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>

<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. A., 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=Cross-clade+protective+immune+responses+to+influenza+viruses+with+H5N1+HA+and+NA+elicited+by+an+influenza+virus-like+particle.">Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle.</a> PLoS One. 3 (1), 1501 (2008).</li><li>Yang, J., 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=Reliability+of+pseudotyped+influenza+viral+particles+in+neutralizing+antibody+detection.">Reliability of pseudotyped influenza viral particles in neutralizing antibody detection.</a> PLoS One. 9 (12), 113629 (2014).</li><li>Wyatt, R., Sodroski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HIV-1+envelope+glycoproteins:+fusogens,+antigens,+and+immunogens.">The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens.</a> Science. 280 (5371), 1884-1888 (1998).</li><li>Joe, A. 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. L., Hewlett, N., Roizman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudotype+formation+between+enveloped+RNA+and+DNA+viruses.">Pseudotype formation between enveloped RNA and DNA viruses.</a> Nature. 252 (5485), 743-745 (1974).</li><li>Rubin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Control+of+Cellular+Susceptibility+to+Pseudotypes+of+Rous+Sarcoma+Virus.">Genetic Control of Cellular Susceptibility to Pseudotypes of Rous Sarcoma Virus.</a> Virology. 26, 270-276 (1965).</li><li>Steffen, I., Simmons, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudotyping+Viral+Vectors+With+Emerging+Virus+Envelope+Proteins.">Pseudotyping Viral Vectors With Emerging Virus Envelope Proteins.</a> Current Gene Therapy. 16 (1), 47-55 (2016).</li><li>Bartosch, B., Dubuisson, J., Cosset, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+hepatitis+C+virus+pseudo-particles+containing+functional+E1-E2+envelope+protein+complexes.">Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes.</a> Journal of Experimental Medicine. 197 (5), 633-642 (2003).</li><li>Bian, T., Zhou, Y., Bi, S., Tan, W., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCV+envelope+protein+function+is+dependent+on+the+peptides+preceding+the+glycoproteins.">HCV envelope protein function is dependent on the peptides preceding the glycoproteins.</a> Biochemical and Biophysical Research Communications. 378 (1), 118-122 (2009).</li><li>Gudima, S., Meier, A., Dunbrack, R., Taylor, J., Bruss, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+potentially+important+elements+of+the+hepatitis+B+virus+large+envelope+protein+are+dispensable+for+the+infectivity+of+hepatitis+delta+virus.">Two potentially important elements of the hepatitis B virus large envelope protein are dispensable for the infectivity of hepatitis delta virus.</a> Journal of Virology. 81 (8), 4343-4347 (2007).</li><li>Yoshida, Y., Emi, N., Hamada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSV-G-pseudotyped+retroviral+packaging+through+adenovirus-mediated+inducible+gene+expression.">VSV-G-pseudotyped retroviral packaging through adenovirus-mediated inducible gene expression.</a> Biochemical and Biophysical Research Communications. 232 (2), 379-382 (1997).</li><li>Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., Yee, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+G+glycoprotein+pseudotyped+retroviral+vectors:+concentration+to+very+high+titer+and+efficient+gene+transfer+into+mammalian+and+nonmammalian+cells.">Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (17), 8033-8037 (1993).</li><li>Zhang, F., 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=Characterization+of+pseudoparticles+paired+with+hemagglutinin+and+neuraminidase+from+highly+pathogenic+H5N1+influenza+and+avian+influenza+A+(H7N9)+viruses.">Characterization of pseudoparticles paired with hemagglutinin and neuraminidase from highly pathogenic H5N1 influenza and avian influenza A (H7N9) viruses.</a> Virus Research. 253, 20-27 (2018).</li><li>Zhang, F., 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=Infectivity+of+Pseudotyped+Particles+Pairing+Hemagglutinin+of+Highly+Pathogenic+Avian+Influenza+a+H5N1+Virus+with+Neuraminidases+of+The+2009+Pandemic+H1N1+and+a+Seasonal+H3N2.">Infectivity of Pseudotyped Particles Pairing Hemagglutinin of Highly Pathogenic Avian Influenza a H5N1 Virus with Neuraminidases of The 2009 Pandemic H1N1 and a Seasonal H3N2.</a> Journal of Bioterrorism &amp; Biodefense. 2, 104 (2011).</li><li>Wu, J., 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=Characterization+of+neuraminidases+from+the+highly+pathogenic+avian+H5N1+and+2009+pandemic+H1N1+influenza+A+viruses.">Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses.</a> PLoS One. 5 (12), 15825 (2010).</li><li>Nefkens, I., 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=Hemagglutinin+pseudotyped+lentiviral+particles:+characterization+of+a+new+method+for+avian+H5N1+influenza+sero-diagnosis.">Hemagglutinin pseudotyped lentiviral particles: characterization of a new method for avian H5N1 influenza sero-diagnosis.</a> Journal of Clinical Virology. 39 (1), 27-33 (2007).</li><li>Zhang, 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=Hemagglutinin+and+neuraminidase+matching+patterns+of+two+influenza+A+virus+strains+related+to+the+1918+and+2009+global+pandemics.">Hemagglutinin and neuraminidase matching patterns of two influenza A virus strains related to the 1918 and 2009 global pandemics.</a> Biochemical and Biophysical Research Communications. 387 (2), 405-408 (2009).</li><li>Lin, X., 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=Oseltamivir+boosts+2009+H1N1+virus+infectivity+in+vitro.">Oseltamivir boosts 2009 H1N1 virus infectivity in vitro.</a> Biochemical and Biophysical Research Communications. 390 (4), 1305-1308 (2009).</li><li>McKay, T., Patel, M., Pickles, R. J., Johnson, L. G., Olsen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+M2+envelope+protein+augments+avian+influenza+hemagglutinin+pseudotyping+of+lentiviral+vectors.">Influenza M2 envelope protein augments avian influenza hemagglutinin pseudotyping of lentiviral vectors.