Molecular characterization of human coronaviruses and their circulation dynamics in Kenya, 2009–2012
Identifieur interne : 000351 ( Pmc/Corpus ); précédent : 000350; suivant : 000352Molecular characterization of human coronaviruses and their circulation dynamics in Kenya, 2009–2012
Auteurs : Lenata A. Sipulwa ; Juliette R. Ongus ; Rodney L. Coldren ; Wallace D. BulimoSource :
- Virology Journal [ 1743-422X ] ; 2016.
Abstract
Human Coronaviruses (HCoV) are a common cause of respiratory illnesses and are responsible for considerable morbidity and hospitalization across all age groups especially in individuals with compromised immunity. There are six known species of HCoV: HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, MERS-CoV and SARS-HCoV. Although studies have shown evidence of global distribution of HCoVs, there is limited information on their presence and distribution in Kenya.
HCoV strains that circulated in Kenya were retrospectively diagnosed and molecularly characterized. A total of 417 nasopharyngeal specimens obtained between January 2009 and December 2012 from around Kenya were analyzed by a real time RT-PCR using HCoV-specific primers. HCoV-positive specimens were subsequently inoculated onto monolayers of LL-CMK2 cells. The isolated viruses were characterized by RT-PCR amplification and sequencing of the partial polymerase (
The prevalence of HCoV infection was as follows: out of the 417 specimens, 35 (8.4 %) were positive for HCoV, comprising 10 (2.4 %) HCoV-NL63, 12 (2.9 %) HCoV-OC43, 9 (2.1 %) HCoV-HKU1, and 4 (1 %) HCoV-229E. The Kenyan HCoV strains displayed high sequence homology to the prototypes and contemporaneous strains. Evolution analysis showed that the Kenyan HCoV-OC43 and HCoV-NL63 isolates were under purifying selection. Phylogenetic evolutionary analyses confirmed the identities of three HCoV-HKU1, five HCoV-NL63, eight HCoV-OC43 and three HCoV-229E.
There were yearly variations in the prevalence and circulation patterns of individual HCoVs in Kenya. This paper reports on the first molecular characterization of human Coronaviruses in Kenya, which play an important role in causing acute respiratory infections among children.
Url:
DOI: 10.1186/s12985-016-0474-x
PubMed: 26833249
PubMed Central: 4736488
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Molecular characterization of human coronaviruses and their circulation dynamics in Kenya, 2009–2012</title><author><name sortKey="Sipulwa, Lenata A" sort="Sipulwa, Lenata A" uniqKey="Sipulwa L" first="Lenata A." last="Sipulwa">Lenata A. Sipulwa</name><affiliation><nlm:aff id="Aff1">College of Health Sciences (COHES), Jomo Kenyatta University of Agriculture and Technology, (JKUAT), Nairobi, Kenya</nlm:aff></affiliation></author><author><name sortKey="Ongus, Juliette R" sort="Ongus, Juliette R" uniqKey="Ongus J" first="Juliette R." last="Ongus">Juliette R. Ongus</name><affiliation><nlm:aff id="Aff1">College of Health Sciences (COHES), Jomo Kenyatta University of Agriculture and Technology, (JKUAT), Nairobi, Kenya</nlm:aff></affiliation></author><author><name sortKey="Coldren, Rodney L" sort="Coldren, Rodney L" uniqKey="Coldren R" first="Rodney L." last="Coldren">Rodney L. Coldren</name><affiliation><nlm:aff id="Aff2">Department of Emerging Infectious Diseases, US Army Medical Research Unit–Kenya, P.O. Box 606 00621, Village Market, Nairobi, Kenya</nlm:aff></affiliation><affiliation><nlm:aff id="Aff3">Special Foreign Activity of the Walter Reed Army Institute of Research, Silver Spring, MD USA</nlm:aff></affiliation></author><author><name sortKey="Bulimo, Wallace D" sort="Bulimo, Wallace D" uniqKey="Bulimo W" first="Wallace D." last="Bulimo">Wallace D. Bulimo</name><affiliation><nlm:aff id="Aff2">Department of Emerging Infectious Diseases, US Army Medical Research Unit–Kenya, P.O. Box 606 00621, Village Market, Nairobi, Kenya</nlm:aff></affiliation><affiliation><nlm:aff id="Aff3">Special Foreign Activity of the Walter Reed Army Institute of Research, Silver Spring, MD USA</nlm:aff></affiliation><affiliation><nlm:aff id="Aff4">Department of Biochemistry, School of Medicine, University of Nairobi, Nairobi, Kenya</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26833249</idno><idno type="pmc">4736488</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736488</idno><idno type="RBID">PMC:4736488</idno><idno type="doi">10.1186/s12985-016-0474-x</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000351</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000351</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Molecular characterization of human coronaviruses and their circulation dynamics in Kenya, 2009–2012</title><author><name sortKey="Sipulwa, Lenata A" sort="Sipulwa, Lenata A" uniqKey="Sipulwa L" first="Lenata A." last="Sipulwa">Lenata A. Sipulwa</name><affiliation><nlm:aff id="Aff1">College of Health Sciences (COHES), Jomo Kenyatta University of Agriculture and Technology, (JKUAT), Nairobi, Kenya</nlm:aff></affiliation></author><author><name sortKey="Ongus, Juliette R" sort="Ongus, Juliette R" uniqKey="Ongus J" first="Juliette R." last="Ongus">Juliette R. Ongus</name><affiliation><nlm:aff id="Aff1">College of Health Sciences (COHES), Jomo Kenyatta University of Agriculture and Technology, (JKUAT), Nairobi, Kenya</nlm:aff></affiliation></author><author><name sortKey="Coldren, Rodney L" sort="Coldren, Rodney L" uniqKey="Coldren R" first="Rodney L." last="Coldren">Rodney L. Coldren</name><affiliation><nlm:aff id="Aff2">Department of Emerging Infectious Diseases, US Army Medical Research Unit–Kenya, P.O. Box 606 00621, Village Market, Nairobi, Kenya</nlm:aff></affiliation><affiliation><nlm:aff id="Aff3">Special Foreign Activity of the Walter Reed Army Institute of Research, Silver Spring, MD USA</nlm:aff></affiliation></author><author><name sortKey="Bulimo, Wallace D" sort="Bulimo, Wallace D" uniqKey="Bulimo W" first="Wallace D." last="Bulimo">Wallace D. Bulimo</name><affiliation><nlm:aff id="Aff2">Department of Emerging Infectious Diseases, US Army Medical Research Unit–Kenya, P.O. Box 606 00621, Village Market, Nairobi, Kenya</nlm:aff></affiliation><affiliation><nlm:aff id="Aff3">Special Foreign Activity of the Walter Reed Army Institute of Research, Silver Spring, MD USA</nlm:aff></affiliation><affiliation><nlm:aff id="Aff4">Department of Biochemistry, School of Medicine, University of Nairobi, Nairobi, Kenya</nlm:aff></affiliation></author></analytic><series><title level="j">Virology Journal</title><idno type="eISSN">1743-422X</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>Human Coronaviruses (HCoV) are a common cause of respiratory illnesses and are responsible for considerable morbidity and hospitalization across all age groups especially in individuals with compromised immunity. There are six known species of HCoV: HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, MERS-CoV and SARS-HCoV. Although studies have shown evidence of global distribution of HCoVs, there is limited information on their presence and distribution in Kenya.