultured cells to be transfected, dilute 8 &#181;L of the transfection reagent to a volume of 150 &#181;L with Reduced Serum Medium (tube 1). Mix gently and incubate for 5 min at room temperature (RT, approximately 20 &#176;C). 
	NOTE: For each transfection sample, prepare two 1.5 mL microcentrifuge tubes, numbered 1 and 2.</li>
	<li>In tube 2, dilute 2.5 &#181;g of plasmid DNA into 150 &#181;L of Reduced Serum Medium. 
	NOTE: In tube 2, dilute each plasmid DNA as shown in Table 1. A plasmid encoding vesicular stomatitis virus (VSV) G glycoprotein (plasmid pLP-VSVG) was used as a positive control, because VSV is able to infect a wide range of cells. Negative control particles that lack influenza envelope glycoproteins (&#916;env pps) were generated using pcDNA-Gag-Pol and pcDNA-GFP plasmids.</li>
	<li>After a 5 min incubation, combine the diluted DNA with diluted transfection reagent. Mix gently and incubate for another 15 min at RT.</li>
	<li>Add the DNA-lipid complex to the corresponding well containing the cells and serum-free medium. Mix gently by rocking the plate back and forth.</li>
	<li>After incubation for 4&#8211;6 h in a 37 &#176;C, 5% CO2 incubator, remove the medium, and replace with 2 mL of DMEM. Incubate for another 36&#8211;48 h in a 37 &#176;C, 5% CO2 incubator. 
	NOTE: Replace with serum-free and antibody-free DMEM. In this protocol, two HAs and two NAs can be used to generate eight types of pps (shown in Table 1).</li>
</ol>
3. Day 3: Susceptible Cells Seeding
<ol>
	<li>For the infectivity assay, seed each type of susceptible cells at 1 x 104 cells per well in a 96 well plate. 
	NOTE: Use two types of target cell to perform the infectivity assay in this article: an alveolar-derived human cell line (A549 cells) and the Madin-Darby Canine Kidney (MDCK) cells. MDCK cells are widely used in influenza research and can be a good control. This step is flexible. Any other target cell lines can be used according to the research requirements.</li>
	<li>Incubate the plate overnight (14&#8211;16 h) in a 37 &#176;C, 5% CO2 incubator.</li>
</ol>
4. Day 4: Pseudotyped Viral Particle Collection, Quantification, and Infectivity Assay
<ol>
	<li>Pseudotyped viral particle collection
	<ol>
		<li>Check the color of the medium. Ideally, it should be light pink or slightly orange. Examine the cells with an inverted fluorescent biological microscope under 440&#8211;460 nm.</li>
		<li>At 36&#8211;48 h posttransfection, harvest the pps by passaging through a 0.45 &#181;m polyvinylidene fluoride (PVDF) membrane filter to eliminate cell debris.</li>
		<li>Divide the pps into small volume aliquots.</li>
		<li>Store the pps at -80 &#176;C. 
		NOTE: The protocol can be paused here. However, this is not encouraged, because the infectivity of the pps will sharply decline after freezing and thawing.</li>
	</ol>
	</li>
	<li>Pseudotyped viral particle quantification
	<ol>
		<li>Transfer 20 &#181;L of purified pps to a 1.5 mL ribonuclease (RNase)-free microcentrifuge tube.</li>
		<li>Add 1 &#181;L of 0.24 U/mL benzonase nuclease. Incubate at 37 &#176;C for 1 h to eliminate any DNA and RNA contamination. 
		NOTE: Target RNA, commonly downregulated cytomegalovirus (CMV)-GFP RNA, is packaged in the pps and can avoid being degraded by benzonase nuclease.</li>
		<li>Freeze the sample at -70 &#176;C to inactivate the benzonase nuclease.</li>
		<li>Add 2 &#181;L of proteinase K. Incubate at 50 &#176;C for 30 min to digest the envelope proteins and release the CMV-GFP RNA. Inactivate the proteinase K at 100 &#176;C for 3 min.</li>
		<li>Quantify the pps by real-time quantitative reverse-transcription (qRT)-PCR with a Universal Probe One-Step RT-qPCR Kit, using the forward primer 5&#39;-AACAAAAGCTGGAGCTCGTTTAA-3&#39;, the reverse primer 5&#39;-GGGTCTCCTCAGAGTGATTGACTAC-3&#39;, and the probe 5&#39;-FAM-CCCCCAAATGAAAGACCCCCGAG-TAM-3&#39;, on a Real-Time PCR thermocycler. Normalize the pps for RNA copy number before infectivity.</li>
	</ol>
	</li>
	<li>Infectivity assay
	<ol>
		<li>Dilute each type of pps in terms of the qRT-PCR data to 4 x 105 copies/mL (pp normalization).</li>
		<li>Add Tosyl-Phenylalanine Chloromethyl-Ketone (TPCK)-trypsin to a final concentration of 40 &#181;g/mL into pps that harbor H7N9 HA. Incubate at 37 &#176;C for 1 h to form its functional subunits HA1 and HA2. 
		NOTE: There is no need to treat the pps that harbor H5 with TPCK-trypsin, because they have multiple arginine and lysine residues at the HA1-HA2 cleavage site. This multi-basic cleavage site can be cleaved by ubiquitous cellular proteases.</li>
		<li>Mix normalized pps with DMEM medium (serum-free) at a 1:1 ratio (volume/volume).</li>
		<li>Bring the plate containing susceptible cells to the biosafety cabinet.</li>
		<li>Aspirate the supernatant and wash the cells once with 0.1 mL of prewarmed PBS.</li>
		<li>Add 0.1 mL of pps-DMEM mixture to one well. Triplicate the infectivity tests of each type of pps to one susceptible cell line (overviewed in Figure 2).</li>
		<li>Incubate the 96 well plate for 4&#8211;6 h in a 37 &#176;C, 5% CO2 incubator.</li>
		<li>Aspirate the supernatant and replace with 0.1 mL of DCM.</li>
		<li>Incubate the 96 well plate for another 24&#8211;36 h in a 37 &#176;C, 5% CO2 incubator.</li>
	</ol>
	</li>
</ol>
5. Day 5 or 6: Infectivity Detection
<ol>
	<li>Bring the 96 well plate to the biosafety cabinet.</li>
	<li>Aspirate the supernatant and wash the cells 1x with 0.2 mL of prewarmed PBS.</li>
	<li>Remove PBS and dissociate the cells with 0.1 mL of 0.25% trypsin-EDTA solution.</li>
	<li>Place the dish in a 37 &#176;C, 5% CO2 incubator for 3 min until the cells are dissociated. 
	NOTE: Avoid incubating for more than 5 min, because this will lead to cell clumping.</li>
	<li>Deactivate trypsin by adding 0.4 mL of DCM.</li>
	<li>Disperse the cells into single-cell suspension by pipetting up and down several times.</li>
	<li>Transfer the cell suspensions to a chilled 1.5 mL microcentrifuge tube.</li>
	<li>Determine the GFP reporter-positive cells with Fluorescence Activated Cell Sorting (FACS). 
	NOTE: To determine the ratio of GFP reporter-positive target cells, set flow cytometer gates using the control samples (pps-untreated A549 cells or MDCK cells), and then count the GFP reporter-positive cells of 10,000 cells per sample.</li>
</ol>

