Studying Evolutionary Adaptation of MERS-CoV
Identifieur interne : 000176 ( Pmc/Corpus ); précédent : 000175; suivant : 000177Studying Evolutionary Adaptation of MERS-CoV
Auteurs :Source :
- MERS Coronavirus ; 2019.
Abstract
Forced viral adaptation is a powerful technique employed to study the ways viruses may overcome various selective pressures that reduce viral replication. Here, we describe methods for in vitro serial passaging of Middle East respiratory syndrome coronavirus (MERS-CoV) to select for mutations which increase replication on semi-permissive cell lines as described in Letko et al., Cell Rep 24, 1730–1737, 2018.
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DOI: 10.1007/978-1-0716-0211-9_1
PubMed: 31883083
PubMed Central: 7121928
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Studying Evolutionary Adaptation of MERS-CoV</title></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31883083</idno><idno type="pmc">7121928</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7121928</idno><idno type="RBID">PMC:7121928</idno><idno type="doi">10.1007/978-1-0716-0211-9_1</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000176</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000176</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Studying Evolutionary Adaptation of MERS-CoV</title></analytic><series><title level="j">MERS Coronavirus</title><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="Par1">Forced viral adaptation is a powerful technique employed to study the ways viruses may overcome various selective pressures that reduce viral replication. Here, we describe methods for in vitro serial passaging of Middle East respiratory syndrome coronavirus (MERS-CoV) to select for mutations which increase replication on semi-permissive cell lines as described in Letko et al., Cell Rep 24, 1730–1737, 2018.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Andino, R" uniqKey="Andino R">R Andino</name></author><author><name sortKey="Domingo, E" uniqKey="Domingo E">E Domingo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Domingo, E" uniqKey="Domingo E">E Domingo</name></author><author><name sortKey="Sheldon, J" uniqKey="Sheldon J">J Sheldon</name></author><author><name sortKey="Perales, C" uniqKey="Perales C">C Perales</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lauring, As" uniqKey="Lauring A">AS Lauring</name></author><author><name sortKey="Andino, R" uniqKey="Andino R">R Andino</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bazmi, Hz" uniqKey="Bazmi H">HZ Bazmi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sato, A" uniqKey="Sato A">A Sato</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mckimm Breschkin, Jl" uniqKey="Mckimm Breschkin J">JL McKimm-Breschkin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shibata, J" uniqKey="Shibata J">J Shibata</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chai, N" uniqKey="Chai N">N Chai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Letko, M" uniqKey="Letko M">M Letko</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ooms, M" uniqKey="Ooms M">M Ooms</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ooms, M" uniqKey="Ooms M">M Ooms</name></author><author><name sortKey="Letko, M" uniqKey="Letko M">M Letko</name></author><author><name sortKey="Simon, V" uniqKey="Simon V">V Simon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Letko, M" uniqKey="Letko M">M Letko</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baric, Rs" uniqKey="Baric R">RS Baric</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mcroy, Wc" uniqKey="Mcroy W">WC McRoy</name></author><author><name sortKey="Baric, Rs" uniqKey="Baric R">RS Baric</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Khasawneh, Ai" uniqKey="Khasawneh A">AI Khasawneh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Das, At" uniqKey="Das A">AT Das</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Westerhout, Em" uniqKey="Westerhout E">EM Westerhout</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Doremalen, N" uniqKey="Van Doremalen N">N van Doremalen</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="chapter-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="publisher-id">978-1-0716-0211-9</journal-id><journal-id journal-id-type="doi">10.1007/978-1-0716-0211-9</journal-id><journal-id journal-id-type="nlm-ta">MERS