</p></sec><sec><title>Methods</title><p>HCoV strains that circulated in Kenya were retrospectively diagnosed and molecularly characterized. A total of 417 nasopharyngeal specimens obtained between January 2009 and December 2012 from around Kenya were analyzed by a real time RT-PCR using HCoV-specific primers. HCoV-positive specimens were subsequently inoculated onto monolayers of LL-CMK2 cells. The isolated viruses were characterized by RT-PCR amplification and sequencing of the partial polymerase (<italic>po</italic>l) gene.</p></sec><sec><title>Results</title><p>The prevalence of HCoV infection was as follows: out of the 417 specimens, 35 (8.4 %) were positive for HCoV, comprising 10 (2.4 %) HCoV-NL63, 12 (2.9 %) HCoV-OC43, 9 (2.1 %) HCoV-HKU1, and 4 (1 %) HCoV-229E. The Kenyan HCoV strains displayed high sequence homology to the prototypes and contemporaneous strains. Evolution analysis showed that the Kenyan HCoV-OC43 and HCoV-NL63 isolates were under purifying selection. Phylogenetic evolutionary analyses confirmed the identities of three HCoV-HKU1, five HCoV-NL63, eight HCoV-OC43 and three HCoV-229E.</p></sec><sec><title>Conclusions</title><p>There were yearly variations in the prevalence and circulation patterns of individual HCoVs in Kenya. This paper reports on the first molecular characterization of human Coronaviruses in Kenya, which play an important role in causing acute respiratory infections among children.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Lai, Mm" uniqKey="Lai M">MM Lai</name></author><author><name sortKey="Cavanagh, D" uniqKey="Cavanagh D">D Cavanagh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, Hj" uniqKey="Lee H">HJ Lee</name></author><author><name sortKey="Shieh, Ck" uniqKey="Shieh C">CK Shieh</name></author><author><name sortKey="Gorbalenya, Ae" uniqKey="Gorbalenya A">AE Gorbalenya</name></author><author><name sortKey="Koonin, Ev" uniqKey="Koonin E">EV Koonin</name></author><author><name sortKey="La Monica, N" uniqKey="La Monica N">N La Monica</name></author><author><name sortKey="Tuler, J" uniqKey="Tuler J">J Tuler</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Xu, Hf" uniqKey="Xu H">HF Xu</name></author><author><name sortKey="Wang, M" uniqKey="Wang M">M Wang</name></author><author><name sortKey="Zhang, Zb" uniqKey="Zhang Z">ZB Zhang</name></author><author><name sortKey="Zou, Xz" uniqKey="Zou X">XZ Zou</name></author><author><name sortKey="Gao, Y" uniqKey="Gao Y">Y Gao</name></author><author><name sortKey="Liu, Xn" uniqKey="Liu X">XN Liu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bredenbeek, Pj" uniqKey="Bredenbeek P">PJ Bredenbeek</name></author><author><name sortKey="Snijder, Ej" uniqKey="Snijder E">EJ Snijder</name></author><author><name sortKey="Noten, Fh" uniqKey="Noten F">FH Noten</name></author><author><name sortKey="Den Boon, Ja" uniqKey="Den Boon J">JA den Boon</name></author><author><name sortKey="Schaaper, Wm" uniqKey="Schaaper W">WM Schaaper</name></author><author><name sortKey="Horzinek, Mc" uniqKey="Horzinek M">MC Horzinek</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woo, Pc" uniqKey="Woo P">PC Woo</name></author><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author><author><name sortKey="Huang, Y" uniqKey="Huang Y">Y Huang</name></author><author><name sortKey="Yuen, Ky" uniqKey="Yuen K">KY Yuen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="King, Amq" uniqKey="King A">AMQ King</name></author><author><name sortKey="Lefkowitz, E" uniqKey="Lefkowitz E">E Lefkowitz</name></author><author><name sortKey="Adams, Mj" uniqKey="Adams M">MJ Adams</name></author><author><name sortKey="Carstens, Eb" uniqKey="Carstens E">EB Carstens</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Der Hoek, L" uniqKey="Van Der Hoek L">L van der Hoek</name></author><author><name sortKey="Pyrc, K" uniqKey="Pyrc K">K Pyrc</name></author><author><name sortKey="Berkhout, B" uniqKey="Berkhout B">B Berkhout</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zaki, Am" uniqKey="Zaki A">AM Zaki</name></author><author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S van Boheemen</name></author><author><name sortKey="Bestebroer, Tm" uniqKey="Bestebroer T">TM Bestebroer</name></author><author><name sortKey="Osterhaus, Ad" uniqKey="Osterhaus A">AD Osterhaus</name></author><author><name sortKey="Fouchier, Ra" uniqKey="Fouchier R">RA Fouchier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vabret, A" uniqKey="Vabret A">A Vabret</name></author><author><name sortKey="Mourez, T" uniqKey="Mourez T">T Mourez</name></author><author><name sortKey="Dina, J" uniqKey="Dina J">J Dina</name></author><author><name sortKey="Van Der Hoek, L" uniqKey="Van Der Hoek L">L van der Hoek</name></author><author><name sortKey="Gouarin, S" uniqKey="Gouarin S">S Gouarin</name></author><author><name sortKey="Petitjean, J" uniqKey="Petitjean J">J Petitjean</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hammitt, Ll" uniqKey="Hammitt L">LL Hammitt</name></author><author><name sortKey="Kazungu, S" uniqKey="Kazungu S">S Kazungu</name></author><author><name sortKey="Welch, S" uniqKey="Welch S">S Welch</name></author><author><name sortKey="Bett, A" uniqKey="Bett A">A Bett</name></author><author><name sortKey="Onyango, Co" uniqKey="Onyango C">CO Onyango</name></author><author><name sortKey="Gunson, Rn" uniqKey="Gunson R">RN Gunson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chiu, Ss" uniqKey="Chiu S">SS Chiu</name></author><author><name sortKey="Chan, Kh" uniqKey="Chan K">KH Chan</name></author><author><name sortKey="Chu, Kw" uniqKey="Chu K">KW Chu</name></author><author><name sortKey="Kwan, Sw" uniqKey="Kwan S">SW Kwan</name></author><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y Guan</name></author><author><name sortKey="Poon, Ll" uniqKey="Poon L">LL Poon</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Bulimo, Wd" uniqKey="Bulimo W">WD Bulimo</name></author><author><name sortKey="Achilla, Ra" uniqKey="Achilla R">RA Achilla</name></author><author><name sortKey="Majanja, J" uniqKey="Majanja J">J Majanja</name></author><author><name sortKey="Mukunzi, S" uniqKey="Mukunzi S">S Mukunzi</name></author><author><name sortKey="Wadegu, M" uniqKey="Wadegu M">M Wadegu</name></author><author><name sortKey="Osunna, F" uniqKey="Osunna F">F Osunna</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sueker, J" uniqKey="Sueker J">J Sueker</name></author><author><name sortKey="Blazes, Dl" uniqKey="Blazes D">DL Blazes</name></author><author><name