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(145), e59010 (2019).</li><li>Ma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+leukemia+virus+pseudotypes+of+La+Crosse+and+Hantaan+Bunyaviruses:+a+system+for+analysis+of+cell+tropism.">Murine leukemia virus pseudotypes of La Crosse and Hantaan Bunyaviruses: a system for analysis of cell tropism.</a> Virus Research. 64 (1), 23-32 (1999).</li><li>Wool-Lewis, R. J., Bates, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Ebola+virus+entry+by+using+pseudotyped+viruses:+identification+of+receptor-deficient+cell+lines.">Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines.</a> Journal of Virology. 72 (4), 3155-3160 (1998).</li><li>Chen, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+design+of+more+effective+avian+replication-incompetent+retroviral+vectors.">Production and design of more effective avian replication-incompetent retroviral vectors.</a> Developmental Biology. 214 (2), 370-384 (1999).</li><li>Kaku, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+generation+of+pseudotype-based+serum+neutralization+assay+for+Nipah+virus+antibodies:+sensitive+and+high-throughput+analysis+utilizing+secreted+alkaline+phosphatase.">Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase.</a> Journal of Virological Methods. 179 (1), 226-232 (2012).</li><li>Rudiger, D., Kupke, S. Y., Laske, T., Zmora, P., Reichl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+modeling+of+influenza+A+virus+replication+in+cell+cultures+predicts+infection+dynamics+for+highly+different+infection+conditions.">Multiscale modeling of influenza A virus replication in cell cultures predicts infection dynamics for highly different infection conditions.</a> PLOS Computational Biology. 15 (2), 1006819 (2019).</li><li>Petiot, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+viruses+production:+Evaluation+of+a+novel+avian+cell+line+DuckCelt(R)-T17.">Influenza viruses production: Evaluation of a novel avian cell line DuckCelt(R)-T17.</a> Vaccine. 36 (22), 3101-3111 (2018).</li></ol>

The authors have nothing to disclose.

