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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">An optimised method for the production of MERS-CoV spike expressing viral pseudotypes</title><author><name sortKey="Grehan, K" sort="Grehan, K" uniqKey="Grehan K" first="K." last="Grehan">K. Grehan</name></author><author><name sortKey="Ferrara, F" sort="Ferrara, F" uniqKey="Ferrara F" first="F." last="Ferrara">F. Ferrara</name></author><author><name sortKey="Temperton, N" sort="Temperton, N" uniqKey="Temperton N" first="N." last="Temperton">N. Temperton</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26587388</idno><idno type="pmc">4625112</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625112</idno><idno type="RBID">PMC:4625112</idno><idno type="doi">10.1016/j.mex.2015.09.003</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000F18</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000F18</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">An optimised method for the production of MERS-CoV spike expressing viral pseudotypes</title><author><name sortKey="Grehan, K" sort="Grehan, K" uniqKey="Grehan K" first="K." last="Grehan">K. Grehan</name></author><author><name sortKey="Ferrara, F" sort="Ferrara, F" uniqKey="Ferrara F" first="F." last="Ferrara">F. Ferrara</name></author><author><name sortKey="Temperton, N" sort="Temperton, N" uniqKey="Temperton N" first="N." last="Temperton">N. Temperton</name></author></analytic><series><title level="j">MethodsX</title><idno type="eISSN">2215-0161</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Graphical abstract</title><fig id="fig0020" position="anchor"><graphic xlink:href="fx1"></graphic></fig></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Molesti, E" uniqKey="Molesti E">E. Molesti</name></author><author><name sortKey="Wright, E" uniqKey="Wright E">E. Wright</name></author><author><name sortKey="Terregino, C" uniqKey="Terregino C">C. Terregino</name></author><author><name sortKey="Rahman, R" uniqKey="Rahman R">R. Rahman</name></author><author><name sortKey="Cattoli, G" uniqKey="Cattoli G">G. Cattoli</name></author><author><name sortKey="Temperton, N J" uniqKey="Temperton N">N.J. Temperton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name></author><author><name sortKey="Qi, J" uniqKey="Qi J">J. Qi</name></author><author><name sortKey="Yuan, Y" uniqKey="Yuan Y">Y. Yuan</name></author><author><name sortKey="Xuan, Y" uniqKey="Xuan Y">Y. Xuan</name></author><author><name sortKey="Han, P" uniqKey="Han P">P. Han</name></author><author><name sortKey="Wan, Y" uniqKey="Wan Y">Y. Wan</name></author><author><name sortKey="Ji, W" uniqKey="Ji W">W. Ji</name></author><author><name sortKey="Li, Y" uniqKey="Li Y">Y. Li</name></author><author><name sortKey="Wu, Y" uniqKey="Wu Y">Y. Wu</name></author><author><name sortKey="Wang, J" uniqKey="Wang J">J. Wang</name></author><author><name sortKey="Iwamoto, A" uniqKey="Iwamoto A">A. Iwamoto</name></author><author><name sortKey="Woo, P C Y" uniqKey="Woo P">P.C.Y. Woo</name></author><author><name sortKey="Yuen, K Y" uniqKey="Yuen K">K.-Y. Yuen</name></author><author><name sortKey="Yan, J" uniqKey="Yan J">J. Yan</name></author><author><name sortKey="Lu, G" uniqKey="Lu G">G. Lu</name></author><author><name sortKey="Gao, G F" uniqKey="Gao G">G.F. Gao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name></author><author><name sortKey="Du, L" uniqKey="Du L">L. Du</name></author><author><name sortKey="Ma, C" uniqKey="Ma C">C. Ma</name></author><author><name sortKey="Li, Y" uniqKey="Li Y">Y. Li</name></author><author><name sortKey="Li, L" uniqKey="Li L">L. Li</name></author><author><name sortKey="Poon, V K M" uniqKey="Poon V">V.K.M. Poon</name></author><author><name sortKey="Wang, L" uniqKey="Wang L">L. Wang</name></author><author><name sortKey="Yu, F" uniqKey="Yu F">F. