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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Reliable typing of <italic>MERS-CoV</italic> variants with a small genome fragment</title><author><name sortKey="Smits, Saskia L" sort="Smits, Saskia L" uniqKey="Smits S" first="Saskia L." last="Smits">Saskia L. Smits</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation><affiliation><nlm:aff id="aff0010">ViroClinics BioSciences BV, Marconistraat 16, 3029 AK Rotterdam, Netherlands</nlm:aff></affiliation></author><author><name sortKey="Raj, V Stalin" sort="Raj, V Stalin" uniqKey="Raj V" first="V. Stalin" last="Raj">V. Stalin Raj</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation></author><author><name sortKey="Pas, Suzan D" sort="Pas, Suzan D" uniqKey="Pas S" first="Suzan D." last="Pas">Suzan D. Pas</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation></author><author><name sortKey="Reusken, Chantal B E M" sort="Reusken, Chantal B E M" uniqKey="Reusken C" first="Chantal B. E. M." last="Reusken">Chantal B. E. M. Reusken</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation></author><author><name sortKey="Mohran, Khaled" sort="Mohran, Khaled" uniqKey="Mohran K" first="Khaled" last="Mohran">Khaled Mohran</name><affiliation><nlm:aff id="aff0015">Ministry of the Environment, Doha, Qatar</nlm:aff></affiliation><affiliation><nlm:aff id="aff0020">Biotechnology Research Department, Animal Health Research Institute, Agricultural Research Center, Egypt</nlm:aff></affiliation></author><author><name sortKey="Farag, Elmoubasher A B A" sort="Farag, Elmoubasher A B A" uniqKey="Farag E" first="Elmoubasher A. B. A." last="Farag">Elmoubasher A. B. A. Farag</name><affiliation><nlm:aff id="aff0025">Supreme Council of Health, Doha, Qatar</nlm:aff></affiliation></author><author><name sortKey="Al Romaihi, Hamad E" sort="Al Romaihi, Hamad E" uniqKey="Al Romaihi H" first="Hamad E." last="Al-Romaihi">Hamad E. Al-Romaihi</name><affiliation><nlm:aff id="aff0025">Supreme Council of Health, Doha, Qatar</nlm:aff></affiliation></author><author><name sortKey="Alhajri, Mohd M" sort="Alhajri, Mohd M" uniqKey="Alhajri M" first="Mohd M." last="Alhajri">Mohd M. Alhajri</name><affiliation><nlm:aff id="aff0025">Supreme Council of Health, Doha, Qatar</nlm:aff></affiliation></author><author><name sortKey="Haagmans, Bart L" sort="Haagmans, Bart L" uniqKey="Haagmans B" first="Bart L." last="Haagmans">Bart L. Haagmans</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation></author><author><name sortKey="Koopmans, Marion P" sort="Koopmans, Marion P" uniqKey="Koopmans M" first="Marion P." last="Koopmans">Marion P. Koopmans</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation><affiliation><nlm:aff id="aff0030">Virology Division, Centre for Infectious Diseases Research, Diagnostics and Screening, National Institute for Public Health and the Environment, Bilthoven 3720BA, Netherlands</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25728084</idno><idno type="pmc">7106551</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106551</idno><idno type="RBID">PMC:7106551</idno><idno type="doi">10.1016/j.jcv.2014.12.006</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000D95</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000D95</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Reliable typing of <italic>MERS-CoV</italic> variants with a small genome fragment</title><author><name sortKey="Smits, Saskia L" sort="Smits, Saskia L" uniqKey="Smits S" first="Saskia L." last="Smits">Saskia L. Smits</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation><affiliation><nlm:aff id="aff0010">ViroClinics BioSciences BV, Marconistraat 16, 3029 AK Rotterdam, Netherlands</nlm:aff></affiliation></author><author><name sortKey="Raj, V Stalin" sort="Raj, V Stalin" uniqKey="Raj V" first="V. Stalin" last="Raj">V. Stalin Raj</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation></author><author><name sortKey="Pas, Suzan D" sort="Pas, Suzan D" uniqKey="Pas S" first="Suzan D." last="Pas">Suzan D. Pas</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation></author><author><name sortKey="Reusken, Chantal B E M" sort="Reusken, Chantal B E M" uniqKey="Reusken C" first="Chantal B. E. M." last="Reusken">Chantal B. E. M. Reusken</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation></author><author><name sortKey="Mohran, Khaled" sort="Mohran, Khaled" uniqKey="Mohran K" first="Khaled" last="Mohran">Khaled Mohran</name><affiliation><nlm:aff id="aff0015">Ministry of the Environment, Doha, Qatar</nlm:aff></affiliation><affiliation><nlm:aff id="aff0020">Biotechnology Research Department, Animal Health Research Institute, Agricultural Research Center, Egypt</nlm:aff></affiliation></author><author><name sortKey="Farag, Elmoubasher A B A" sort="Farag, Elmoubasher A B A" uniqKey="Farag E" first="Elmoubasher A. B. A." last="Farag">Elmoubasher A. B. A. Farag</name><affiliation><nlm:aff id="aff0025">Supreme Council of Health, Doha, Qatar</nlm:aff></affiliation></author><author><name sortKey="Al Romaihi, Hamad E" sort="Al Romaihi, Hamad E" uniqKey="Al Romaihi H" first="Hamad E." last="Al-Romaihi">Hamad E. Al-Romaihi</name><affiliation><nlm:aff id="aff0025">Supreme Council of Health, Doha, Qatar</nlm:aff></affiliation></author><author><name sortKey="Alhajri, Mohd M" sort="Alhajri, Mohd M" uniqKey="Alhajri M" first="Mohd M." last="Alhajri">Mohd M. Alhajri</name><affiliation><nlm:aff id="aff0025">Supreme Council of Health, Doha, Qatar</nlm:aff></affiliation></author><author><name