Coronavirus</journal-id><journal-title-group><journal-title>MERS Coronavirus</journal-title><journal-subtitle>Methods and Protocols </journal-subtitle></journal-title-group><isbn publication-format="print">978-1-0716-0210-2</isbn><isbn publication-format="electronic">978-1-0716-0211-9</isbn></journal-meta><article-meta><article-id pub-id-type="pmid">31883083</article-id><article-id pub-id-type="pmc">7121928</article-id><article-id pub-id-type="publisher-id">1</article-id><article-id pub-id-type="doi">10.1007/978-1-0716-0211-9_1</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Studying Evolutionary Adaptation of MERS-CoV</article-title></title-group><contrib-group content-type="book editors"><contrib contrib-type="editor"><name><surname>Vijay</surname><given-names>Rahul</given-names></name><address><email>rahul-vijay@uiowa.edu</email></address><xref ref-type="aff" rid="Aff2"></xref></contrib><aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.214572.7</institution-id><institution-id institution-id-type="ISNI">0000 0004 1936 8294</institution-id><institution>Department of Microbiology and Immunology,</institution><institution>University of Iowa,</institution></institution-wrap>Iowa City, IA USA</aff></contrib-group><contrib-group><contrib contrib-type="author" corresp="yes"><name><surname>Letko</surname><given-names>Michael</given-names></name><address><email>michael.letko@nih.gov</email></address><xref ref-type="aff" rid="Aff3"></xref></contrib><contrib contrib-type="author"><name><surname>Munster</surname><given-names>Vincent</given-names></name><xref ref-type="aff" rid="Aff3"></xref></contrib><aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.419681.3</institution-id><institution-id institution-id-type="ISNI">0000 0001 2164 9667</institution-id><institution>Laboratory of Virology, Division of Intramural Research,</institution><institution>National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, National Institutes of Health,</institution></institution-wrap>Hamilton, MT USA</aff></contrib-group><pub-date pub-type="epub"><day>14</day><month>9</month><year>2019</year></pub-date><pub-date pub-type="collection"><year>2020</year></pub-date><volume>2099</volume><fpage>3</fpage><lpage>8</lpage><permissions><copyright-statement>© Springer Science+Business Media, LLC, part of Springer Nature 2020</copyright-statement><license><license-p>This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract id="Abs1"><p id="Par1">Forced viral adaptation is a powerful technique employed to study the ways viruses may overcome various selective pressures that reduce viral replication. Here, we describe methods for in vitro serial passaging of Middle East respiratory syndrome coronavirus (MERS-CoV) to select for mutations which increase replication on semi-permissive cell lines as described in Letko et al., Cell Rep 24, 1730–1737, 2018.</p></abstract><kwd-group xml:lang="en"><title>Key words</title><kwd>MERS-CoV</kwd><kwd>Forced adaptation</kwd><kwd>Experimental evolution</kwd><kwd>Cell culture</kwd><kwd>Semi-permissive cell line</kwd><kwd>Host restriction</kwd><kwd>Species barrier</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© Springer Science+Business Media, LLC, part of Springer Nature 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1"><title>Introduction</title><p id="Par2">RNA viruses are ideal model organisms to study evolutionary genetics under selection. This is due to their large population sizes and short generation times, which are characterized by rapid accumulation of mutations relative to other organisms. Given the error-prone nature of viral RNA-dependent RNA polymerases, viral replication leads to the formation of a quasispecies [<xref ref-type="bibr" rid="CR1">1</xref>–<xref ref-type="bibr" rid="CR3">3</xref>]. Rather than one virus producing identical progeny during replication, a population of viruses is produced, each differing from one another by nucleotide substitutions or deletions as a result of errors incorporated by the RNA polymerase. While the majority of these mutations will have neutral or negative effects on viral fitness, a small subset of these mutations may prove beneficial and enhance the ability for certain variants to replicate despite selective pressures of interest such as the host immune response or an antiviral drug. Forced adaptation experiments have been used to determine viral mutations that facilitate escape from drugs [<xref ref-type="bibr" rid="CR4">4</xref>–<xref ref-type="bibr" rid="CR6">6</xref>], monoclonal antibodies [<xref ref-type="bibr" rid="CR7">7</xref>, <xref ref-type="bibr" rid="CR8">8</xref>], host restriction factors [<xref ref-type="bibr" rid="CR9">9</xref>–<xref ref-type="bibr" rid="CR11">11</xref>], and species variation in host receptors [<xref ref-type="bibr" rid="CR12">12</xref>–<xref ref-type="bibr" rid="CR14">14</xref>] and to elucidate various viral mechanisms of infection and replication [<xref ref-type="bibr" rid="CR15">15</xref>–<xref ref-type="bibr" rid="CR17">17</xref>].