sortKey="Johns, Mc" uniqKey="Johns M">MC Johns</name></author><author><name sortKey="Blair, Pj" uniqKey="Blair P">PJ Blair</name></author><author><name sortKey="Sjoberg, Pa" uniqKey="Sjoberg P">PA Sjoberg</name></author><author><name sortKey="Tjaden, Ja" uniqKey="Tjaden J">JA Tjaden</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fischers, A" uniqKey="Fischers A">A FISCHERS</name></author><author><name sortKey="Andrew, A" uniqKey="Andrew A">A ANDREW</name></author><author><name sortKey="Townsend, W" uniqKey="Townsend W">W TOWNSEND</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kuypers, J" uniqKey="Kuypers J">J Kuypers</name></author><author><name sortKey="Martin, Et" uniqKey="Martin E">ET Martin</name></author><author><name sortKey="Heugel, J" uniqKey="Heugel J">J Heugel</name></author><author><name sortKey="Wright, N" uniqKey="Wright N">N Wright</name></author><author><name sortKey="Morrow, R" uniqKey="Morrow R">R Morrow</name></author><author><name sortKey="Englund, Ja" uniqKey="Englund J">JA Englund</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woo, Pc" uniqKey="Woo P">PC Woo</name></author><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author><author><name sortKey="Chu, Cm" uniqKey="Chu C">CM Chu</name></author><author><name sortKey="Chan, Kh" uniqKey="Chan K">KH Chan</name></author><author><name sortKey="Tsoi, Hw" uniqKey="Tsoi H">HW Tsoi</name></author><author><name sortKey="Huang, Y" uniqKey="Huang Y">Y Huang</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Altschul, Sf" uniqKey="Altschul S">SF Altschul</name></author><author><name sortKey="Gish, W" uniqKey="Gish W">W Gish</name></author><author><name sortKey="Miller, W" uniqKey="Miller W">W Miller</name></author><author><name sortKey="Myers, Ew" uniqKey="Myers E">EW Myers</name></author><author><name sortKey="Lipman, Dj" uniqKey="Lipman D">DJ Lipman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Edgar, Rc" uniqKey="Edgar R">RC Edgar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ronquist, F" uniqKey="Ronquist F">F Ronquist</name></author><author><name sortKey="Teslenko, M" uniqKey="Teslenko M">M Teslenko</name></author><author><name sortKey="Van Der Mark, P" uniqKey="Van Der Mark P">P van der Mark</name></author><author><name sortKey="Ayres, Dl" uniqKey="Ayres D">DL Ayres</name></author><author><name sortKey="Darling, A" uniqKey="Darling A">A Darling</name></author><author><name sortKey="Hohna, S" uniqKey="Hohna S">S Hohna</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author><author><name sortKey="Woo, Pc" uniqKey="Woo P">PC Woo</name></author><author><name sortKey="Yip, Cc" uniqKey="Yip C">CC Yip</name></author><author><name sortKey="Tse, H" uniqKey="Tse H">H Tse</name></author><author><name sortKey="Tsoi, Hw" uniqKey="Tsoi H">HW Tsoi</name></author><author><name sortKey="Cheng, Vc" uniqKey="Cheng V">VC Cheng</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Owusu, M" uniqKey="Owusu M">M Owusu</name></author><author><name sortKey="Annan, A" uniqKey="Annan A">A Annan</name></author><author><name sortKey="Corman, Vm" uniqKey="Corman V">VM Corman</name></author><author><name sortKey="Larbi, R" uniqKey="Larbi R">R Larbi</name></author><author><name sortKey="Anti, P" uniqKey="Anti P">P Anti</name></author><author><name sortKey="Drexler, Jf" uniqKey="Drexler J">JF Drexler</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kuypers, J" uniqKey="Kuypers J">J Kuypers</name></author><author><name sortKey="Wright, N" uniqKey="Wright N">N Wright</name></author><author><name sortKey="Ferrenberg, J" uniqKey="Ferrenberg J">J Ferrenberg</name></author><author><name sortKey="Huang, Ml" uniqKey="Huang M">ML Huang</name></author><author><name sortKey="Cent, A" uniqKey="Cent A">A Cent</name></author><author><name sortKey="Corey, L" uniqKey="Corey L">L Corey</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Berkley, Ja" uniqKey="Berkley J">JA Berkley</name></author><author><name sortKey="Munywoki, P" uniqKey="Munywoki P">P Munywoki</name></author><author><name sortKey="Ngama, M" uniqKey="Ngama M">M Ngama</name></author><author><name sortKey="Kazungu, S" uniqKey="Kazungu S">S Kazungu</name></author><author><name sortKey="Abwao, J" uniqKey="Abwao J">J Abwao</name></author><author><name sortKey="Bett, A" uniqKey="Bett A">A Bett</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dijkman, R" uniqKey="Dijkman R">R Dijkman</name></author><author><name sortKey="Jebbink, Mf" uniqKey="Jebbink M">MF Jebbink</name></author><author><name sortKey="Gaunt, E" uniqKey="Gaunt E">E Gaunt</name></author><author><name sortKey="Rossen, Jw" uniqKey="Rossen J">JW Rossen</name></author><author><name sortKey="Templeton, Ke" uniqKey="Templeton K">KE Templeton</name></author><author><name sortKey="Kuijpers, Tw" uniqKey="Kuijpers T">TW Kuijpers</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Smuts, He" uniqKey="Smuts H">HE Smuts</name></author><author><name sortKey="Workman, Lj" uniqKey="Workman L">LJ Workman</name></author><author><name sortKey="Zar, Hj" uniqKey="Zar H">HJ Zar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lu, R" uniqKey="Lu R">R Lu</name></author><author><name sortKey="Yu, X" uniqKey="Yu X">X Yu</name></author><author><name sortKey="Wang, W" uniqKey="Wang W">W Wang</name></author><author><name sortKey="Duan, X" uniqKey="Duan X">X Duan</name></author><author><name sortKey="Zhang, L" uniqKey="Zhang L">L Zhang</name></author><author><name sortKey="Zhou, W" uniqKey="Zhou W">W Zhou</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mclntosh, K" uniqKey="Mclntosh K">K Mclntosh</name></author><author><name sortKey="Englund, Ja" uniqKey="Englund J">JA Englund</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Elden, Lj" uniqKey="Van Elden L">LJ van Elden</name></author><author><name sortKey="Van Loon, Am" uniqKey="Van Loon A">AM van Loon</name></author><author><name sortKey="Van Alphen, F" uniqKey="Van Alphen F">F van Alphen</name></author><author><name sortKey="Hendriksen, Ka" uniqKey="Hendriksen K">KA Hendriksen</name></author><author><name sortKey="Hoepelman, Ai" uniqKey="Hoepelman A">AI Hoepelman</name></author><author><name sortKey="Van Kraaij, Mg" uniqKey="Van Kraaij M">MG van Kraaij</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Kraaij, Mg" uniqKey="Van Kraaij M">MG van Kraaij</name></author><author><name sortKey="Van Elden, Lj" uniqKey="Van Elden L">LJ van Elden</name></author><author><name sortKey="Van Loon, Am" uniqKey="Van Loon A">AM van Loon</name></author><author><name sortKey="Hendriksen, Ka" uniqKey="Hendriksen K">KA Hendriksen</name></author><author><name sortKey="Laterveer, L" uniqKey="Laterveer L">L Laterveer</name></author><author><name sortKey="Dekker, Aw" uniqKey="Dekker A">AW Dekker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pyrc, K" uniqKey="Pyrc