</a> Gene Therapy. 13 (8), 715-724 (2006).</li><li>Pan, H., 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=Autophagy+mediates+avian+influenza+H5N1+pseudotyped+particle-induced+lung+inflammation+through+NF-kappaB+and+p38+MAPK+signaling+pathways.">Autophagy mediates avian influenza H5N1 pseudotyped particle-induced lung inflammation through NF-kappaB and p38 MAPK signaling pathways.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 306 (2), 183-195 (2014).</li><li>Szecsi, J., 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=Induction+of+neutralising+antibodies+by+virus-like+particles+harbouring+surface+proteins+from+highly+pathogenic+H5N1+and+H7N1+influenza+viruses.">Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses.</a> Virology Journal. 3, 70 (2006).</li><li>Garcia, J. M., Lagarde, N., Ma, E. S., de Jong, M. D., Peiris, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+evaluation+of+an+influenza+A+(H5)+pseudotyped+lentiviral+particle-based+serological+assay.">Optimization and evaluation of an influenza A (H5) pseudotyped lentiviral particle-based serological assay.</a> Journal of Clinical Virology. 47 (1), 29-33 (2010).</li><li>Garcia, J. M., Lai, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+influenza+pseudotyped+lentiviral+particles+and+their+use+in+influenza+research+and+diagnosis:+an+update.">Production of influenza pseudotyped lentiviral particles and their use in influenza research and diagnosis: an update.</a> Expert Review of Anti-infective Therapy. 9 (4), 443-455 (2011).</li><li>Haynes, J. R., 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-pseudotyped+Gag+virus-like+particle+vaccines+provide+broad+protection+against+highly+pathogenic+avian+influenza+challenge.">Influenza-pseudotyped Gag virus-like particle vaccines provide broad protection against highly pathogenic avian influenza challenge.</a> Vaccine. 27 (4), 530-541 (2009).</li><li>Schmeisser, F., 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+characterization+of+mammalian+virus-like+particles+from+modified+vaccinia+virus+Ankara+vectors+expressing+influenza+H5N1+hemagglutinin+and+neuraminidase.">Production and characterization of mammalian virus-like particles from modified vaccinia virus Ankara vectors expressing influenza H5N1 hemagglutinin and neuraminidase.</a> Vaccine. 30 (23), 3413-3422 (2012).</li><li>Liu, Y. V., 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=Chimeric+severe+acute+respiratory+syndrome+coronavirus+(SARS-CoV)+S+glycoprotein+and+influenza+matrix+1+efficiently+form+virus-like+particles+(VLPs)+that+protect+mice+against+challenge+with+SARS-CoV.">Chimeric severe acute respiratory syndrome coronavirus (SARS-CoV) S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV.</a> Vaccine. 29 (38), 6606-6613 (2011).</li><li>Moeschler, S., Locher, S., Conzelmann, K. K., Kramer, B., Zimmer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Lyssavirus-Neutralizing+Antibodies+Using+Vesicular+Stomatitis+Virus+Pseudotype+Particles.">Quantification of Lyssavirus-Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles.</a> Viruses. 8 (9), 254 (2016).</li><li>Lai, A. L., Millet, J. K., Daniel, S., Freed, J. H., Whittaker, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SARS-CoV+Fusion+Peptide+Forms+an+Extended+Bipartite+Fusion+Platform+that+Perturbs+Membrane+Order+in+a+Calcium-Dependent+Manner.">The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner.</a> Journal of Molecular Biology. 429 (24), 3875-3892 (2017).</li><li>Millet, J. K., 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=Middle+East+respiratory+syndrome+coronavirus+infection+is+inhibited+by+griffithsin.">Middle East respiratory syndrome coronavirus infection is inhibited by griffithsin.</a> Antiviral Research. 133, 1-8 (2016).</li><li>Millet, J. K., 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+of+Pseudotyped+Particles+to+Study+Highly+Pathogenic+Coronaviruses+in+a+Biosafety+Level+2+Setting.">Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting.</a> Journal of Visualized Experiments. (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 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...

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

Research

JoVE Journal

Immunology and Infection

8.1K Views.  Hang Zhou Red Cross Hospital. 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.

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.

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 ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

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 ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. Products are routinely stored at 22 &#176;C on an agitating shaker, a condition that can promote bacterial growth. Although the total number of bacteria believed to be introduced into a platelet product is extremely low, these bacteria can multiply to a very high titer prior to transfusion, potentially resulting in serious adverse events. The aim of this study was to evaluate a riboflavin based pathogen reduction process against a panel of bacteria that have been identified as common contaminants of platelet products. This panel included the following organisms: S. epidermidis, S. aureus, S. mitis, S. pyogenes, S. marcescens, Y. enterocolitica, B. neotomae, B. cereus, E. coli, P. aeruginosa and K. pneumoniae. Each platelet unit was inoculated with a high bacterial load and samples were removed both before and after treatment. A colony forming assay, using an end point dilution scheme, was used to determine the pre-treatment and post-treatment bacterial titers. Log reduction was calculated by subtracting the post-treatment titer from the pre-treatment titer. The following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805;5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%). The results from this study suggest the process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...