</p><p id="Par3">Within the laboratory setting, the strength of selective pressure can be adjusted by increasing or decreasing the levels of the restrictive factor, thus facilitating the rapid expansion of viral variants within the population of quasispecies that can overcome the applied selective pressure. The ideal environment is “semi-permissive”—allowing only low levels of wild-type virus replication. Below is the method employed to adapt MERS-CoV to a semi-permissive host receptor, <italic>Desmodus rotundus</italic> DPP4 . The techniques described below could be applicable to a wide range of experiments to better understand the adaptive capacity of various coronaviruses under specific selective pressures.</p></sec><sec id="Sec2"><title>Materials</title><sec id="Sec3"><title>Cell Culture</title><p id="Par4"><list list-type="order"><list-item><p id="Par5">Semi-permissive cells: baby hamster kidney (BHK) cells which have been transduced to stably express <italic>Desmodus rotundus</italic> DPP4 (<italic>dr</italic>DPP4 [<xref ref-type="bibr" rid="CR12">12</xref>]. Briefly, the coding sequence for <italic>dr</italic>DPP4 was cloned into a lentiviral expression cassette also encoding for mcherry-T2A-puromycin-N-acetyltransferase-P2A (System Biosciences) and used to generate lentiviral particles [<xref ref-type="bibr" rid="CR9">9</xref>] (<italic>see</italic><bold>Note 1</bold>). BHK cells were infected with lentiviral particles and then grown in DMEM containing puromycin at a final concentration of 1 ug/mL.</p></list-item><list-item><p id="Par6">Cell culture media: Dulbecco’s Modified Eagles Medium (DMEM), 10% fetal bovine serum, 1% <sc>l</sc>-glutamine, 1% penicillin and streptomycin, and 1 μg/mL puromycin.</p></list-item><list-item><p id="Par7">Passaging culture media: Dulbecco’s Modified Eagles Medium (DMEM), 2% fetal bovine serum, 1% <sc>l</sc>-glutamine, 1% penicillin and streptomycin, and 1 μg/mL puromycin.</p></list-item><list-item><p id="Par8">Light microscope to check cell cultures for cytopathic effects .</p></list-item></list></p></sec><sec id="Sec4"><title>Passaging Experiment</title><p id="Par9"><list list-type="order"><list-item><p id="Par10">6-Well cell-culture cluster plates.</p></list-item><list-item><p id="Par11">MERS-CoV/EMC2012, passage 6. This virus stock was grown in-house and titered by standard endpoint titration on Vero cells [<xref ref-type="bibr" rid="CR18">18</xref>].</p></list-item></list></p></sec><sec id="Sec5"><title>Directed Sequencing of MERS-CoV Spike</title><p id="Par12"><list list-type="order"><list-item><p id="Par13">Viral RNA extraction mini kit.</p></list-item><list-item><p id="Par14">Superscript IV reverse transcriptase cDNA production kit.</p></list-item><list-item><p id="Par15">iProof High-fidelity PCR kit.</p></list-item><list-item><p id="Par16">Agarose gel purification kit.</p></list-item><list-item><p id="Par17">MERS-CoV Spike receptor-binding domain sequencing primers (<italic>see</italic> Table <xref rid="Tab1" ref-type="table">1</xref>).</p></list-item><list-item><p id="Par18">Sequence analysis software capable of multiple sequence alignment and viewing chromatograms.</p></list-item></list><table-wrap id="Tab1"><label>Table 1</label><caption><p>Primers for sequencing MERS-CoV spike</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Primer number</th><th>Primer sequence</th><th>Primer orientation</th></tr></thead><tbody><tr><td>1</td><td>ATGATACACTCAGTGTTTCT</td><td>Forward</td></tr><tr><td>2</td><td>TAGAAGGCAGCCCAAGCTTTT</td><td>Reverse</td></tr><tr><td>3</td><td>TTACGTAACTGCACCTTTATG</td><td>Forward</td></tr><tr><td>4</td><td>CATTTCACCTGGAACAGAGC</td><td>Reverse</td></tr><tr><td>5</td><td>AGATTCTACATATGGCCCCCT</td><td>Forward</td></tr><tr><td>6</td><td>TTAGTGAACATGAACCTTATGCGGC</td><td>Reverse</td></tr></tbody></table></table-wrap></p></sec></sec><sec id="Sec6"><title>Methods</title><sec id="Sec7"><title>Prepare Cells for Viral Passaging</title><p id="Par19"><list list-type="order"><list-item><p id="Par20">Plan number of conditions. At least three replicates (well of semi-permissive cells) for each forced adaptation experiment should be performed in parallel. Critically, parental cells or a cell line stably expressing an irrelevant protein should be included to control for any nonspecific cell culture mutations.