K">K Pyrc</name></author><author><name sortKey="Sims, Ac" uniqKey="Sims A">AC Sims</name></author><author><name sortKey="Dijkman, R" uniqKey="Dijkman R">R Dijkman</name></author><author><name sortKey="Jebbink, M" uniqKey="Jebbink M">M Jebbink</name></author><author><name sortKey="Long, C" uniqKey="Long C">C Long</name></author><author><name sortKey="Deming, D" uniqKey="Deming D">D Deming</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Virol J</journal-id><journal-id journal-id-type="iso-abbrev">Virol. J</journal-id><journal-title-group><journal-title>Virology Journal</journal-title></journal-title-group><issn pub-type="epub">1743-422X</issn><publisher><publisher-name>BioMed Central</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26833249</article-id><article-id pub-id-type="pmc">4736488</article-id><article-id pub-id-type="publisher-id">474</article-id><article-id pub-id-type="doi">10.1186/s12985-016-0474-x</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research</subject></subj-group></article-categories><title-group><article-title>Molecular characterization of human coronaviruses and their circulation dynamics in Kenya, 2009–2012</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Sipulwa</surname><given-names>Lenata A.</given-names></name><address><email>lenata7@gmail.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Ongus</surname><given-names>Juliette R.</given-names></name><address><email>julietteongus@jkuat.ac.ke</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Coldren</surname><given-names>Rodney L.</given-names></name><address><email>Rodney.Coldren@usamru-k.org</email></address><xref ref-type="aff" rid="Aff2"></xref><xref ref-type="aff" rid="Aff3"></xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Bulimo</surname><given-names>Wallace D.</given-names></name><address><phone>+254733616602</phone><email>Wallace.bulimo@uonbi.ac.ke</email></address><xref ref-type="aff" rid="Aff2"></xref><xref ref-type="aff" rid="Aff3"></xref><xref ref-type="aff" rid="Aff4"></xref></contrib><aff id="Aff1"><label></label>College of Health Sciences (COHES), Jomo Kenyatta University of Agriculture and Technology, (JKUAT), Nairobi, Kenya</aff><aff id="Aff2"><label></label>Department of Emerging Infectious Diseases, US Army Medical Research Unit–Kenya, P.O. Box 606 00621, Village Market, Nairobi, Kenya</aff><aff id="Aff3"><label></label>Special Foreign Activity of the Walter Reed Army Institute of Research, Silver Spring, MD USA</aff><aff id="Aff4"><label></label>Department of Biochemistry, School of Medicine, University of Nairobi, Nairobi, Kenya</aff></contrib-group><pub-date pub-type="epub"><day>1</day><month>2</month><year>2016</year></pub-date><pub-date pub-type="pmc-release"><day>1</day><month>2</month><year>2016</year></pub-date><pub-date pub-type="collection"><year>2016</year></pub-date><volume>13</volume><elocation-id>18</elocation-id><history><date date-type="received"><day>21</day><month>10</month><year>2015</year></date><date date-type="accepted"><day>20</day><month>1</month><year>2016</year></date></history><permissions><copyright-statement>© Sipulwa et al. 2016</copyright-statement><license license-type="OpenAccess"><license-p><bold>Open Access</bold>This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">http://creativecommons.org/publicdomain/zero/1.0/</ext-link>) applies to the data made available in this article, unless otherwise stated.</license-p></license></permissions><abstract id="Abs1"><sec><title>Background</title><p>Human Coronaviruses (HCoV) are a common cause of respiratory illnesses and are responsible for considerable morbidity and hospitalization across all age groups especially in individuals with compromised immunity. There are six known species of HCoV: HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, MERS-CoV and SARS-HCoV. Although studies have shown evidence of global distribution of HCoVs, there is limited information on their presence and distribution in Kenya.</p></sec><sec><title>Methods</title><p>HCoV strains that circulated in Kenya were retrospectively diagnosed and molecularly characterized. A total of 417 nasopharyngeal specimens obtained between January 2009 and December 2012 from around Kenya were analyzed by a real time RT-PCR using HCoV-specific primers. HCoV-positive specimens were subsequently inoculated onto monolayers of LL-CMK2 cells. The isolated viruses were characterized by RT-PCR amplification and sequencing of the partial polymerase (<italic>po</italic>l) gene.</p></sec><sec><title>Results</title><p>The prevalence of HCoV infection was as follows: out of the 417 specimens, 35 (8.4 %) were positive for HCoV, comprising 10 (2.4 %) HCoV-NL63, 12 (2.9 %) HCoV-OC43, 9 (2.1 %) HCoV-HKU1, and 4 (1 %) HCoV-229E. The Kenyan HCoV strains displayed high sequence homology to the prototypes and contemporaneous strains. Evolution analysis showed that the Kenyan HCoV-OC43 and HCoV-NL63 isolates were under purifying selection. Phylogenetic evolutionary analyses confirmed the identities of three HCoV-HKU1, five HCoV-NL63, eight HCoV-OC43 and three HCoV-229E.</p></sec><sec><title>Conclusions</title><p>There were yearly variations in the prevalence and circulation patterns of individual HCoVs in Kenya. This paper reports on the first molecular characterization of human Coronaviruses in Kenya, which play an important role in causing acute respiratory infections among children.</p></sec></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd>Molecular characterization</kwd><kwd>Human coronaviruses</kwd><kwd>Prevalence</kwd><kwd>Kenya</kwd><kwd>HCOV-NL63</kwd><kwd>HCoV-OC43</kwd><kwd>HCoV-229E</kwd><kwd>HCoV-HKU1</kwd></kwd-group><funding-group><award-group><funding-source><institution>Funding was provided by the US Armed Forces Health Surveillance Center Division of GEIS Operations</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2016</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1"><title>Background</title><p>Coronaviruses are enveloped viruses with a linear, non-segmented, positive-sense, single-stranded RNA genome of about 27–32 kb [<xref ref-type="bibr" rid="CR1">1</xref>]. They are round and sometimes pleiomorphic virions of approximately 80-120 nm in diameter. Their genome is the largest of all RNA viruses [<xref ref-type="bibr" rid="CR2">2</xref>, <xref ref-type="bibr" rid="CR3">3</xref>]. Coronaviruses share features of genome organization and replication strategy, but have different virion morphology and genome lengths. The 20 kb polymerase (<italic>pol</italic>) gene, which covers the 5′ two-thirds of the coronavirus genome, contains two large open reading frames (ORFs), ORF 1a and ORF 1b [<xref ref-type="bibr" rid="CR1">1</xref>]. ORF 1b hosts the most highly conserved genomic sequences, encoding conserved functions such as polymerase and helicase activity [<xref ref-type="bibr" rid="CR4">4</xref>]. These functions combined with similarities in replication and expression strategies, exhibit an evolutionary link among coronaviruses, arteriviruses, and toroviruses, thus forming the rationale for placing these viruses in the order <italic>Nidovirales</italic> [<xref ref-type="bibr" rid="CR1">1</xref>]. Therefore, the highly conserved structure and function of viral polymerases make the <italic>pol</italic> a logical region for making phylogenetic comparisons.