sortKey="Haagmans, Bart L" sort="Haagmans, Bart L" uniqKey="Haagmans B" first="Bart L." last="Haagmans">Bart L. Haagmans</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation></author><author><name sortKey="Koopmans, Marion P" sort="Koopmans, Marion P" uniqKey="Koopmans M" first="Marion P." last="Koopmans">Marion P. Koopmans</name><affiliation><nlm:aff id="aff0005">Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</nlm:aff></affiliation><affiliation><nlm:aff id="aff0030">Virology Division, Centre for Infectious Diseases Research, Diagnostics and Screening, National Institute for Public Health and the Environment, Bilthoven 3720BA, Netherlands</nlm:aff></affiliation></author></analytic><series><title level="j">Journal of Clinical Virology</title><idno type="ISSN">1386-6532</idno><idno type="eISSN">1873-5967</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Highlights</title><p><list list-type="simple" id="lis0005"><list-item id="lsti0005"><label>•</label><p id="par0005">A simple typing of <italic>MERS-CoV</italic> variants is crucial in monitoring the <italic>MERS-CoV</italic> outbreak.</p></list-item><list-item id="lsti0010"><label>•</label><p id="par0010">The developed <italic>MERS-CoV</italic> typing assay provides an indication of the <italic>MERS-CoV</italic> variant.</p></list-item><list-item id="lsti0015"><label>•</label><p id="par0015">It allows more informative initial screening in labs with basic sequencing capacity.</p></list-item><list-item id="lsti0020"><label>•</label><p id="par0020">The data aid in informing effective international preparedness and response.</p></list-item></list></p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="The Who Mers Cov Research Group" uniqKey="The Who Mers Cov Research Group">The Who Mers-Cov Research Group</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zaki, A M" uniqKey="Zaki A">A.M. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Clin Virol</journal-id><journal-id journal-id-type="iso-abbrev">J. Clin. Virol</journal-id><journal-title-group><journal-title>Journal of Clinical Virology</journal-title></journal-title-group><issn pub-type="ppub">1386-6532</issn><issn pub-type="epub">1873-5967</issn><publisher><publisher-name>The Authors. Published by Elsevier B.V.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25728084</article-id><article-id pub-id-type="pmc">7106551</article-id><article-id pub-id-type="publisher-id">S1386-6532(14)00468-5</article-id><article-id pub-id-type="doi">10.1016/j.jcv.2014.12.006</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Reliable typing of <italic>MERS-CoV</italic> variants with a small genome fragment</article-title></title-group><contrib-group><contrib contrib-type="author" id="aut0005"><name><surname>Smits</surname><given-names>Saskia L.</given-names></name><xref rid="aff0005" ref-type="aff">a</xref><xref rid="aff0010" ref-type="aff">b</xref></contrib><contrib contrib-type="author" id="aut0010"><name><surname>Raj</surname><given-names>V. Stalin</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="aut0015"><name><surname>Pas</surname><given-names>Suzan D.</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="aut0020"><name><surname>Reusken</surname><given-names>Chantal B.E.M.</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="aut0025"><name><surname>Mohran</surname><given-names>Khaled</given-names></name><xref rid="aff0015" ref-type="aff">c</xref><xref rid="aff0020" ref-type="aff">d</xref></contrib><contrib contrib-type="author" id="aut0030"><name><surname>Farag</surname><given-names>Elmoubasher A.B.A.</given-names></name><xref rid="aff0025" ref-type="aff">e</xref></contrib><contrib contrib-type="author" id="aut0035"><name><surname>Al-Romaihi</surname><given-names>Hamad E.</given-names></name><xref rid="aff0025" ref-type="aff">e</xref></contrib><contrib contrib-type="author" id="aut0040"><name><surname>AlHajri</surname><given-names>Mohd M.</given-names></name><xref rid="aff0025" ref-type="aff">e</xref></contrib><contrib contrib-type="author" id="aut0045"><name><surname>Haagmans</surname><given-names>Bart L.</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="aut0050"><name><surname>Koopmans</surname><given-names>Marion P.</given-names></name><email>m.koopmans@erasmusmc.nl</email><xref rid="aff0005" ref-type="aff">a</xref><xref rid="aff0030" ref-type="aff">f</xref><xref rid="cor0005" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff0005"><label>a</label>Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands</aff><aff id="aff0010"><label>b</label>ViroClinics BioSciences BV, Marconistraat 16, 3029 AK Rotterdam, Netherlands</aff><aff id="aff0015"><label>c</label>Ministry of the Environment, Doha, Qatar</aff><aff id="aff0020"><label>d</label>Biotechnology Research Department, Animal Health Research Institute, Agricultural Research Center, Egypt</aff><aff id="aff0025"><label>e</label>Supreme Council of Health, Doha, Qatar</aff><aff id="aff0030"><label>f</label>Virology Division, Centre for Infectious Diseases Research, Diagnostics and Screening, National Institute for Public Health and the Environment, Bilthoven 3720BA, Netherlands</aff><author-notes><corresp id="cor0005"><label>⁎</label>Corresponding author at: Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA, Rotterdam, the Netherlands. Tel.: +31 10 7044515; fax: +31 10 7044760. <email>m.koopmans@erasmusmc.nl</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>15</day><month>12</month><year>2014</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"><month>3</month><year>2015</year></pub-date><pub-date