In this protocol, we describe a method to produce influenza virus pseudotyped particles (pp) in a BSL-2 setting. The reporter plasmid pcDNA-GFP is incorporated into the pps and can be used to quantify pps by FACS in an infectivity assay. We chose two types of susceptible cell lines because they are widely used in influenza research. MDCK cells would provide a good control to the variable immortalized human cells used in these studies.
This protocol is based on the retrovirus MLV, which can incorporate a GFP reporter and produce buds on the cellular membrane. This technique has been widely developed for packaging influenza virus particles4,22,23,24. Some other systems are based on the lentiviral HIV-1 pseudotyping system19,25,26 or the baculovirus-insect cell expression system27,28,29. Many other reporter genes have also been used for pseudoparticle production, such as luciferase (luminescence)30,31,32,33, &#946;-gal/LacZ (by colorimetry)34,35, and secreted alkaline phosphatase36,37. Millet et al. used the luciferase assay to quantify the infectivity of coronavirus pseudotyped particles33. Compared with luciferase, GFP&#39;s green fluorescence is easily observed with an inverted fluorescent biological microscope. This is a convenient way to check transfection and infection efficiencies. For this study, the infectivity can be determined directly by detecting the GFP-positive cells with FACS.
In this method, several steps critically affect the results. Optimized cell density is a critical factor for successful transfection. In this protocol, a cell density in the range of 80&#8211;90% confluent was found to be optimal for influenza virus pp production. Higher or lower cell density will result in lower transfection efficiency. Good physiological status of producer cells is also very important for high particle production. This is why lower cell passage HEK 293T/17 cells are recommended to produce pps in this protocol. Also, the volume of transfection reagent and the ratio of the four plasmids quantities can also affect the transfection efficiency. To obtain the highest transfection efficiency, it is recommended to optimize these factors by varying and testing their amount. We set the ratio of four plasmids quantities as 16:16:1:1 and the volume of transfection reagent as 8 &#181;L for one well tranfection in a 6 well plate. Another issue is the handling of the cells. The HEK 293T/17producer cells are low-adhesion, so manual handling of the cells must be very gentle. Lipofection can lead to a cell permeability increase, and serum and antibodies in the medium may increase cytotoxicity. It is best to use serum-free and antibody-free DMEM to culture post-transfected HEK 293T/17 cells. Furthermore, collection time is important. At 36&#8211;48 h post-transfection, the color of the cell supernatant must be checked to make sure that it is pink or orange-pink before pp collection. Yellow supernatant indicates that the medium is too acidic to support cell growth and usually leads to poor pp yields.
We performed the transfection assay in a 6 well plate. To obtain more pp volume the number of transfection wells can be increased and supernatants that contain the same pps can be pooled together. Alternatively, some other kind of vessels can be used to increase pp yield. For instance, 18 &#181;L of transfection reagent and 5.5 &#181;g of plasmid DNA can be used to perform transfection with 2.2 x 106 cells in one 60 mm dish. Similarly, an infectivity assay can be carried out in a 24 well plate.
The success of this technique can be influenced by many factors. Therefore, the procedure should be optimized for particle packaging of different kinds of viruses.
In this protocol, pseudotyped viral particles of HPAI H5N1 and H7N9, as well as their reassortants, were generated. In this method, we use qRT-PCR of the GFP-RNA in the infectivity assay at the single-cell level (as genome copies per cell) instead of Tissue Culture Infectious Dose 50 (TCID50) or traditional MOI23,38,39. Virus infectivity titration methods are based on plaque assays or TCID50 assays, which are both time-consuming and labor-intensive. Both assays rely on the measurement of visible cytopathic effects (CPE) caused by virus infection in cell lines, so it may be hard to titrate viruses of low infectivity (i.e., H7pp in this study). We think it is a convenient, effective, and rapid method to normalize pps with qRT-PCR of the GFP RNA contained in influenza pps.
Taken together, our technique is safe and adaptable, and can be used to study enveloped viruses, including their receptors, tropisms, envelope glycoprotein function, neutralizing antibodies, antivirus drug development, diagnosis, and vaccine design and evaluation.