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">MethodsX</journal-id><journal-id journal-id-type="iso-abbrev">MethodsX</journal-id><journal-title-group><journal-title>MethodsX</journal-title></journal-title-group><issn pub-type="epub">2215-0161</issn><publisher><publisher-name>Elsevier</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26587388</article-id><article-id pub-id-type="pmc">4625112</article-id><article-id pub-id-type="publisher-id">S2215-0161(15)00046-1</article-id><article-id pub-id-type="doi">10.1016/j.mex.2015.09.003</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>An optimised method for the production of MERS-CoV spike expressing viral pseudotypes</article-title></title-group><contrib-group><contrib contrib-type="author" id="aut0005"><name><surname>Grehan</surname><given-names>K.</given-names></name></contrib><contrib contrib-type="author" id="aut0010"><name><surname>Ferrara</surname><given-names>F.</given-names></name></contrib><contrib contrib-type="author" id="aut0015"><name><surname>Temperton</surname><given-names>N.</given-names></name><email>n.temperton@kent.ac.uk</email><xref rid="cor0005" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff0005">Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent, Chatham Maritime, Kent, United Kingdom</aff><author-notes><corresp id="cor0005"><label>⁎</label>Corresponding author. <email>n.temperton@kent.ac.uk</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>13</day><month>10</month><year>2015</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><year>2015</year></pub-date><pub-date pub-type="epub"><day>13</day><month>10</month><year>2015</year></pub-date><volume>2</volume><fpage>379</fpage><lpage>384</lpage><history><date date-type="received"><day>24</day><month>6</month><year>2015</year></date><date date-type="accepted"><day>28</day><month>9</month><year>2015</year></date></history><permissions><copyright-statement>© 2015 The Authors</copyright-statement><copyright-year>2015</copyright-year><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract abstract-type="graphical" id="abs0005"><title>Graphical abstract</title><fig id="fig0020" position="anchor"><graphic xlink:href="fx1"></graphic></fig></abstract><abstract id="abs0010"><p>The production and use of pseudotyped viral particles are widely established for many viruses, and applications in the fields of serology and vaccine development are manifold. Viral pseudotypes have proven to be powerful tools to study the effects of viral evolution on serological outcomes, viral tropism and immunogenicity studies. Pseudotyped viruses are chimeric constructs in which the outer (surface) glycoprotein(s) of one virus is combined with the replication-defective viral “core” of another virus. Pseudotypes allow for accurate, sequence-directed, sensitive antibody neutralisation assays and antiviral screening to be conducted within a low biosecurity facility and offer a safe and efficient alternative to wildtype virus use.</p><p>The protocol outlined here represents a rapid and reliable method for the generation of high-titre pseudotype viral particles with the MERS-CoV spike protein on a lentiviral core, and is adapted from previously published protocols. This protocol is optimised for transfection in a 100 mm Petri dish with 7 ml of supernatant harvested, however it can be readily scaled to different production volumes.</p><p>This protocol has a number of advantages including:<list list-type="simple" id="lis0005"><list-item id="lsti0005"><label>•</label><p id="par0005">Use of readily available reagents.</p></list-item><list-item id="lsti0010"><label>•</label><p id="par0010">Consistent, high virus titres.