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:...

<ol>
	<li>Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology (6th). , (2013).</li><li>White, J. M., Delos, S. E., Brecher, M., Schornberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+and+mechanisms+of+viral+membrane+fusion+proteins:+multiple+variations+on+a+common+theme.">Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme.</a> Critical Reviews in Biochemistry and Molecular Biology. 43 (3), 189-219 (2008).</li><li>Bright, R. 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K., Foo, H. H., Kleeman, L., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transmembrane+domains+of+Sindbis+virus+envelope+glycoproteins+induce+cell+death.">The transmembrane domains of Sindbis virus envelope glycoproteins induce cell death.</a> Journal of Virology. 72 (5), 3935-3943 (1998).</li><li>Albecka, A., Laine, R. F., Janssen, A. F., Kaminski, C. F., Crump, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSV-1+Glycoproteins+Are+Delivered+to+Virus+Assembly+Sites+Through+Dynamin-Dependent+Endocytosis.">HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.</a> Traffic. 17 (1), 21-39 (2016).</li><li>Huang, A. S., Palma, E. 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J., Johnson, L. G., Olsen, J. 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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 inc...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 
			Sterilized by gamma radiation and certified nonpyrogenic 
			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 
			Treated for optimal cell attachment 
			Sterilized by gamma irradiation</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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 
			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 &#181;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 
			Temperature range from -80 &#176;C to 120 &#176;C 
			RNase-/DNase-free 
			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 
			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 
			TPCK Treated, essentially salt-free, lyophilized powder, &#8805;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</s...

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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 ...

This technique of producing influenza viral PP and determining its infectivity can simplify the research of influenza virus in a BSL-2 city. We normalized the PPS for the infections based on qRT-PCR data of PPS. Two glycoprotein expression plasmid are encoded, which can simplify the research on virus reassortment.One day after cell seeding, check the cell morphology and density under an inverted light microscope. Ideally the cell should be approximately 85%confluent at transfection. Replace the medium with one milliliter of serum-free DMEM per well, and put the plate back in the incubator.For each well of cells to be transfected, dilute eight microliters of the transfection reagent to a volume of 150 microliters with reduced serum medium. Mix the solution gently and let it sit at room temperature for five minutes. Meanwhile, dilute 2.5 micrograms of plasmid DNA into 158 microliters of reduced serum medium.After the five minute incubation, combine the diluted DNA with diluted transfection reagent, gently mix the solution and leave it at room temperature for 15 minutes. Add the DNA lipid complex to the corresponding wells with the cells in serum-free medium. Then mix the plate gently by rocking back and forth.Incubate the plate at 37 degree Celsius and 5%carbon dioxide for four to six hours. After the incubation, remove the medium and replace it with two milliliters of DMEM per well. Then incubate it for another 36 to 48 hours.Seed each type of susceptible cell at 10, 000 cells per well and a 96 well plate. Then leave the plate over night in a 37 degree Celsius and five percent carbon dioxide incubator. On the third day after transfection, check the color of the medium which should light pink or slightly orange and examine the cells with an inverted florescent biological microscope under 440 to 460 nanometer wavelength.At 38 to 48 hours after post-transfection, harvest the pseuotyped particles or PPS by passaging them through a 0.45 micrometer polyvinylidene fluoride membrane filter to eliminate cell debris, then divide them into small volume aliquots and store them at minus 80 degree Celsius. To quantify the PPS, transfer 30 microliters of the purified viral particles to a 1.5 microliter RNase-free tube and add one microliter of Benzonase Nuclease. Incubate the tube at 37 degrees Celsius for one hour to eliminate any DNA and RNA contamination.After the incubation, freeze the sample to minus 70 degrees Celsius to active the nuclease and add two microliters of Proteinase K.Incubate the mixture for 30 minutes to digest the envelope proteins and release the CMV-GFP RNA. Then inactive the Proteinase K at 100 degrees Celsius for three minutes. Quantify the PPS with quantitative reverse transcription RTPCR using the Universal Probe One-Step RT-qPCR Kit in the primers and probes specified in the text manuscript.Normalize the PPS to four times 10 to fifth RNA copies per milliliter. Then add TPCK-trypsin to a final concentration of 10 micrograms per milliliter into the PPS that harbors H7N9 hemagglutinin. Incubate the PPS at 37 degrees Celsius for one hour to form the functional subunits HA1 and HA2.Mix the normalized PPS with DMEM medium at a one to one ratio, then bring the plate containing susceptible cells to the biosafety cabinet. Aspirate the supernatant and wash the cells once with 100 microliters of pre-warmed PBS. Add 100 microliters of PPS-DMEM mixture into each well, triplicating the infectivity tests of each type of PPS to one susceptible cell line.Incubate the plate for four to six hours at 37 degrees Celsius and 5%carbon dioxide. Then aspirate the supernatant and replace it with 100 microliters of DCM Incubate the plate for another 24 to 36 hours before infectivity detection. Our results are PPS are considered now infectious to people.So necessary safe guard procedures are recommended to take such as wearing a mask and performing the infectivity assays in a biosafety cabinet. This protocol was generate to generate 10 types of pseudotyped particles or PPS with two grouped hemaglutinin and neuraminidase vesicular stomatitis virus G glycoprotein or no envelope glycoproteins. The PPS were imaged with transmission electron microscopy and their infectivity were evaluated in two cell lines, A549 and MDCK.In the PPS group harboring H5, the infectivity of H5N1, H5 plus N9 and H5 was approximately 90%18%and 10%for cell line A549 and 40%5%and 5%for MDCK, respectively. In the PPS group harboring H7, the infectivity of H7N9, H7 plus N1 and H7 was approximately 10%7%and 1%for cell line A549 and 8%4%and 1%for MDCK, respectively. The infectivity of vesicular stomatitis virus G glycoprotein PP was about 21%for A549 and 16%for MDCK cells.While the delta envelope glycoproteins PP showed no infectivity. It's critical to remember that the PP producer HEK-293T/17 cells constitute to the foundation of this procedure. So the optimal growth teach us HEK-293T/17 cells is most important.Following this procedure or some other test such as L2 recept the bounding assay can be performed. This assay can almost give rid of the biological peculiarities of the receptor preference and the pattern will reveal the tropism of the PPS. Since they took the glycoproteins from influenza virus are cloned into two plus max.HA and the NA proteins can be studied separately so we reassortment between them. The mutants and the mature effects can be explored.

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Research

JoVE Journal

Immunology and Infection

8.1K vistas.  Hang Zhou Red Cross Hospital. Esta técnica de producción de PP viral de gripe y determinar su infectividad puede simplificar la investigación del virus de la gripe en una ciudad BSL-2. Normalizamos el PPS para las infecciones basado en datos qRT-PCR de PPS. Se codifican dos plásmidos de expresión de glicoproteína, lo que puede simplificar la investigación sobre la reordenación de virus.Un día después de la siembra celular, compruebe la morfología celular y la densidad bajo un microscopio de luz invertido...

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