</p><p>Coronaviruses cause various diseases in different hosts, such as respiratory tract infections, gastroenteritis, hepatitis, and encephalomyelitis in birds and mammals [<xref ref-type="bibr" rid="CR5">5</xref>]. Coronaviruses are classified into four distinct phylogenetic groups. Whereas alphacoronaviruses, betacoronaviruses and gammacoronaviruses infect mammals, deltacoronaviruses infect avian species [<xref ref-type="bibr" rid="CR6">6</xref>]. All known human coronaviruses (HCoV) belong to the alphacoronavirus and betacoronaviruses. HCoV cause 30 % of common colds as well as severe infections in infants, children, and immunocompromised persons and the elderly [<xref ref-type="bibr" rid="CR7">7</xref>]. HCoVs have a worldwide circulation, with reports published in populations of different countries such as Saudi Arabia [<xref ref-type="bibr" rid="CR8">8</xref>], France [<xref ref-type="bibr" rid="CR9">9</xref>], Kenya [<xref ref-type="bibr" rid="CR10">10</xref>] and China [<xref ref-type="bibr" rid="CR11">11</xref>] among others. However in 2012, a novel betacoronavirus Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was isolated in Jeddah from a patient who was suffering from pneumonia and later died [<xref ref-type="bibr" rid="CR8">8</xref>]. This MERS-CoV continues to cause fatal severe lower respiratory tract illnesses in various parts of the world, especially in the Middle East. As of February 2015, the World Health Organization had reported 971 laboratory confirmed cases with 356 deaths [<xref ref-type="bibr" rid="CR12">12</xref>]. In light of the finding of this previously unknown coronavirus, it is probable that coronaviruses, other than those recognized to date, may be circulating in human populations in Kenya. As human coronaviruses are not routinely analyzed in Kenya, this study sought to identify and characterize HCoV strains circulating in different parts of Kenya and thus provide a knowledge base for epidemiological importance of human coronaviruses.</p></sec><sec id="Sec2"><title>Methods</title><sec id="Sec3"><title>Study sites, inclusion criteria, clinical parameters</title><p>Nasopharyngeal (NP) specimens were retrieved from archives of the respiratory virus surveillance program in the Department of Emerging Infectious Diseases (DEID) of the United States Army Medical Research Unit-Kenya (USAMRU-K) [<xref ref-type="bibr" rid="CR13">13</xref>]. These approximately 10000 archived NP specimens were collected from a study population comprising persons from two months of age onwards who attended outpatient clinics in the hospitals involved in the USAMRU-K respiratory virus surveillance; presenting with influenza like illnesses symptoms. These symptoms were: cough, sore throat, and fever (≥38 °C) within 72 h after onset of illness. The nasoopharyngeal swabs were suspended in virus transport medium (VTM) and stored at −80 °C. These samples had been screened for other respiratory viruses including influenza viruses, HAdV, RSV, HMPV, HSV1, rhinoviruses and non-polio enteroviruses including coxsackie viruses [<xref ref-type="bibr" rid="CR14">14</xref>].</p><p>The surveillance network comprised Mbagathi, Isiolo, Alupe, Kericho, Kisi, Malindi, and Port Reitz district hospitals, as well as the New Nyanza General provincial hospital [<xref ref-type="bibr" rid="CR13">13</xref>]. These hospitals represent disparate geographic regions and population demographics across the country and were delineated into Central, Northern, Western, Highlands and Coastal regions of Kenya, respectively. Inclusion criteria consisted of being an outpatient aged at least 2 months and having influenza-like-illness (ILI) symptoms [<xref ref-type="bibr" rid="CR13">13</xref>]. Clinical parameters and symptoms including recent history of ILI, cough, sore throat, difficulty in breathing, chills, nasal stuffiness, runny nose, sputum production, headache, joint pain, fatigue, diarrhea, vomiting and bleeding were documented. Only those samples collected from January 2009 to December 2012 for which specific consent was granted for future use were analyzed. The minimum sample size was 173 as determined by Fischer’s formula [<xref ref-type="bibr" rid="CR15">15</xref>]. Based on available resources, the final number of samples was determined by using the randomized sampling technique. This involved systematic sampling, where the first sample was picked randomly and thereafter the k<sup>th</sup> sample selected from a list of samples for each site.</p></sec><sec id="Sec4"><title>Ethical approval statement</title><p>The Walter Reed Army Institute of Research (WRAIR) Institutional Review Board (IRB) and the Kenya Medical Research Institute (KEMRI) Scientific and Ethics Review Unit (SERU) reviewed and approved this work under protocol numbers WRAIR# 2075 and KEMRI SSC#2673 respectively.</p></sec><sec id="Sec5"><title>Molecular screening of the virus isolates</title><p>RNA was extracted from 140 μl of nasopharyngeal (NP) specimens using the QIAmp Viral RNA Mini Kit (Qiagen, Inc.,USA) according to the manufacturer’s specifications. Viral RNA was used as a template for real time RT-PCR using HCoV-specific primers as previously described by Kuypers et al. [<xref ref-type="bibr" rid="CR16">16</xref>]. For MERS-CoV detection, samples were analysed using NCV-2012rRT-PCR assay according to the CDC protocols. Control RNAs used were obtained from ATCC (VR-1558D for OC43 and VR740D for 229E) as well as NL63 RNA from the KEMRI-Welcome Trust research program. The MERS-CoV control RNA came along with the test kit from CDC. Samples that tested positive by real time RT-PCR were inoculated onto monolayers of LL-CMK2 cells (ATCC strain CCL-7) in culture tubes (Nunc, Denmark) to contain 100 μl of clinical specimen. The culture medium consisted of Dulbecco’s minimum essential medium (DMEM, Life Technologies, NY, USA) supplemented with 0.04 μg/ml gentamycin, 100U/mL of penicillin & 100 μg/mL streptomycin (Sigma-Aldrich Co. MO, USA). These were incubated at 33 °C under 5 % CO<sub>2</sub>, and virus growth was monitored for cytopathic effects (CPEs) for up to 10 days. Culture controls were obtained from ATCC and included betacoronavirus 1 (ATCC® VR-1558™) and human coronavirus 229E (ATCC® VR-740™).