</p></list-item><list-item id="lsti0015"><label>•</label><p id="par0015">Rapid generation of novel glycoproteins for research into strain variation.</p></list-item></list></p></abstract><kwd-group id="kwd0010"><title>Keywords</title><kwd>MERS coronavirus</kwd><kwd>Lentiviral pseudotype</kwd><kwd>Virus neutralisation</kwd></kwd-group></article-meta></front><body><sec id="sec0005"><title>Method details</title><sec id="sec0010"><title>Materials and equipment</title><p id="par0020"><list list-type="simple" id="lis0010"><list-item id="lsti0020"><label>•</label><p id="par0025">HEK 293T/17 cells (ATCC<sup>®</sup> CRL-11268™).</p></list-item><list-item id="lsti0025"><label>•</label><p id="par0030">Dulbecco's modified Eagle medium with Glutamax (Cat. No. 31966-021) supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (P/S).</p></list-item><list-item id="lsti0030"><label>•</label><p id="par0035">Trypsin–EDTA (0.05%), phenol red (Cat. No. 25300-054).</p></list-item><list-item id="lsti0035"><label>•</label><p id="par0040">Gibco reduced serum media Opti-MEM<sup>®</sup> (Cat. No. 31985-047).</p></list-item><list-item id="lsti0040"><label>•</label><p id="par0045">Optional: TC20™ Automated Cell Counter (Cat. No. 145-0102EDU).</p></list-item><list-item id="lsti0045"><label>•</label><p id="par0050">Branched polyethyleneimine solution at concentration of 1 mg/ml (Cat. No. 408727).</p></list-item><list-item id="lsti0050"><label>•</label><p id="par0055">Sterile syringes (10 ml).</p></list-item><list-item id="lsti0055"><label>•</label><p id="par0060">Millex-HA 0.45 μm filters (Cat. No. SLHAM33SS).</p></list-item><list-item id="lsti0060"><label>•</label><p id="par0065">Rabbit polyclonal antibody to novel coronavirus (HCoV-EMC/2012) spike protein (SinoBiological Cat. No. 40069-RP02).</p></list-item><list-item id="lsti0065"><label>•</label><p id="par0070">Nunc<sup>®</sup> UpCell™ surface cell culture dish (Manufacturer No. 174902).</p></list-item><list-item id="lsti0070"><label>•</label><p id="par0075">Microcentrifuge tube Safe-Lock write-on graduated with lid latch 1.5 ml.</p></list-item></list></p><p id="par0080"><italic>Note</italic>: All steps should be carried out in a class II biosafety cabinet to avoid contamination.</p></sec><sec id="sec0015"><title>Plasmids</title><p id="par0085">Glycoprotein expression plasmid: pCAGGS-MERS-CoV spike. (<italic>Note</italic>: The MERS-CoV spike protein should be codon optimised) <xref rid="bib0075" ref-type="bibr">[3]</xref>, <xref rid="bib0085" ref-type="bibr">[5]</xref>.</p><p id="par0090">Lentiviral vector plasmid expressing firefly luciferase: pCSFLW <xref rid="bib0100" ref-type="bibr">[8]</xref>, <xref rid="bib0115" ref-type="bibr">[11]</xref>.</p><p id="par0095">Second-generation lentiviral packaging construct plasmid: p8.91 (expressing HIV-gag) <xref rid="bib0100" ref-type="bibr">[8]</xref>, <xref rid="bib0120" ref-type="bibr">[12]</xref>.</p></sec><sec id="sec0030"><title>Transfection steps</title><p id="par0330"><list list-type="simple" id="lis0015"><list-item id="lsti0075"><p id="par0105"><bold>Timeline: Transfection – 24</bold>
<bold>h</bold><list list-type="simple" id="lis0020"><list-item id="lsti0080"><label>1.</label><p id="par0110">293T/17 cells should be subcultured into 100 mm Petri dishes at a ratio that will yield 70–90% confluence at the time of transfection. In our hands this protocol yields similar results regardless of Petri dish size when supernatant yield is equivalent.</p></list-item></list></p></list-item><list-item id="lsti0085"><p id="par0115"><bold>Timeline: Day of transfection</bold><list list-type="simple" id="lis0025"><list-item id="lsti0090"><label>2.</label><p id="par0120">DMEM/10% FBS/1% P/S and Opti-MEM<sup>®</sup> should be pre-warmed to 37 °C using a water bath or similar.</p></list-item><list-item id="lsti0095"><label>3.