</p></list-item><list-item><p id="Par21">Grow semi-permissive BHK cells to confluency in appropriate format. One 75 cm<sup>2</sup> flask should be sufficient to seed at least three 6-well cluster plates.</p></list-item><list-item><p id="Par22">Wash, trypsinize, count, and seed BHK cell lines (parental controls and semi-permissive) in cell culture media (10% FBS) at a density of 1.5 × 10<sup>5</sup> cells/mL in a 2 mL volume in each well of 6-well plates (<italic>see</italic><bold>Note 2</bold>).</p></list-item></list></p></sec><sec id="Sec8"><title>Infect Cells</title><p id="Par23"><list list-type="order"><list-item><p id="Par24">Twenty-four hours later, replace media on seeded cell lines with 2 mL of fresh passaging culture media (2% FBS).</p></list-item><list-item><p id="Par25">Infect cells with MERS-CoV/EMC2012 at a final MOI of 0.01 (Fig. <xref rid="Fig1" ref-type="fig">1</xref>).</p></list-item></list><fig id="Fig1"><label>Fig. 1</label><caption><p>Transduced cells are infected with wild-type stock. Approximately 72 hours later, supernatant from the infected cells is used to infect fresh cells as passage one. The process is repeated until the formation of cytopathic effects in culture. Supernatant from each passage is sequenced to detect the presence of adaptive mutations</p></caption><graphic xlink:href="468732_1_En_1_Fig1_HTML" id="MO1"></graphic></fig></p></sec><sec id="Sec9"><title>Prepare Cells for Subsequent Passage and Passage Virus</title><p id="Par26"><list list-type="order"><list-item><p id="Par27">After 48 h postinfection, prepare new cell culture plates to passage virus. Follow initial seeding conditions and plate at a density of 1.5 × 10<sup>5</sup> cells/mL in a 2 mL volume in each well of 6-well plates.</p></list-item><list-item><p id="Par28">Twenty-four hours after seeding the new cells (72 h postinfection of previous culture), replace media on seeded cell lines with 2 mL of fresh passaging culture media (2% FBS).</p></list-item><list-item><p id="Par29">After 72 h postinfection for previous culture, take a 500 μL of supernatant sample from the infected culture and store for downstream viral sequencing. Store supernatants at −80 °C.</p></list-item><list-item><p id="Par30">Check previously infected cells for emergence of cytopathic effects (cell death, rounding-up, and detachment from cell culture plate in more than 50% of individual cultures) (<italic>see</italic><bold>Note 3</bold>). If cytopathic effects are observed, this is strongly suggestive of viral adaptation to the semi-permissive cells. Proceed with step 3.4. Subsequent passages may be performed to select for further mutations that enhance viral replication in the semi-permissive cells (<italic>see</italic><bold>Note 4</bold>).</p></list-item><list-item><p id="Par31">If no cytopathic effects are observed, then begin next viral passage: from the previously infected culture, transfer 250 μL of supernatant to the new cell cultures seeded the day before.</p></list-item><list-item><p id="Par32">Discard previously infected culture.</p></list-item><list-item><p id="Par33">Repeat steps 1–6 until cytopathic effects are observed, indicative of viral adaptation.</p></list-item></list></p></sec><sec id="Sec10"><title>Extract Viral RNA and Sequence Spike</title><p id="Par34"><list list-type="order"><list-item><p id="Par35">Extract RNA from stored supernatants using the Qiagen viral RNA miniprep kit (Qiagen), following manufacturer’s instructions.</p></list-item><list-item><p id="Par36">Generate cDNA from extracted RNA using Superscript IV, following manufacturer’s instructions.</p></list-item><list-item><p id="Par37">Amplify select regions from viral cDNA using iProof high-fidelity PCR polymerase kit (Bio-Rad). Below are example PCR conditions for amplifying the MERS-CoV receptor-binding domain following the primer numbers listed in Subheading 2.2.5 of [<xref ref-type="bibr" rid="CR12">12</xref>] (see Table <xref rid="Tab2" ref-type="table">2</xref>).