The mission of a viral particle is to transport its genome from an infected host cell to a non-infected host cell and to deliver it into the cytoplasm or the nucleus in a replication-competent form1. This process is initially triggered by binding to host cell receptors, followed by fusion of virion and cellular membranes. For enveloped viruses, like influenza viruses, the spike glycoproteins are responsible for receptor binding and fusion1,2. Viral envelope glycoproteins (e.g., pyrogens, antigens), are involved in many important properties and events, such as virus lifecycle initiation (binding and fusion), viral pathogenesis, immunogenicity, host cell apoptosis and cellular tropism, the cellular endocytic pathway, as well as interspecies transmission and reassortment1,3,4,5,6,7. Research on viral envelope glycoproteins will help us understand many aspects of the viral infection process. Pseudotyped viral particles (pp), also termed pseudovirions or pseudoparticles, can be generated through a pseudotyping technique8,9,10. This technology has been used to develop pseudotyped particles of many viruses, including hepatitis C11,12, hepatitis B13, vesicular stomatitis virus (VSV)14,15, and influenza virus16,17,18,19. This technology is based on the Gag-Pol protein of lentiviruses or other retroviruses.
Pseudotyped viral particles can be obtained using a three-plasmid system by cotransfecting a viral envelope glycoprotein expression plasmid, a retroviral packaging plasmid missing the envelope env gene, and a separate reporter plasmid into pp producer cells. The retrovirus is assembled by its Gag protein, and it buds from an infected cell membrane that expresses the virus envelope protein1. Therefore, it is possible to obtain high titer influenza pps using the retrovirus Gag protein to produce buds on a cellular membrane expressing influenza HA and NA. In our previous studies, HAs/NAs in all combinations were functional and able to perform their corresponding functions in the viral life cycle16,17,18,20,21. These pps are used to investigate influenza biological characteristics, including hemagglutination, neuraminidase activity, HA-receptor binding tropism, and infectivity. Because HA and NA are both important surface functional proteins in the viral life cycle, mismatched HAs and NAs derived from different strains of influenza can partly demonstrate reassortment between them. Here, we generate eight types of influenza pps by combining two HAs and two NAs (derived from the HPAI H5N1 strain and the H7N9 stain), using a three-plasmid pseudotyping system. These eight types of pps include two native pps, H5N1pp, H7N9pp; two mismatched pps, (H5+N9)pp, (H7+N1)pp; and four pps only harboring a single glycoprotein (HA or NA), H5pp, N1pp, H7pp, N9pp. Studies on the influenza virus, such as H5N1 and H7N9, are limited by biosafety requirements. All studies of the wild influenza virus strains should be performed in a biosafety level 3 (BSL-3) laboratory. Pseudotyped viral particle technology can be used to package an artificial virion in a biosafety level 2 (BSL-2) setting. Therefore, pps represent a safer and useful tool to study the influenza virus processes depending on its two major glycoproteins: hemagglutinin (HA) and neuraminidase (NA).
This protocol describes the generation of these pps with a three-plasmid cotransfection strategy (overviewed in Figure 1), how to quantify pps, and infectivity detection. The pp production involves three kinds of plasmids (Figure 1). The gag-pol gene, which encodes the retrovirus Gag-Pol protein, was cloned from a retrovirus packaging kit and inserted into the pcDNA 3.1 plasmid and named pcDNA-Gag-Pol. The enhanced green fluorescent protein (eGFP) gene, which encodes Green Fluorescent Protein, was cloned from pTRE-EGFP vector, inserted into the pcDNA 3.1 plasmid, and called pcDNA-GFP. During cloning, a packaging signal (&#968;) sequence was added via a primer. The HA and NA genes were cloned into a pVRC plasmid, named pVRC-HA and pVRC-NA, respectively. The last plasmid encodes the fusion protein and can be replaced with any other fusion protein of interest. Our pseudotyping platform includes two glycoprotein expression plasmids: pVRC-HA and pVRC-NA. This can simplify the research on reassortment between different virus strains in a BSL-2 setting.