</label><p id="par0125">Prepare and label two sterile 1.5 ml microcentrifuge tubes (tube 1 and tube 2) per transfection.</p></list-item><list-item id="lsti0100"><label>4.</label><p id="par0130">Add the following plasmids (0.9:1:1.5 envelope:core:vector ratio) for transfection to tube 1:<list list-type="simple" id="lis0030"><list-item id="lsti0105"><label>a.</label><p id="par0135">pCAGGS-MERS-CoV spike: 0.9 μg.</p></list-item><list-item id="lsti0110"><label>b.</label><p id="par0140">p8.91-lentiviral vector: 1.0 μg.</p></list-item><list-item id="lsti0115"><label>c.</label><p id="par0145">pCSFLW: 1.5 μg.</p></list-item></list></p></list-item><list-item id="lsti0120"><label>5.</label><p id="par0150">Add 200 μl Opti-MEM<sup>®</sup> to the plasmid DNA mix (tube 1).</p></list-item><list-item id="lsti0125"><label>6.</label><p id="par0155">Add 200 μl Opti-MEM<sup>®</sup> and 35 μl of 1 mg/ml PEI to tube 2.</p></list-item><list-item id="lsti0130"><label>7.</label><p id="par0160"><italic>Incubation step</italic>. Mix both tubes by gently flicking and incubate for 5 min at room temperature (RT).</p></list-item><list-item id="lsti0135"><label>8.</label><p id="par0165">After incubation, pipette the Opti-MEM<sup>®</sup>/PEI solution from tube 2 into the Opti-MEM<sup>®</sup>/DNA solution in tube 1.</p></list-item><list-item id="lsti0140"><label>9.</label><p id="par0170"><italic>Incubation step</italic>. Gently flicking the tube to mix every 3–4 min, incubate the tube at RT for 20 min.</p></list-item><list-item id="lsti0145"><label>10.</label><p id="par0175">While transfection mix is incubating, the culture media on the 293T/17 cells should be removed and 7 ml of fresh DMEM/10% FBS/1% P/S added. It is important at this point to add media slowly to one side of the dish to avoid detaching adherent cells.</p></list-item><list-item id="lsti0150"><label>11.</label><p id="par0180">After 20 min incubation, pipette the DNA/Opti-MEM<sup>®</sup>/PEI solution onto the 293T/17 cells by adding dropwise over the complete area of the plate. Swirl the plates gently to ensure even dispersal.</p></list-item><list-item id="lsti0155"><label>12.</label><p id="par0185"><italic>Incubation step</italic>. Incubate the plate at 37 °C, 5% CO<sub>2</sub> overnight (o/n). In our hands incubation times of between 12 and 16 h result in equivalent final pseudotype production titres.</p></list-item></list></p></list-item><list-item id="lsti0160"><p id="par0190"><bold>Timeline: 12–16 h post transfection</bold><list list-type="simple" id="lis0035"><list-item id="lsti0165"><label>13.</label><p id="par0195">Post o/n incubation the media on the cells should be changed and 7 ml fresh DMEM/10% FBS/1% P/S added. Add media slowly to one side of the plate to avoid cell detachment.</p></list-item><list-item id="lsti0170"><label>14.</label><p id="par0200">Incubate the plates 37 °C 5% CO<sub>2</sub> o/n for 32–36 h.</p></list-item></list></p></list-item><list-item id="lsti0175"><p id="par0205"><bold>Timeline: 44–52 h post transfection</bold><list list-type="simple" id="lis0040"><list-item id="lsti0180"><label>15.</label><p id="par0210">Supernatant containing the viral pseudotype particles are harvested using a 10 ml sterile syringe and then filtered into falcon tubes via a syringe driven Millex HA-0.45 μm filter.</p></list-item><list-item id="lsti0185"><label>16.</label><p id="par0215">Store all filtered supernatant at −80 °C. It is recommended that supernatant is stored as aliquots to avoid multiple freeze thaw cycles. <italic>Note</italic>: Supernatant may be stored at 4 °C for up to one week with no detectable loss of titre.</p></list-item><list-item id="lsti0190"><label>17.