pub-type="epub"><day>15</day><month>12</month><year>2014</year></pub-date><volume>64</volume><fpage>83</fpage><lpage>87</lpage><history><date date-type="received"><day>19</day><month>8</month><year>2014</year></date><date date-type="rev-recd"><day>6</day><month>11</month><year>2014</year></date><date date-type="accepted"><day>8</day><month>12</month><year>2014</year></date></history><permissions><copyright-statement>© 2014 The Authors</copyright-statement><copyright-year>2014</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="author-highlights" id="abs0005"><title>Highlights</title><p><list list-type="simple" id="lis0005"><list-item id="lsti0005"><label>•</label><p id="par0005">A simple typing of <italic>MERS-CoV</italic> variants is crucial in monitoring the <italic>MERS-CoV</italic> outbreak.</p></list-item><list-item id="lsti0010"><label>•</label><p id="par0010">The developed <italic>MERS-CoV</italic> typing assay provides an indication of the <italic>MERS-CoV</italic> variant.</p></list-item><list-item id="lsti0015"><label>•</label><p id="par0015">It allows more informative initial screening in labs with basic sequencing capacity.</p></list-item><list-item id="lsti0020"><label>•</label><p id="par0020">The data aid in informing effective international preparedness and response.</p></list-item></list></p></abstract><abstract id="abs0010"><sec><title>Background</title><p><italic>Middle East Respiratory Syndrome coronavirus</italic> (<italic>MERS-CoV</italic>) is an emerging pathogen that causes lower respiratory tract infection in humans. Camels are the likely animal source for zoonotic infection, although exact transmission modes remain to be determined. Human-to-human transmission occurs sporadically. The wide geographic distribution of <italic>MERS-CoV</italic> among dromedary camels and ongoing transmissions to humans provides concern for the evolution of a <italic>MERS-CoV</italic> variant with efficient human-to-human transmission capabilities. Phylogenetic analysis of <italic>MERS-CoV</italic> has occurred by analysis of full-length genomes or multiple concatenated genome fragments, which is time-consuming, costly and limited to high viral load samples.</p></sec><sec><title>Objective</title><p>To develop a simple, reliable <italic>MERS-CoV</italic> variant typing assay to facilitate monitoring of <italic>MERS-CoV</italic> diversity in animals and humans.</p></sec><sec><title>Study Design</title><p>Phylogenetic analysis of presently known full-length <italic>MERS-CoV</italic> genomes was performed to identify genomic regions with sufficient phylogenetic content to allow reliable <italic>MERS-CoV</italic> variant typing. RT-PCR assays targeting these regions were designed and optimized.</p></sec><sec><title>Results</title><p>A reverse-transcription PCR assay for <italic>MERS-CoV</italic> targeting a 615 bp spike fragment provides a phylogenetic clustering of <italic>MERS-CoV</italic> variants comparable to that of full-length genomes. The detection limit corresponds to a cycle treshold value of ∼35 with standard upE real time PCR assays on RNA isolated from <italic>MERS-CoV</italic> EMC. Nasal swabs from RT-PCR positive camels (Ct values 12.9–32.2) yielded reliable sequence information in 14 samples.</p></sec><sec><title>Conclusions</title><p>We developed a simple, reliable <italic>MERS-CoV</italic> variant typing assay which is crucial in monitoring <italic>MERS-CoV</italic> circulation in real time with relatively little investment on location.</p></sec></abstract><kwd-group id="kwd0005"><title>Keywords</title><kwd><italic>MERS-CoV</italic></kwd><kwd>Diversity</kwd><kwd>Camel</kwd><kwd>Human</kwd><kwd>Type</kwd><kwd>Surveillance</kwd></kwd-group></article-meta></front><body><sec id="sec0005"><label>1</label><title>Background</title><p id="par0025"><italic>Middle East Respiratory Syndrome coronavirus</italic> (<italic>MERS-CoV; Family Coronaviridae</italic>) may cause severe lower respiratory tract infection in humans <xref rid="bib0005" ref-type="bibr">[1]</xref>, <xref rid="bib0010" ref-type="bibr">[2]</xref>. Camels are considered the likely animal source for zoonotic infection; sporadic human-to-human transmission does occur, but is considered to be inefficient based on currently available data <xref rid="bib0015" ref-type="bibr">[3]</xref>, <xref rid="bib0020" ref-type="bibr">[4]</xref>, <xref rid="bib0025" ref-type="bibr">[5]</xref>, <xref rid="bib0030" ref-type="bibr">[6]</xref>, <xref rid="bib0035" ref-type="bibr">[7]</xref>, <xref rid="bib0040" ref-type="bibr">[8]</xref>, <xref rid="bib0045" ref-type="bibr">[9]</xref>, <xref rid="bib0050" ref-type="bibr">[10]</xref>. Until recently, new <italic>MERS-CoV</italic> infections in humans were reported at a steady low rate reaching ∼200 confirmed human cases early 2014. In March and April 2014, however, a surge of <italic>MERS-CoV</italic> infections occurred mainly in hospitals around Jeddah, Kingdom of Saudi Arabia (KSA) and United Arab Emirates (UAE). This increased case load was in part attributed to an increase in community cases, but mostly to transmission within hospitals, with no evidence for evolution of a <italic>MERS-CoV</italic> variant with more efficient human-to-human transmission capabilities (WHO;<ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/disease/coronavirus_infections/MERS_CoV_Update_09_May_2014.pdf%3Fua" id="intr0255">http://www.who.int/csr/disease/coronavirus_infections/MERS_CoV_Update_09_May_2014.pdf?ua</ext-link><ext-link ext-link-type="uri" xlink:href="http://=" id="intr0260">=</ext-link><ext-link ext-link-type="uri" xlink:href="http://1" id="intr0265">1</ext-link>). The ongoing occurrence of new cases, however, and the finding that <italic>MERS-CoV</italic> is endemic in dromedary camels in a wide geographic region <xref rid="bib0015" ref-type="bibr">[3]</xref>, <xref rid="bib0050" ref-type="bibr">[10]</xref>, <xref rid="bib0055" ref-type="bibr">[11]</xref>, stresses the need for surveillance of strain diversity, to help unravel the epidemiology of this newly identified pathogen, and to provide a reference for studies into <italic>MERS-CoV</italic> evolution.