<table><tbody><tr><td>Benzonase Nuclease</td><td>Millipore</td><td>70664</td><td>Effective viscosity reduction and removal of nucleic acids from protein solutions</td></tr><tr><td>Clear Flat Bottom Polystyrene TC-treated Microplates (96-well)</td><td>Corning</td><td>3599</td><td>Treated for optimal cell attachment&lt;br/&gt; Sterilized by gamma radiation and certified nonpyrogenic&lt;br/&gt; Individual alphanumeric codes for well identification</td></tr><tr><td>Clear TC-treated Multiple Well Plates (6-wells)</td><td>Costar</td><td>3516</td><td>Individual alphanumerical codes for well identification&lt;br/&gt; Treated for optimal cell attachment&lt;br/&gt; Sterilized by gamma irradiation</td></tr><tr><td>Dulbecco&#039;s modified essential medium (DMEM)</td><td>Gibco</td><td>11965092</td><td>A widely used basal medium for supporting the growth of many different mammalian cells</td></tr><tr><td>Fetal bovine serum</td><td>Excell</td><td>FND500</td><td>fetal bovine sera that can offer excellent value for basic cell culture, specialty research, and specific assays</td></tr><tr><td>Fluorescence Activated Cell Sorting (FACS)</td><td>Beckman coulter</td><td>cytoflex</td><td></td></tr><tr><td>Human alveolar adenocarcinoma A549 cells</td><td>ATCC</td><td>CRM-CCL-185</td><td></td></tr><tr><td>Human embryonic kidney (HEK) HEK-293T/17 cells</td><td>ATCC</td><td>CRL-11268</td><td>A versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines</td></tr><tr><td>Inverted fluorescent biological microscope</td><td>Olympus</td><td>BX51-32P01-FLB3</td><td></td></tr><tr><td>Inverted light microscope</td><td>Olympus</td><td>CKX31-12PHP</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Invitrogen</td><td>11668019</td><td>Rapid, sensitive and precise probe-based qPCR detection and quantitation of target RNA targets.</td></tr><tr><td>Luna Universal Probe One-Step RT-qPCR Kit</td><td>NEB</td><td>E3006L</td><td>Will withstand up to 14,000 RCF&lt;br/&gt; RNase-/DNase-free Nonpyrogenic</td></tr><tr><td>Madin-Darby Canine Kidney (MDCK) cells</td><td>ATCC</td><td>CCL-34</td><td></td></tr><tr><td>MaxyClear Snaplock Microcentrifuge Tube (1.5 mL)</td><td>Axygen</td><td>MCT-150-C</td><td>33 mm, gamma sterilized</td></tr><tr><td>Millex-HV Syringe Filter Unit, 0.45 &amp;micro;m, PVDF</td><td>Millipore</td><td>SLHV033RS</td><td>an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in cell growth rate or morphology. Opti-MEM I medium is also recommended for use with cationic lipid transfection reagents, such as Lipofectamine reagent.</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>11058021</td><td>The antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective combined action against gram-positive and gram-negative bacteria.</td></tr><tr><td>penicillin-streptomycin</td><td>Gibco</td><td>15140122</td><td>Maximum RCF is 12,500 xg&lt;br/&gt; Temperature range from -80 &amp;deg;C to 120 &amp;deg;C&lt;br/&gt; RNase-/DNase-free&lt;br/&gt; Sterile</td></tr><tr><td>PP Centrifuge Tubes (15 mL)</td><td>Corning</td><td>430791</td><td>a stable and highly reactive serine protease</td></tr><tr><td>Proteinase K</td><td>Beyotime</td><td>ST532</td><td>Treated for optimal cell attachment&lt;br/&gt; Sterilized by gamma radiation and certified nonpyrogenic</td></tr><tr><td>TC-treated Culture Dish (60mm)</td><td>Corning</td><td>430166</td><td>Trypsin from bovine pancreas&lt;br/&gt; TPCK Treated, essentially salt-free, lyophilized powder, &amp;ge;10,000 BAEE units/mg protein</td></tr><tr><td>TPCK-trypsin</td><td>Sigma</td><td>T1426</td><td>This liquid formulation of trypsin contains EDTA and phenol red. Gibco Trypsin-EDTA is made from trypsin powder, an irradiated mixture of proteases derived from porcine pancreas. Due to its digestive strength, trypsin is widely used for cell dissociation, routine cell culture passaging, and primary tissue dissociation. The trypsin concentration required for dissociation varies with cell type and experimental requirements.</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Gibco</td><td>25200056</td><td></td></tr></tbody></table>

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.