</p></sec><sec id="Sec6"><title>Amplification and nucleotide sequencing of RdRp (pol) gene</title><p>A 440 bp portion of the intergenic region of ORF1a and ORF1b of the RNA-dependent RNA polymerase (pol) gene of HCoVs was amplified by RT-PCR following the method described by Woo et al. [<xref ref-type="bibr" rid="CR17">17</xref>] . RT was performed using the SuperScript III kit (Invitrogen, San Diego, USA) following manufacturer’s instructions. The forward primer was 5′-GGTTGGGACTATCCTAAGTGTGA-3′ whereas the reverse primer was 5′ -CCATCATCAGATAGAATCATCATA-3′. The PCR mixture (50 μl) contained RNA (300 ng), PCR buffer containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 3 mM MgCl<sub>2</sub>, 0.01 % gelatin, 200 μM each of deoxynucleoside triphosphates, and 1.0 U of Taq polymerase (Boehringer, Mannheim, Germany). The amplification was carried out as described by Woo et al. [<xref ref-type="bibr" rid="CR17">17</xref>]. using the ABI 9700 automated thermal cycler (Applied Biosystems, Foster city, USA). The PCR amplicons were purified using Exonuclease I/Shrimp Alkaline Phosphatase (ExoSap-IT) enzyme (Affymetrix, California, USA) and sequenced directly on both strands with the same primers used in the PCR on an automated ABI 3500XL Genetic Analyzer (Applied Biosystems, Foster city, USA). Cycle sequencing was performed using the Big Dye Terminator v3.1 sequencing kit (Applied Biosystems, Foster city, USA) according to manufacturer’s instructions.</p></sec><sec id="Sec7"><title>Sequence analysis</title><p>The nucleotide sequence fragments were edited and assembled into consensus contigs using DNA baser version 3.2 [<xref ref-type="bibr" rid="CR18">18</xref>]. The nucleotide sequences were compared against known sequences of coronavirus pol genes in the GenBank database using the BLAST [<xref ref-type="bibr" rid="CR19">19</xref>]. Prototype nucleotide sequences and sequences showing highest sequence homologies were retrieved from GenBank for further analyses. Multiple sequence alignment of these was performed using Muscle v3.8 software [<xref ref-type="bibr" rid="CR20">20</xref>]. Phylogenetic relationships were inferred using MrBayes software v3.2 [<xref ref-type="bibr" rid="CR21">21</xref>] and the tree visualized using Fig Tree v1.4.0 software [<xref ref-type="bibr" rid="CR22">22</xref>]. Nucleotide sequences of the partial pol genes of the Kenyan HCoV isolates reported in this study are available in GenBank under accession numbers:[GenBank: KP112147 to KP112161].</p></sec></sec><sec id="Sec8"><title>Results</title><p>A total of 417 nasopharyngeal samples were screened. Among these, 35 tested positive for human coronaviruses, displaying Ct values ranging from 21 to 39. Upon molecular typing the 35 positives, 14 (37 %) belonged to alphacoronaviruses and 21 (63 %) were betacoronaviruses. Amongst the alphacoronaviruses, 4 were HCoV-229E and 10 were HCoV-NL63. Betacoronaviruses consisted of 12 HCoV-OC43 and 9 HCoV-HKU1 species. Thus the prevalence of the various HCoVs was 1 % for 229E, 2 % for NL63, 3 % OC43 and 2 % for HKU1.</p><p>HCoV-OC43 was detected in most months in 2009–2011, but was not detected in 2012. The peak circulation was observed in February 2011 (Fig. <xref rid="Fig1" ref-type="fig">1</xref>) affecting patients aged 5 months to 5 years. It was most commonly detected in Central Kenya but never in Northern Kenya (Fig. <xref rid="Fig2" ref-type="fig">2</xref>). HCoV-NL63 was detected mostly from samples obtained in Northern and Western Kenya and amongst patients aged 3 months to 1.75 years (Fig. <xref rid="Fig1" ref-type="fig">1</xref>). HCoV-NL63 predominated from February to June 2009, then disappeared and resurfaced in September 2012.<fig id="Fig1"><label>Fig. 1</label><caption><p>Temporal distribution of HCoVs in Kenya between 2009 to 2012</p></caption><graphic xlink:href="12985_2016_474_Fig1_HTML" id="MO1"></graphic></fig><fig id="Fig2"><label>Fig. 2</label><caption><p>Spatial Distribution of HCoVs in Kenya between 2009 and 2012. Mbagathi, Isiolo, Alupe, Kericho, Kisii, Malindi, and Port Reitz District Hospitals are abbreviated as MBG, IDH, ALH, KCH, KSI, MDH and PDH respectively. New Nyanza General provincial hospital is abbreviated as NNY</p></caption><graphic xlink:href="12985_2016_474_Fig2_HTML" id="MO2"></graphic></fig></p><p>HCoV-HKU1 was detected all over Kenya, but mostly in the warmer Western and Coastal regions, with peaks observed in June 2009, November 2010, February 2011 and December 2012 (Figs. <xref rid="Fig1" ref-type="fig">1</xref> and <xref rid="Fig2" ref-type="fig">2</xref>). It was detected in patients aged 4 months to 6 years. HCoV 229E was only detected in the Western Kenya, from patients aged 9 months to 2 years with peaks detected in July and December (Fig. <xref rid="Fig2" ref-type="fig">2</xref>).</p><p>The range of ages of the patients in the study was between 2 months and 67 years, with a mean age of 1.5 years and median of 1.67 years. Overall, 94 % of the HCoV were from patients aged <5 years and; 4555 % of the patients were female and 55 % were male. All the patients from whom HCoVs were detected presented with a cough and runny nose, with 50 % of the patients reporting nasal stuffiness. We observed five co-infections with a HCoV. These included two triple co-infection involving HKU1 with influenza A (H3N2) & human adenovirus and NL63 with influenza A (H3N2) & human adenovirus. Three double co-infections were observed. They included HKU1 with RSV, OC43 with influenza A (H1N1)pdm and OC43 with influenza B (Brisbane-like). All the co-infections, except that consisting of HKU1 & RSV presented with vomiting as a symptom. One patient with HCoV-NL63 presented with neurological symptoms. Amongst the patients positive for HCoV-OC43, 90 % presented with vomiting and malaise.</p><p>Characterization by cell culture showed that of the 35 positive samples, 12 (34 %) produced cytopathic effects (CPE) in cell culture after incubation for 10 days. PCR amplification of a 440 bp fragment using CoV-specific primers confirmed that the CPE was due to HCoVs. To determine whether there were any HCoVs in the supernatants of the 23 cultures that did not yield CPE, RT-PCR using HCoV-specific primers generated the expected 440 bp amplicon from 3 of these samples.</p><p>Homology analyses of the partial nucleotide sequences of the RdRp (<italic>pol</italic>) gene of the Kenyan HCoV against the prototype strains ranged from 59 to 100 % (Table <xref rid="Tab1" ref-type="table">1</xref>). Nucleotide sequence homology within groups ranged from 99 to 100 % for HCoV-OC43, 97–99 % for HCoV-HKU1 and 98–99 % for HCoV-NL63. The Kenya HCoV-229E species had 99 % nucleotide sequence identity compared to the prototype strain.