</p><p id="par0030">All currently sequenced human and camel <italic>MERS-CoV</italic> genomes share >99% nucleotide identity across the ∼30 kb genome. Phylogenetic analysis has occurred mainly by analysing full-length genomes or multiple concatenated genome fragments, to provide reliable phylogenetic information <xref rid="bib0025" ref-type="bibr">[5]</xref>, <xref rid="bib0060" ref-type="bibr">[12]</xref>, <xref rid="bib0065" ref-type="bibr">[13]</xref>, <xref rid="bib0070" ref-type="bibr">[14]</xref>. However, full length genome sequencing capacity is not widely available, and requires relatively high viral load, leading to limited success when trying to sequence animal or human samples <xref rid="bib0025" ref-type="bibr">[5]</xref>.</p></sec><sec id="sec0010"><label>2</label><title>Objectives</title><p id="par0035">An accurate typing of <italic>MERS-CoV</italic> variants, preferably with a simple assay encompassing a short region of the <italic>MERS-CoV</italic> genome, is crucial in monitoring the <italic>MERS-CoV</italic> outbreak in real time with relatively little investments on location. In this study, we describe the development of a <italic>MERS-CoV</italic> variant typing assay, which can be used in monitoring <italic>MERS-CoV</italic> circulation, especially when more information on virus type is required rapidly from a large number of viruses from animals/humans.</p></sec><sec id="sec0015"><label>3</label><title>Study design</title><sec id="sec0020"><label>3.1</label><title>In silico analysis</title><p id="par0040">Full or near full-length <italic>MERS-CoV</italic> genomes encompassing nucleotides 215–29770 (numbering corresponding to <italic>MERS-CoV</italic> EMC genome <ext-link ext-link-type="uri" xlink:href="ncbi-n:JX869059" id="intr0270">JX869059</ext-link>) were aligned with MAFFT version 7 (<ext-link ext-link-type="uri" xlink:href="http://mafft.cbrc.jp/alignment/server/" id="intr0275">http://mafft.cbrc.jp/alignment/server/</ext-link>) (<xref rid="tbl0005" ref-type="table">Table 1</xref>). To remove the redundancy from the dataset, FastGroupII analysis (<ext-link ext-link-type="uri" xlink:href="http://fastgroup.sdsu.edu/fg_tools.htm" id="intr0280">http://fastgroup.sdsu.edu/fg_tools.htm</ext-link>) was performed grouping all currently available viral genomes based on nucleotide composition, resulting in 15 groups (<xref rid="tbl0005" ref-type="table">Table 1</xref>). One viral genome from each group was taken as representative in subsequent analyses. A summary of nucleotide positions that vary across the genomes was created using BioEdit v7.2.0 <xref rid="bib0075" ref-type="bibr">[15]</xref> and the number of nucleotide positions with variations was plotted over 1000 nucleotide windows. PhyML trees were generated using Seaview 4 software with the approximate likelihood ratio test based on a Shimodaira–Hasegawa-like procedure which used general time reversible as substitution model. Nearest neighbor interchange, subtree pruning, and regrafting-based tree search algorithms were used to estimate tree topologies, as described previously <xref rid="bib0025" ref-type="bibr">[5]</xref>.<table-wrap position="float" id="tbl0005"><label>Table 1</label><caption><p>Viral genomes used in this study.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left">Group name</th><th align="left">Sequences</th><th align="left">Accession number</th></tr></thead><tbody><tr><td align="left">RIYADH_9_2013</td><td align="left">RIYADH_9_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ156869" id="intr0010">KJ156869</ext-link></td></tr><tr><td align="left"></td><td align="left">RIYADH_2_2012</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600652" id="intr0015">KF600652</ext-link></td></tr><tr><td align="left">AL-HASA_15_2013</td><td align="left">AL-HASA_19_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600632" id="intr0020">KF600632</ext-link></td></tr><tr><td align="left"></td><td align="left">BURAIDAH_1_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600630" id="intr0025">KF600630</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_2_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF186566" id="intr0030">KF186566</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_21_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600634" id="intr0035">KF600634</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_25_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ156866" id="intr0040">KJ156866</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_17_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600647" id="intr0045">KF600647</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_15_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600645" id="intr0050">KF600645</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_16_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600644" id="intr0055">KF600644</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_12_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600627" id="intr0060">KF600627</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_3_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF186565" id="intr0065">KF186565</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_4_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF186564" id="intr0070">KF186564</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_18_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600651" id="intr0075">KF600651</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_1_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF186567" id="intr0080">KF186567</ext-link></td></tr><tr><td