</label><p id="par0220"><italic>Optional step</italic>: Additional media may be added to cells to allow a second harvest 18–24 h later by adding further DMEM/10% FBS/1% P/S. In this case extreme care must taken in initial pseudotype collection (step 15) to avoid damage to cell monolayer. We have observed that cells in poor health after first harvest yield significantly less viral particles upon second harvest.</p></list-item></list></p></list-item></list></p><p id="par0225"><italic>Note</italic>: A control pseudotype virus may be created by following the steps outlined above but leaving out the pCAGGS-MERS-CoV spike construct. This produces particles that do not express the viral surface glycoprotein and therefore should be unable to transduce target cells (Δ-env control) <xref rid="bib0075" ref-type="bibr">[3]</xref>.</p></sec><sec id="sec0020"><title>Titration steps (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>)</title><p id="par0230"><italic>Note</italic>: Titration consists of transduction of reporter (in this case firefly luciferase) into target cells mediated by the viral glycoprotein expressed on the viral pseudotype. Controls for titrations are provided via the inclusion of “cell only” and “Δ-env” columns.
<list list-type="simple" id="lis0045"><list-item id="lsti0195"><label>1.</label><p id="par0235">In a 96 well white plate add 50 μl of DMEM/10% FBS/1% P/S to the entire column of “cell only” control (see <xref rid="fig0005" ref-type="fig">Fig. 1</xref> column 12).</p></list-item><list-item id="lsti0200"><label>2.</label><p id="par0240">Add 50 μl of DMEM/10% FBS/1% P/S from row B to H that are to contain pseudotyped virus or Δ-env control.</p></list-item><list-item id="lsti0205"><label>3.</label><p id="par0245">Add 100 μl of MERS pseudotype virus supernatant to each well of row A (excluding control columns) and add 100 μl of Δ-env to column 11 (see <xref rid="fig0005" ref-type="fig">Fig. 1</xref>).</p></list-item><list-item id="lsti0210"><label>4.</label><p id="par0250">Remove 50 μl from row 1 virus-containing wells and perform 1:2 serial dilutions down all wells below.</p></list-item><list-item id="lsti0215"><label>5.</label><p id="par0255">With each dilution step use pipette to mix 8 times up and down.</p></list-item><list-item id="lsti0220"><label>6.</label><p id="par0260">After completing serial dilution the final 50 μl from the last well of each column should be discarded. <italic>Note</italic>: at this point each well should contain 50 μl of mixed DMEM and viral supernatant.</p></list-item><list-item id="lsti0225"><label>7.</label><p id="par0265">Prepare a plate of susceptible target cells (Huh-7) (preferentially subcultured 1:4 48 h before):<list list-type="simple" id="lis0050"><list-item id="lsti0230"><label>a.</label><p id="par0270">Remove media from plate.</p></list-item><list-item id="lsti0235"><label>b.</label><p id="par0275">Wash the plate with 2 ml of trypsin and discard trypsin.</p></list-item><list-item id="lsti0240"><label>c.</label><p id="par0280">Add additional 2 ml of trypsin to the plate to detach cells.</p></list-item><list-item id="lsti0245"><label>d.</label><p id="par0285">After cells have detached add DMEM/10% FBS/1% P/S to the plate to quench trypsin activity.</p></list-item><list-item id="lsti0250"><label>e.</label><p id="par0290">Count cells using TC20™ Automated Cell Counter or haemocytometer and add 1x10<sup>4</sup> cells in a total volume of 50 μl to each well.</p></list-item></list></p></list-item><list-item id="lsti0255"><label>8.</label><p id="par0295">Centrifuge plate for 1 min at 500 rpm if there are droplets on the sides of the wells.</p></list-item><list-item id="lsti0260"><label>9.</label><p id="par0300">Incubate the plate for 48 h at 37 °C 5% CO<sub>2</sub>.</p></list-item><list-item id="lsti0265"><label>10.</label><p id="par0305">Read plate using Bright Glo™ luciferase assay system or equivalent.