<table-wrap id="Taba"><table frame="hsides" rules="groups"><tbody><tr><td>31.5 μL</td><td>diH<sub>2</sub>O</td></tr><tr><td>10 μL</td><td>iProof buffer</td></tr><tr><td>5 μL</td><td>dNTP mix</td></tr><tr><td>1 μL</td><td>forward primer (10 μM)</td></tr><tr><td>1 μL</td><td>reverse primer (10 μM)</td></tr><tr><td>0.5 μL</td><td>iProof enzyme</td></tr><tr><td>1 μL</td><td>cDNA (from Subheading <xref rid="Sec10" ref-type="sec">3.4</xref>, <bold>step 2</bold>)</td></tr></tbody></table></table-wrap></p><p id="Par38"><italic>PCR</italic><italic>Cycling conditions</italic><table-wrap id="Tabb"><table frame="hsides" rules="groups"><thead><tr><th>Temperature</th><th>Time</th><th></th></tr></thead><tbody><tr><td>98 °C</td><td>3 min</td><td></td></tr><tr><td>98 °C</td><td>10 s</td><td rowspan="3">1.1.40 cycles</td></tr><tr><td>50 °C</td><td>30 s</td></tr><tr><td>72 °C</td><td>30 s</td></tr><tr><td>72 °C</td><td>5 min</td><td></td></tr><tr><td>10 °C</td><td>Hold</td><td></td></tr></tbody></table></table-wrap></p></list-item><list-item><p id="Par39">Gel purify PCR amplicons from 1% agarose using gel purification kit and following manufacturer’s instructions.</p></list-item><list-item><p id="Par40">Send each product for Sanger sequencing.</p></list-item><list-item><p id="Par41">Check Sanger sequencing chromatograms for overlapping peaks, indicative of mutations within a mixed viral population, as further described in [<xref ref-type="bibr" rid="CR12">12</xref>]</p></list-item></list><table-wrap id="Tab2"><label>Table 2</label><caption><p>Primer pairs and expected product sizes for tiled MERS-CoV spike PCR amplification</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Forward primer</th><th>Reverse primer</th><th>Expected PCR product size (bp)</th></tr></thead><tbody><tr><td>1</td><td>2</td><td>940</td></tr><tr><td>3</td><td>4</td><td>1571</td></tr><tr><td>5</td><td>6</td><td>2447</td></tr></tbody></table></table-wrap></p></sec></sec><sec id="Sec11"><title>Notes</title><p id="Par42"><list list-type="order"><list-item><p id="Par43">Importantly, this specific lentivector cassette is expressed under the Ef1α promoter, which allows for mid-level expression of the transgene as compared to other popular lentiviral transgene promoters such as CMV or CAGGS. This midlevel expression is ideal for semi-permissive selective pressure created by the transgene, in this case, <italic>dr</italic>DPP4 .</p></list-item><list-item><p id="Par44">The plating density of cells may vary from this suggested value, depending on growth kinetics. In general, cells should be plated to achieve approximately 80–90% confluency on the day of infection.</p></list-item><list-item><p id="Par45">Cytopathic effects may be gradual to appear. To increase selective pressures on a viral population which is beginning to show signs of adaptation, one can apply a population bottleneck in the subsequent passage by reducing the amount of viral supernatant passaged to the next cell culture. In this case, we recommend reducing the passage volume by approximately tenfold.</p></list-item><list-item><p id="Par46">In our initial study [<xref ref-type="bibr" rid="CR12">12</xref>], cytopathic effects were observed by the eighth passage; however, sequencing from earlier passages showed adaptive mutations emerging in the culture by the third passage. Depending on the strength of selection, the number of passages required to elicit adaptive mutations will vary.</p></list-item></list></p></sec></body><back><ack><title>Acknowledgment</title><p>Funding was provided by the Intramural research Program of the NIAID.</p></ack><ref-list id="Bib1"><title>References</title><ref id="CR1"><label>1.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Andino</surname><given-names>R</given-names></name><name><surname>Domingo</surname><given-names>E</given-names></name></person-group><article-title>Viral quasispecies</article-title><source>Virology</source><year>2015</year><volume>479-480</volume><fpage>46</fpage><lpage>51</lpage><pub-id pub-id-type="doi">10.1016/j.virol.2015.03.022</pub-id><pub-id pub-id-type="pmid">25824477</pub-id></element-citation></ref><ref id="CR2"><label>2.</label><element-citation publication-type="journal"><person-group 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