<table-wrap id="Tab1"><label>Table 1</label><caption><p>Nucleotide sequence homology of the Kenyan HCoV isolates</p></caption><table frame="hsides" rules="groups"><thead><tr><th></th><th>NC_005147_OC43</th><th>NC_006577_HKU1</th><th>NC_002645_229E</th><th>NC_005831_NL63</th><th>HCoV_016_2010</th><th>HCoV_018_2010</th><th>HCoV017_2010</th><th>HCoV_019_2010</th><th>HCoV_014_2010</th><th>HCoV_012_2010</th><th>HCoV_007_2010</th><th>HCoV_013_2010</th><th>HCoV_001_2010</th><th>HCoV_010_2009</th><th>HCoV_008_2009</th><th>HCoV_002_2012</th><th>HCoV_006_2009</th><th>HCoV_009_2010</th><th>HCoV_005_2009</th><th>HCoV_011_2009</th><th>HCoV_015_2009</th><th>HCoV_004_2012</th><th>HCoV_003_2009</th></tr></thead><tbody><tr><td>NC_005147_OC43</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>NC_006577_HKU1</td><td>81</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>NC_002645_229E</td><td>59</td><td>62</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>NC_005831_NL63</td><td>60</td><td>61</td><td>75</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_016_2010</td><td>100</td><td>81</td><td>59</td><td>60</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_018_2010</td><td>99</td><td>82</td><td>59</td><td>60</td><td>99</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_017_2010</td><td>99</td><td>82</td><td>59</td><td>60</td><td>99</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_019_2010</td><td>99</td><td>82</td><td>59</td><td>60</td><td>99</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_14_2010</td><td>99</td><td>82</td><td>59</td><td>60</td><td>99</td><td>100</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_012_2010</td><td>99</td><td>82</td><td>59</td><td>60</td><td>99</td><td>100</td><td>100</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_007_2010</td><td>99</td><td>82</td><td>59</td><td>60</td><td>99</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_013_2010</td><td>99</td><td>82</td><td>59</td><td>60</td><td>99</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_001_2010</td><td>80</td><td>97</td><td>61</td><td>60</td><td>80</td><td>81</td><td>81</td><td>81</td><td>81</td><td>81</td><td>81</td><td>81</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_010_2009</td><td>81</td><td>99</td><td>63</td><td>61</td><td>81</td><td>82</td><td>82</td><td>82</td><td>82</td><td>82</td><td>82</td><td>82</td><td>96</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_008_2009</td><td>81</td><td>99</td><td>62</td><td>61</td><td>81</td><td>81</td><td>81</td><td>81</td><td>81</td><td>81</td><td>81</td><td>81</td><td>97</td><td>99</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_002_2012</td><td>59</td><td>62</td><td>99</td><td>75</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>62</td><td>63</td><td>63</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_006_2009</td><td>59</td><td>62</td><td>99</td><td>75</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>62</td><td>63</td><td>63</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_009_2010</td><td>59</td><td>62</td><td>99</td><td>75</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>59</td><td>62</td><td>63</td><td>63</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_005_2009</td><td>60</td><td>60</td><td>76</td><td>99</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>61</td><td>61</td><td>76</td><td>76</td><td>76</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_011_2009</td><td>59</td><td>60</td><td>75</td><td>98</td><td>59</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>59</td><td>60</td><td>60</td><td>75</td><td>75</td><td>75</td><td>99</td><td></td><td></td><td></td><td></td></tr><tr><td>HCoV_015_2009</td><td>60</td><td>60</td><td>76</td><td>99</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>61</td><td>61</td><td>76</td><td>76</td><td>76</td><td>100</td><td>99</td><td></td><td></td><td></td></tr><tr><td>HCoV_004_2012</td><td>60</td><td>60</td><td>76</td><td>99</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>61</td><td>61</td><td>76</td><td>76</td><td>76</td><td>100</td><td>99</td><td>100</td><td></td><td></td></tr><tr><td>HCoV_003_2009</td><td>60</td><td>60</td><td>76</td><td>99</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>60</td><td>61</td><td>61</td><td>76</td><td>76</td><td>76</td><td>100</td><td>99</td><td>100</td><td>100</td><td></td></tr></tbody></table><table-wrap-foot><p>The numbers represent % nucleotide identities among the strains</p></table-wrap-foot></table-wrap></p><p>Phylogenetic analyses of the Kenyan isolates based on the nucleotide sequences with representative reference strains revealed separation of the viruses into the two α and β corona-virus groups with further resolution into four species, corresponding to HCoV-OC43, HCoV-NL63, HCoV-229E and HCoV-HKU1 (Fig. <xref rid="Fig3" ref-type="fig">3</xref>). The majority (42 %) of the Kenyan isolates clustered within the HCoV-OC43 group, followed by HCoV-NL63 (26 %) and HCoV-HKUI & HCoV-229E (16 % each). All the Kenyan HCoV-OC43 isolates formed a cluster on their own except isolate HCOV-016 which grouped with the other HCoV-OC43 viruses from other countries across the globe. The Kenyan HCoV-NL63 isolates clustered with other HCoV-NL63 viruses. The Kenyan HCoV-HKU1 isolates grouped close to the French virus whose accession number is EF507794 and the Kenyan HCoV-229E isolates were identical to the reference strains used.<fig id="Fig3"><label>Fig. 3</label><caption><p>Phylogenetic relationships of partial sequences from the HCoV <italic>pol</italic> gene of the Kenyan isolates with representatives from different species. The Kenyan HCoV sequences are in blue color, the prototype strains are in red and representative strains in black; each sequence is represented by the GenBank accession number. The PEDV in green was used as an out-group. The tree was constructed by Mr. Bayes v3.2, using the general time-reversible nucleotide substitution model. Posterior probability support values are shown as percentages at the nodes</p></caption><graphic xlink:href="12985_2016_474_Fig3_HTML" id="MO3"></graphic></fig></p><p>In establishing the evolution rates of the Kenyan HCoVs, Tajima’s test showed an overall value of Tajima’s D statistic for the eight HCoV-OC43 pol gene segments as −1.31009 with p > 0.10 at 95 % CI. For HCoV-NL63, the overall value of Tajima’s D statistic for the 5 sequences was −0.97256 with p > 0.10 at 95 % CI. The evolutionary rates of the Kenyan HCoVs were also analyzed using Fu and Li’s D as well as Fu and Li’s F tests as these can accommodate small sample sizes. The overall Fu and Li’s D value for the HCoV-OC43 pol gene segments was −1.40980 and −0.97256 for HCoV-NL63, p > 0.10. The overall Fu and Li’s F statistic was −1.51361 for HCoV-OC43 and −0.95440 for HCoV-NL63, p > 0.10.