align="left"></td><td align="left">AL-HASA_15_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600645" id="intr0085">KF600645</ext-link></td></tr><tr><td align="left">FRA/UAE_2012</td><td align="left">KFU_HKU1_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ650297" id="intr0090">KJ650297</ext-link></td></tr><tr><td align="left"></td><td align="left">KFU_HKU19DAM2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ650296" id="intr0095">KJ650296</ext-link></td></tr><tr><td align="left"></td><td align="left">KFU_HKU13_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ650295" id="intr0100">KJ650295</ext-link></td></tr><tr><td align="left"></td><td align="left">FRA/UAE_2012</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF745068" id="intr0105">KF745068</ext-link></td></tr><tr><td align="left">HAFR-AL-BATIN_6_2013</td><td align="left">HAFR-AL-BATIN_6_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ156874" id="intr0110">KJ156874</ext-link></td></tr><tr><td align="left"></td><td align="left">HAFR-AL-BATIN_2_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ156910" id="intr0115">KJ156910</ext-link></td></tr><tr><td align="left"></td><td align="left">HAFR-AL-BATIN_1_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600628" id="intr0120">KF600628</ext-link></td></tr><tr><td align="left">RIYADH_5_2013</td><td align="left">RIYADH_5_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ156944" id="intr0125">KJ156944</ext-link></td></tr><tr><td align="left"></td><td align="left">RIYADH_4_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ156952" id="intr0130">KJ156952</ext-link></td></tr><tr><td align="left"></td><td align="left">TAIF_1_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ156949" id="intr0135">KJ156949</ext-link></td></tr><tr><td align="left"></td><td align="left">JEDDAH_1_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ556336" id="intr0140">KJ556336</ext-link></td></tr><tr><td align="left"></td><td align="left">WADI-AD-DAWASIR_1_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ156881" id="intr0145">KJ156881</ext-link></td></tr><tr><td align="left">QATAR_3_2013</td><td align="left">QATAR_3_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF961221" id="intr0150">KF961221</ext-link></td></tr><tr><td align="left"></td><td align="left">QATAR_4_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF961222" id="intr0155">KF961222</ext-link></td></tr><tr><td align="left">RIYADH_1_2012</td><td align="left">RIYADH_1_2012</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600612" id="intr0160">KF600612</ext-link></td></tr><tr><td align="left"></td><td align="left">BISHA_1_2012</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600620" id="intr0165">KF600620</ext-link></td></tr><tr><td align="left">RIYADH_3_2013</td><td align="left">RIYADH_3_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF600613" id="intr0170">KF600613</ext-link></td></tr><tr><td align="left">Egypt_Camel_NRCE-HKU205_2014</td><td align="left">Egypt_Camel_NRCE-HKU205_2014</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ477102" id="intr0175">KJ477102</ext-link></td></tr><tr><td align="left">ENGLAND/QATAR_2012</td><td align="left">ENGLAND/QATAR_2012</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KC667074" id="intr0180">KC667074</ext-link></td></tr><tr><td align="left">EMC_2012</td><td align="left">EMC_2012</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:JX869059" id="intr0185">JX869059</ext-link></td></tr><tr><td align="left">RIYADH_14_2013</td><td align="left">RIYADH_14_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ156934" id="intr0190">KJ156934</ext-link></td></tr><tr><td align="left">Munich/AbuDhabi_2013</td><td align="left">Munich/AbuDhabi_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF192507" id="intr0195">KF192507</ext-link></td></tr><tr><td align="left">Qatar_Camel_2_2014</td><td align="left">Qatar_Camel_2_2014</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ650098" id="intr0200">KJ650098</ext-link></td></tr><tr><td align="left">JORDAN/N3_2012</td><td align="left">JORDAN/N3_2012</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KC776174" id="intr0205">KC776174</ext-link></td></tr><tr><td align="left"></td><td align="left">Not in variant analysis</td><td align="left"></td></tr><tr><td align="left"></td><td align="left">KSA_CAMEL_363_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ713298" id="intr0210">KJ713298</ext-link></td></tr><tr><td align="left"></td><td align="left">KSA_CAMEL_376_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ713299" id="intr0215">KJ713299</ext-link></td></tr><tr><td align="left"></td><td