</p></list-item></list><fig id="fig0005"><label>Fig. 1</label><caption><p>Serial dilution step showing addition of 100 μl of pseudotype virus supernatant to each well of row A and dilution of 50 μl taken from this well to row B. This process is continued to end of plate (row H) at which point the final 50 μl is discarded. Δ-Env control is indicated in red (column 11) and cell only control is indicated in green (column 12). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</p></caption><graphic xlink:href="gr1"></graphic></fig></p></sec><sec id="sec0025"><title>Method validation and transfection results</title><p id="par0310"><xref rid="fig0010" ref-type="fig">Fig. 2</xref>displays data recorded from multiple transfections indicating consistency of results. Results are measured in Relative Luminescence Units (RLUs) as measured using a GloMax<sup>®</sup> 96 Microplate Luminometer and the Bright Glo™ luciferase assay system. The pseudotype particles generated in the absence of viral envelope (Delta) show increased luciferase activity compared to cell only in part due to transformation method used to discern RLU per ml. The presence of some carry-over luciferase within viral particles is also likely to generate an increase in RLU values recorded (<xref rid="tbl0005" ref-type="table">Table 1</xref>).<fig id="fig0010"><label>Fig. 2</label><caption><p>Pseudotype production titres from three replicates of optimised transfection protocol using codon optimised MERS-CoV Spike. Delta envelope titre overestimation in comparison to cell only control is related to the mathematical method that is used to calculate pseudotype titre and that cannot be applied to cell only.</p></caption><graphic xlink:href="gr2"></graphic></fig><table-wrap position="float" id="tbl0005"><label>Table 1</label><caption><p>Mean RLU calculated per ml of viral supernatant for three pseudotype production runs.</p></caption><table frame="hsides" rules="groups"><thead><tr><th></th><th align="left">Transfection 1</th><th align="left">Transfection 2</th><th align="left">Transfection 3</th><th align="left">Cell only</th></tr></thead><tbody><tr><td align="left">Mean/ml titre</td><td align="char">2.2E+08</td><td align="char">2.3E+08</td><td align="char">2.6E+08</td><td align="char">91</td></tr></tbody></table></table-wrap></p><p id="par0315"><xref rid="fig0015" ref-type="fig">Fig. 3</xref>shows percentage neutralisation of the MERS-CoV pseudotype with commercially produced anti-MERS spike antibody. Figure clearly indicates that as the dilution factor increases, so the percentage neutralisation decreases, 100% neutralisation indicates that RLU values at this concentration are equivalent to a delta envelope control.<fig id="fig0015"><label>Fig. 3</label><caption><p>Anti-MERS-spike antibody (rabbit polyclonal antibody to novel coronavirus (HCoV-EMC/2012) spike protein) neutralises MERS viral pseudotype entry into Huh7 cells.</p></caption><graphic xlink:href="gr3"></graphic></fig></p><p id="par0320">The protocol outlined here provides a rapid and consistent method for the generation of high-titre viral pseudotype particles expressing the MERS-CoV spike protein suitable for further downstream applications <xref rid="bib0070" ref-type="bibr">[2]</xref>, <xref rid="bib0080" ref-type="bibr">[4]</xref>, <xref rid="bib0085" ref-type="bibr">[5]</xref>, <xref rid="bib0095" ref-type="bibr">[7]</xref>. Efficient knock-down of pseudotype virus entry using a polyclonal antibody directed against the spike glycoprotein (<xref rid="fig0015" ref-type="fig">Fig. 3</xref>) demonstrates potential utility for vaccine immunogenicity and Mab/antiviral screening <xref rid="bib0075" ref-type="bibr">[3]</xref>. 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