Depending on the general procedure described above, we have generated 10 types of pps combining two group HAs/NAs or VSV-G glycoprotein or no-envelope glycoproteins (shown in Table 1). Seven of them are infectious. The pps that harbor no-envelope glycoprotein or only harbor NA did not show any infectivity here. The influenza pp production procedure is overviewed in Figure 1. Transmission electron micrographs of pps (e.g., H5N1pp) are shown in Figure 3. The results of infectivity assays of these pps are shown in Figure 4. The infectivity of the seven types of pps was evaluated at a genome copies per cell (GCP) [similar to multiplicity of infection (MOI)] value of 20. In the pps group harboring H5, the infectivity of H5N1, (H5+N9), and H5 was 90.05 &#177; 4.05%, 17.78 &#177; 1.58%, 10.15 &#177; 2.85% for cell line A549 and 40.37 &#177; 4.92%, 5.24 &#177; 1.32%, 4.88 &#177; 0.27% for MDCK, respectively. In the pps group harboring H7, the infectivity of H7N9, (H7+N1), and H7 was 10.45 &#177; 2.35%, 6.75 &#177; 1.37%, 1.23 &#177; 0.33% for cell line A549 and 7.61 &#177; 1.04%, 4.12 &#177; 1.29%, 1.08 &#177; 0.02% for MDCK, respectively. For the infectivity assays of the pps, especially HApp (H5pp, H7pp), exogenous neuraminidase was not added. In this infectivity assay, the MDCK cells were also used as the control cell line to test pps infectivity. The infectivity of VSVGpp was 20.9 &#177; 2.00% for A549 and 16.02 &#177; 2.41% for MDCK cells. The delta-envelope glycoproteins pp (&#916;env pp) showed no infectivity in our study. These data also showed that HAs/NAs from diverse virus strains are capable of successfully generate infectious viral particles. Taken together, our pseudotyping platform can develop infectious pseudotyped viral particles used in influenza research.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60663/60663fig1a.jpg" /> 
Figure 1: Overview of influenza pp production procedure. (A) The packaging plasmid pcDNA-Gag-pol, the reporter plasmid pcDNA-GFP, and the envelope glycoprotein expression plasmids pVRC-HA and pVRC-NA, are cotransfected into pp-producing HEK 293/17 cells. (B) In HEK 293/17 cells, the Gag-Pol polyproteins are synthesized and transported by an unknown mechanism to the cell membrane. Glycoproteins HA and NA are transported and anchored onto the cell membrane via the secretory pathway. Reporter plasmid pcDNA-GFP is transcribed into the single-stranded &#968;-GFP genomic RNA. (C) During or after transport, the Gag-Pol protein recruits the single-stranded &#968;-GFP genomic RNA via the psi-RNA packaging signals, thereby forming the pre-budding complexes. (D) The assembled Gag-pol-&#968;-RNA complex induces membrane curvature, leading to the formation of a bud. During budding, the viral envelope glycoproteins HAs/NAs are incorporated into the nascent particles. Budding is completed as the particle pinches off from the cell membrane. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60663/60663fig1alarge.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/60663/60663fig2a.jpg" /> 
Figure 2: Overall arrangement in infectivity assay. The infectivity tests of each type of pps to one susceptible cell line were measured in triplicate. Alveolar-derived human cells A549 and MDCK are used as susceptible cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60663/60663fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60663/60663fig3a.jpg" /> 
Figure 3: Transmission electron micrographs of pps. Unconcentrated (left) and concentrated (right) supernatant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60663/60663fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60663/60663fig4a.jpg" /> 
Figure 4: Infectivity of normalized pps. Infectivity is presented as the mean &#177; standard deviation (SD) percentage of infected cells (n = 3). Infectivity of pps was assessed in two cell lines, A549 and MDCK cells. Seven types of pps displayed various infectivity profiles in two target cells. Pps that only harbor NA are not shown here as they did not manifest any infectivity. Pps that harbor no-envelope glycoproteins (&#916;env) also showed no infectivity in our study. The percent of infectivity denoted the ratio of GFP reporter-positive cells in 10,000 cells per sample. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60663/60663fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="10">plasmid preparations</td>
		</tr>
		<tr>
			<td>plasmid (&#181;L, 0.1 &#181;g/&#181;L)</td>
			<td>H5N1</td>
			<td>(H5+N9)</td>
			<td>H7N9</td>
			<td>(H7+N1)</td>
			<td>H5</td>
			<td>N1</td>
			<td>H7</td>
			<td>N9</td>
			<td>VSVG</td>
			<td>&#916;env</td>
		</tr>
		<tr>
			<td>pcDNA-Gag-pol</td>
			<td>10.53</td>
			<td>10.53</td>
			<td>10.53</td>
			<td>10.53</td>
			<td>11.43</td>
			<td>11.43</td>
			<td>11.43</td>
			<td>11.43</td>
			<td>11.43</td>
			<td>12.5</td>
		</tr>
		<tr>
			<td>pcDNA-GFP</td>
			<td>10.53</td>
			<td>10.53</td>
			<td>10.53</td>
			<td>10.53</td>
			<td>11.43</td>
			<td>11.43</td>
			<td>11.43</td>
			<td>11.43</td>
			<td>11.43</td>
			<td>12.5</td>
		</tr>
		<tr>
			<td>pVRC-HA</td>
			<td>1.98</td>
			<td>1.98</td>
			<td>1.98</td>
			<td>1.98</td>
			<td>2.14</td>
			<td>-</td>
			<td>2.14</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>pVRC-NA</td>
			<td>1.98</td>
			<td>1.98</td>
			<td>1.98</td>
			<td>1.98</td>
			<td>-</td>
			<td>2.14</td>
			<td>-</td>
			<td>2.14</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>pcDNA-VSVG</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>2.14</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 1: Combinations of HA and NA proteins derived from H5N1 and H7N9 viruses and the required quantities (volume) of plasmid DNA for one well transfection of a 6 well plate. According to the concentration of plasmid preparations, calculate the required volume of each plasmid DNA. The total amount of plasmid DNA for tranfection of one well is 2.5 &#181;g. To obtain the highest transfection efficiency and lowest non-specific effects, we optimized the transfection conditions by varying plasmid DNA and tranfection reagent concentrations. After optimizing, the ratio of the four plasmids quantities was set as 16:16:1:1. The volume of transfection reagent used in one well transfection of a 6 well plate is 8 &#181;L (not shown in the table). Two HAs and two NAs from H5N1 and H7N9 viruses can be used to generate eight types of pps: H5N1pp, (H5 + N9)pp, H5pp, N1pp, H7N9pp, (H7 + N1)pp, H7pp and N9pp. The pps that harbor no-envelope glycoproteins (&#916;env) or only harbor NA glycoproteins showed no infectivity in our study.