align="left">KSA_Camel_378_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ713296" id="intr0220">KJ713296</ext-link></td></tr><tr><td align="left"></td><td align="left">KSA_CAMEL_503_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ713297" id="intr0225">KJ713297</ext-link></td></tr><tr><td align="left"></td><td align="left">KSA_CAMEL_505_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ713295" id="intr0230">KJ713295</ext-link></td></tr><tr><td align="left"></td><td align="left">Jeddah_Camel_1_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF917527" id="intr0235">KF917527</ext-link></td></tr><tr><td align="left"></td><td align="left">Jeddah_Human_1_2013</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KF958702" id="intr0240">KF958702</ext-link></td></tr><tr><td align="left"></td><td align="left">Florida/USA_2_Saudi Arabia_2014</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ829365" id="intr0245">KJ829365</ext-link></td></tr><tr><td align="left"></td><td align="left">Indiana/USA-1_Saudi Arabia_2014</td><td align="left"><ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ813439" id="intr0250">KJ813439</ext-link></td></tr></tbody></table></table-wrap></p></sec><sec id="sec0025"><label>3.2</label><title><italic>MERS-CoV</italic> typing RT-PCR</title><p id="par0045">Total nucleic acids were isolated using an automated MagNAPure 96 extraction with the total nucleic acid isolation kit (Roche, Mannheim, Germany) as described previously <xref rid="bib0065" ref-type="bibr">[13]</xref>. The partial S2 domain of <italic>MERS-CoV</italic> spike (corresponding to nucleotides 23781–24395 of <italic>MERS-CoV</italic> EMC genome <ext-link ext-link-type="uri" xlink:href="ncbi-n:JX869059" id="intr0285">JX869059</ext-link>) was amplified with the OneStep RT-PCR Kit (Qiagen) using 1x QIAGEN OneStep RT-PCR Buffer, 400 μM of each dNTP, 2 μl QIAGEN OneStep RT-PCR Enzyme Mix and 0.6 μM of primers VS804 (5′-TCAGGTTGATCAACTTAATAGT-3′) and VS805 (5′-TTGAGTAATGCCAACACCGTT-3′) in a volume of 25 μl; 30 min 50 °C, 15 min 95 °C, 40 cycles of 0.5 min 94 °C, 0.5 min 50 °C, 1 min 72 °C, and a final extension of 10 min 72 °C. A nested PCR was performed using 1x PCR Buffer, 2 mM MgCl2, 200 μM of each dNTP, 20 μM primers VS804 and VS805 and 2.5 units HotStarTaq DNA polymerase (Qiagen) in a volume of 50 μl; 15 min 95 °C, 40 cycles of 0.5 min 94 °C, 0.5 min 50 °C, 1 min 72 °C, and a final extension of 10 min 72 °C. Amplicons were sequenced directly on both strands with the BigDye Terminator version 3.1 cycle sequencing kit on an ABI PRISM 3100 genetic analyser (Applied Biosystems).</p></sec><sec id="sec0030"><label>3.3</label><title>Samples</title><p id="par0050">The detection limit of the assay was determined on RNA isolated from 10x dilutions of cell culture derived <italic>MERS-CoV</italic> EMC_2012 (<ext-link ext-link-type="uri" xlink:href="ncbi-n:JX869059" id="intr0290">JX869059</ext-link>), as described above. Virus stocks were prepared as described previously <xref rid="bib0080" ref-type="bibr">[16]</xref>. RNA was isolated from serial 10-fold dilutions of <italic>MERS-CoV</italic> EMC_2012 <xref rid="bib0065" ref-type="bibr">[13]</xref>. Serial 10-fold dilutions of this RNA were amplified in parallel with the <italic>MERS-CoV</italic> variant typing assay described above and the upE and N gene real time PCR assays <xref rid="bib0085" ref-type="bibr">[17]</xref>, <xref rid="bib0090" ref-type="bibr">[18]</xref>. The sensitivity of the <italic>MERS-CoV</italic> variant typing assay was expressed as cycle threshold value based on the upE real time PCR assay.</p><p id="par0055">On May 13 and 15, 2014, the first two <italic>MERS-CoV</italic> infected patients in the Netherlands who became infected upon travel to Saudi Arabia were reported to WHO; throat swabs from these patients were available <xref rid="bib0095" ref-type="bibr">[19]</xref>.</p><p id="par0060">In February and April 2014, nasal swabs were taken from dromedary camels of different age and sex from a slaughterhouse in Doha, Qatar <xref rid="bib0065" ref-type="bibr">[13]</xref>, which were available for this study.</p></sec></sec><sec id="sec0035"><label>4</label><title>Results</title><p id="par0065">To identify genome regions for reliable phylogenetic analysis comparable to that of full-length genome, (near) full-length <italic>MERS-CoV</italic> genomes (<xref rid="tbl0005" ref-type="table">Table 1</xref>) were aligned. Viral genomes that were 99.9% identical were grouped and one viral genome from each group was taken as representative in the analysis (<xref rid="tbl0005" ref-type="table">Table 1</xref>) and a PhyML tree was generated (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>A). Four major <italic>MERS-CoV</italic> clusters can be discerned, represented by <italic>MERS-CoV</italic> Qatar_3_2014, England/Qatar_2012, EMC_2012, and Egypt_Camel_NRCE-HKU205_2014 (<ext-link ext-link-type="uri" xlink:href="ncbi-n:KF961221" id="intr0295">KF961221</ext-link>, <ext-link ext-link-type="uri" xlink:href="ncbi-n:KC667074" id="intr0300">KC667074</ext-link>, <ext-link ext-link-type="uri" xlink:href="ncbi-n:JX869059" id="intr0305">JX869059</ext-link>, <ext-link ext-link-type="uri" xlink:href="ncbi-n:KJ477102" id="intr0310">KJ477102</ext-link>), which have been identified previously in analyses based on full-length genomes or concatenated genome fragments <xref rid="bib0025" ref-type="bibr">[5]</xref>, <xref rid="bib0060" ref-type="bibr">[12]</xref>, <xref rid="bib0065" ref-type="bibr">[13]</xref>, <xref rid="bib0070" ref-type="bibr">[14]</xref>. The number of single nucleotide polymorphisms (SNPs) across the 15 representative <italic>MERS-CoV</italic> genomes was plotted over 1000 nt windows (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>B). Four genome fragments, two located in ORF1a, one in S and one in ORF4b, showed a relatively high number of SNPs (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>B) and for this reason already had been used as concatenated genome fragment for phylogenetic analysis <xref rid="bib0060" ref-type="bibr">[12]</xref>. However, phylogenetic trees created for these four fragments separately did not accurately reflect the phylogenetic positions of the currently known full-length <italic>MERS-CoV</italic> genomes (data not shown).<fig id="fig0005"><label>Fig. 1</label><caption><p>Characterization of <italic>MERS-CoV</italic> variation. (A) PhyML tree of full-length genome sequences of 15 distinct MERS coronavirus variants based on analysis of nucleotide sequence diversity across the genome. Four major <italic>MERS-CoV</italic> clusters are indicated (A, B1, B2, C). (B) The number of nucleotide positions with variations over 15 representative <italic>MERS-CoV</italic> genomes (Supplementary Table 1) was plotted over 1000 nucleotide windows. A schematic diagram of the <italic>MERS-CoV</italic> genome is depicted below the graph, and four fragments with the highest number of single nucleotide polymorphisms (SNPs) are indicated with black boxes. The dotted line indicates the average number of SNPs per 1000 nt window added with 1x standard deviation. In red, nucleotide positions displaying considerable phylogenetic information regarding four identified <italic>MERS-CoV</italic> variant clusters and the red box indicates the 615 bp fragment of the here-described <italic>MERS-CoV</italic> variant typing assay. (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_lrg"></graphic></fig></p><p id="par0070">In a subsequent analysis all identified SNPs were inspected visually and regions containing SNPs with phylogenetic information regarding the previously identified four clusters of viruses were identified (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>A). The S2 domain of the spike protein contains a number of these mutations (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>B). A fragment of 615 bp, containing three of these SNPs, provided a phylogenetic tree similar to the one obtained upon full-genome analysis regarding the previously identified four <italic>MERS-CoV</italic> clusters (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>), whereas other <italic>MERS-CoV</italic> genome regions did not provide similar results. As observed for the full-length genomes, human, and camel <italic>MERS-CoV</italic> genomes shared >99% nucleotide identity across the 615 bp S2 domain fragment. <italic>MERS-CoV</italic> genomes that were released recently and not taken along in the variation analysis were typed using the 615 bp S2 domain and clustered similar to their phylogenetic positions as upon full genome analysis (<xref rid="fig0010" ref-type="fig">Fig. 2</xref> and <xref rid="tbl0005" ref-type="table">Table 1</xref>), thereby validating the assay.<fig id="fig0010"><label>Fig. 2</label><caption><p>Phylogenetic analysis of human and camel <italic>MERS-CoVs</italic> using the <italic>MERS-CoV</italic> variant typing assay. A PhyML tree was generated from a spike S2 domain genome fragment corresponding to nt 23781–24395 of <italic>MERS-CoV</italic> EMC genome (accession number <ext-link ext-link-type="uri" xlink:href="ncbi-n:JX869059" id="intr0005">JX869059</ext-link>) for known <italic>MERS-CoV</italic> genomes (<xref rid="tbl0005" ref-type="table">Table 1</xref>) and the first two Dutch <italic>MERS-CoV</italic> patients (boxed). In red are newly identified camel sequences from a slaughterhouse in Qatar. (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="gr2_lrg"></graphic></fig></p><p id="par0075">A sequencing RT-PCR targeting the identified genome fragment was developed and optimized using RNA isolated from cell culture-derived <italic>MERS-CoV</italic>. In limiting dilution experiments, the <italic>MERS-CoV</italic> variant typing assay amplified the 615 bp fragment down to a cycle treshold value of ∼35 as determined by diagnostic upE real time PCR assays <xref rid="bib0085" ref-type="bibr">[17]</xref>, <xref rid="bib0090" ref-type="bibr">[18]</xref>. The <italic>MERS-CoV</italic> variant typing assay was used to type RT-PCR positive nose swabs from the first two human Dutch <italic>MERS-CoV</italic> cases in 2014. The results showed grouping consistent with previous findings based on long sequence fragments <xref rid="bib0095" ref-type="bibr">[19]</xref> (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>). In addition, the <italic>MERS-CoV</italic> variant typing assay was performed on camel samples from a slaughterhouse in Qatar <xref rid="bib0065" ref-type="bibr">[13]</xref> and sequences for 14 <italic>MERS-CoV</italic> positive animals with cycle threshold values ranging from 12.9 to 32.2 as determined by UpE real time RT-PCR <xref rid="bib0085" ref-type="bibr">[17]</xref>, <xref rid="bib0090" ref-type="bibr">[18]</xref> were obtained (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>). Five different camel <italic>MERS-CoV</italic> variants in clusters B1 and B2 were detected, without the need for full genome sequencing.