Watch this Scientific Journal Video about Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses at JoVE.com

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

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 ...

production-high-titer-infectious-influenza-pseudotyped-particles-with

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.2K Views.  Hang Zhou Red Cross Hospital. Deze techniek van het produceren van influenza virale PP en het bepalen van de infectiviteit kan het onderzoek van influenza virus in een BSL-2 stad te vereenvoudigen. We normaliseerden de PPS voor de infecties op basis van qRT-PCR gegevens van PPS. Twee glycoproteïne expressie plasmi zijn gecodeerd, die het onderzoek naar virus reassortment kan vereenvoudigen.Een dag na het zaaien van cellen, controleer de celmorfologie en dichtheid onder een omgekeerde lichtmicroscoop. Idealiter moe...

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

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

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 ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

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

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

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

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.

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 ...

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. ...

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

A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System

Source: Arevalo, M. T., et al.&#160; <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54041">Expression and Purification of Virus-like Particles for Vaccination.</a> J. Vis. Exp. (2016).
This video demonstrates the generation of influenza virus-like particles (VLPs) in a mammalian system. Upon expressing three eukaryotic expression vectors &#8212; encoding different viral structural proteins &#8212; in suitable mammalian host cells, the expressed viral proteins self-assemble into VLPs on the host plasma membrane and release via budding from the cell surface.

Source: Arevalo, M. T., et al.&#160; Expression and Purification of Virus-like Particles for Vaccination. J. Vis. Exp. (2016).
This video demonstrates the ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

In Vitro Models

Generating  Recombinant Live-Attenuated Influenza Viruses for Vaccines

Source: P&#233;rez-Gir&#243;n, J. V. et al, <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52803">Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity.</a>&#160;J. Vis. Exp. (2015)
The video showcases a method to create cold-adapted recombinant influenza viruses. It involves transfecting human cells with bi-directional plasmids, followed by incubation at lower temperature incubation to yield viruses suitable for vaccine production, characterized by reduced replication in warmer conditions.

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

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