</p></sec><sec id="sec0040"><label>5</label><title>Discussion</title><p id="par0080">Phylogenetic analysis of representative <italic>MERS-CoV</italic> full-length genomes indicated that four regions in the <italic>MERS-CoV</italic> genome exist with a substantially higher nucleotide variation across genomes. However, phylogenetic analysis of these genome regions separately did not provide reliable phylogenetic information, in contrast to an analysis of the concatenated fragments <xref rid="bib0025" ref-type="bibr">[5]</xref>, <xref rid="bib0060" ref-type="bibr">[12]</xref>, <xref rid="bib0095" ref-type="bibr">[19]</xref>. Subsequent analyses revealed a region in the open reading frame that encodes the spike protein with a number of positions in which nucleotide variation occurs between <italic>MERS-CoV</italic> variants with a strong phylogenetic signal regarding previously identified clusters of viruses based on full-length <italic>MERS-CoV</italic> genomes. The here-described <italic>MERS-CoV</italic> variant typing assay based on a 615 bp spike fragment provides a crude indication of the <italic>MERS-CoV</italic> variant under study on the basis of four identified clusters of <italic>MERS-CoV</italic> variants, exemplified by <italic>MERS-CoV</italic> Qatar_3_2014, England/Qatar_2012, EMC_2012, and Egypt_Camel_NRCE-HKU205_2014. In silico, the assay accurately typed all currently known <italic>MERS-CoVs</italic> for which full-length genomes are available, including the recently released Florida/USA_2_KSA_2014 and Indiana/USA-1_KSA_2014 strains. This was confirmed by typing of samples available in our laboratory from the first two Dutch <italic>MERS-CoV</italic> cases that phylogenetically grouped with Indiana/USA-1_KSA_2014 and other viruses in cluster I <xref rid="bib0095" ref-type="bibr">[19]</xref> and from camels from a slaughterhouse in Doha, Qatar <xref rid="bib0065" ref-type="bibr">[13]</xref>. The observed detection limit of a cycle treshold value of ∼35 allows variant typing in clinical samples obtained from humans and animals with relatively low viral loads. It enables inclusion of samples in phylogenetic analysis that would not have been included when only full length genomes would have been accepted.</p><p id="par0085">This <italic>MERS-CoV</italic> variant typing assay, targeting a part of the <italic>MERS-CoV</italic> spike gene, is a relatively simple RT-PCR sequencing assay that could be performed more widely as initial screening assay in laboratories with basic sequencing capacity. It provides accurate crude <italic>MERS-CoV</italic> type information, applicable in monitoring viral variants in real time. New variants identified through this initial screening could then be sent to a reference laboratory for further characterization. The continued occurrence of transmission between humans in health care and family settings is an ongoing concern as stated by the World Health Organization (<ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/disease/coronavirus_infections/MERS_CoV_Update_27_March_2014.pdf%3Fua=1" id="intr0315">http://www.who.int/csr/disease/coronavirus_infections/MERS_CoV_Update_27_March_2014.pdf?ua=1</ext-link>), although the outbreaks appear to be self‐limiting or extinguishable with rigorous implementation of appropriate infection control guidelines at present. However, as the primary route of transmission to humans is uninterrupted, human-to-human transmissions will continue to occur. The data obtained from the <italic>MERS-CoV</italic> variant typing assay would aid in informing the most effective international preparedness and response, allowing ad hoc risk assessment and implementation of containment strategies if necessary.</p></sec><sec id="sec0045"><title>Conflict of interest</title><p id="par0090">Saskia L. Smits is part time employed by Viroclinics Biosciences BV. This does not alter our adherence to all the policies on sharing data and materials.</p><p id="par0095">Bart Haagmans has a patent on <italic>MERS-CoV</italic> field.</p></sec><sec id="sec0050"><title>Funding</title><p id="par0100">This work was funded by ZonMW TOP project 91213058.</p></sec><sec id="sec0055"><title>Ethical approval</title><p id="par0105">All procedures were performed in compliance with relevant laws and institutional guidelines and in accordance with the Declaration of Helsinki.</p></sec><sec id="sec0060"><title>Authors’ contributions</title><p id="par0110">All authors contributed to gathering and analysis of the information. 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Eurosurveillance 19:20817.</mixed-citation></ref></ref-list><sec id="sec0070" sec-type="supplementary-material"><label>Appendix A</label><title>Supplementary data</title><p id="par0125">The following are Supplementary data to this article:<supplementary-material content-type="local-data" id="upi0005"><media xlink:href="mmc1.xlsx"></media></supplementary-material></p></sec><ack id="ack0005"><title>Acknowledgment</title><p>This work was funded by <funding-source id="gs0005">ZonMW TOP</funding-source> project 91213058Z. This does not alter our adherence to all the policies on sharing data and materials.</p></ack><fn-group><fn id="sec0065" fn-type="supplementary-material"><label>Appendix A</label><p id="par0120">Supplementary data associated with this article can be found, in the online version, at <ext-link ext-link-type="doi" xlink:href="10.1016/j.jcv.2014.12.006" id="intr0320">http://dx.doi.org/10.1016/j.jcv.2014.12.006</ext-link>.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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