</p></sec><sec id="Sec9"><title>Discussion</title><p>HCoVs have a global distribution and are endemic to many countries [<xref ref-type="bibr" rid="CR5">5</xref>, <xref ref-type="bibr" rid="CR8">8</xref>, <xref ref-type="bibr" rid="CR23">23</xref>]. However, in Africa and especially sub-Saharan Africa, there is a dearth of information about these viruses in regard to their circulation dynamics, diversity, molecular characteristics and epidemiology [<xref ref-type="bibr" rid="CR24">24</xref>].</p><p>Four out of the six known human coronavirus species were detected by RT-PCR assay, which has been shown to detect respiratory viruses with high sensitivity and specificity [<xref ref-type="bibr" rid="CR25">25</xref>]. This finding echoes previous studies in Kenya [<xref ref-type="bibr" rid="CR26">26</xref>] and globally where HCoVs were identified as significant etiological agents of ILI and acute respiratory illnesses (ARI) [<xref ref-type="bibr" rid="CR9">9</xref>, <xref ref-type="bibr" rid="CR23">23</xref>]. HCoV-OC43 was the most commonly observed HCoV and this agrees with many previous studies that have reported HCoV-OC43 predominance [<xref ref-type="bibr" rid="CR27">27</xref>]. HCoV-NL63 contributed to 2 % of ILI cases in our study period. This finding echoes the findings in South Africa by Smuts, [<xref ref-type="bibr" rid="CR28">28</xref>] where they showed that HCoV-NL63, contributed to 2.4 % of ILI cases in the period of one and a half years. HCoV-229E contributed to 1 % of ILI cases in the study period, echoing results from other studies worldwide that show low detection of this HCoV [<xref ref-type="bibr" rid="CR27">27</xref>]. This is comparable to what has been observed globally, where HCoV activity is sporadic throughout the year with no obvious distinct seasonality [<xref ref-type="bibr" rid="CR29">29</xref>]. Overall, the symptoms associated with coronavirus infection were similar to what has been reported elsewhere [<xref ref-type="bibr" rid="CR1">1</xref>, <xref ref-type="bibr" rid="CR7">7</xref>, <xref ref-type="bibr" rid="CR24">24</xref>] and it was difficult to distinguish an infection involving HCoV using symptoms alone. This justifies the WHO classification of respiratory viral infections into influenza-like-illnesses without clear distinction of a specific respiratory virus infection based on symptoms alone.</p><p>Out of 35 PCR-positive cases for HCoV, only 12 (34 %) yielded virus in LLCMK2 [<xref ref-type="bibr" rid="CR30">30</xref>] and were confirmed by PCR. This was expected because molecular assays including PCR are more sensitive and specific at detecting pathogens [<xref ref-type="bibr" rid="CR31">31</xref>, <xref ref-type="bibr" rid="CR32">32</xref>] than cell culture or serology. The low isolation rate may be attributed to low viral loads in the patients or the presence of inactivated viruses, potentially due to poor specimen handling and/or storage. Nonetheless, this finding agrees with reports of poor growth and a lack of cytopathic effect in cell cultures by HCoVs [<xref ref-type="bibr" rid="CR33">33</xref>].</p><p>Phylogenetic analysis confirmed that the Kenyan isolates belonged to the <italic>alpha</italic> and <italic>betacoronavirus</italic> groups [<xref ref-type="bibr" rid="CR6">6</xref>]. The Kenyan isolates clustered together with reference strains indicating that the Kenyan HCoVs are strain variants of those circulating elsewhere. Natural selection analyses revealed that the Kenyan HCoV-OC43 and HCoV-NL63 viruses were under negative selection, albeit not statistically significantly so. We did not detect any MERS-CoV or SARS-CoV.</p><p>This study had several shortcomings. The small sample size made it difficult to analyze evolutionary rates using the robust SLAC and FEL methods. Only a portion of the virus genome was analyzed and the evolution rates based on this small portion of the genome may be misleading. These shortcomings notwithstanding, it was demonstrated that four HCoV subtype strains were in circulation in Kenya from 2009 to 2012 and were similar to those circulating in other countries. However, sustained respiratory virus surveillance is necessary to monitor introductions of any of these dangerous species. Furthermore, full genome studies are required to provide comprehensive insight into the genetics and evolutionary characteristics of HCoVs in Kenya.</p></sec><sec id="Sec10"><title>Conclusions</title><p>There were yearly variations in the prevalence and circulation patterns of individual HCoVs in Kenya. This paper reports on the first molecular characterization of human Coronaviruses in Kenya, which play an important role in causing acute respiratory infections among children.</p></sec></body><back><fn-group><fn><p><bold>Competing interests</bold></p><p>The authors declare that they have no competing interests. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.</p></fn><fn><p><bold>Authors’ contributions</bold></p><p>LAS, JRO, WDB conceived and designed the experiments; LAS performed the experiments; LAS and WDB analyzed the data; WDB contributed reagents/materials/analysis tools; LAS, JRO, RLC, AND WDB wrote the paper. All authors read and approved the final manuscript.</p></fn></fn-group><ack><p>We thank the patients who provided specimens for this work. We also thank the following individuals: Ms Rachel Achilla, Mr. Benjamin Opot and Silvanos Mukunzi of the US Army Medical Research Unit-Kenya, for laboratory management, nucleotide sequencing and analysis respectively. Funding was provided by the US Armed Forces Health Surveillance Center Division of GEIS Operations. The control RNA for HCoV-NL63 was provided by Prof. James Nokes of KWTRP-Kilifi.</p><sec id="FPar1"><title>Disclaimer</title><p>The views expressed in this work belong to the authors and should not be construed to represent the US Department of Defense, the Department of the Army, or the US Army Medical Department.</p></sec></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>Lai</surname><given-names>MM</given-names></name><name><surname>Cavanagh</surname><given-names>D</given-names></name></person-group><article-title>The molecular biology of coronaviruses</article-title><source>Adv Virus Res</source><year>1997</year><volume>48</volume><fpage>1</fpage><lpage>100</lpage><pub-id pub-id-type="doi">10.1016/S0065-3527(08)60286-9</pub-id><pub-id pub-id-type="pmid">9233431</pub-id></element-citation></ref><ref id="CR2"><label>2.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>HJ</given-names></name><name><surname>Shieh</surname><given-names>CK</given-names></name><name><surname>Gorbalenya</surname><given-names>AE</given-names></name><name><surname>Koonin</surname><given-names>EV</given-names></name><name><surname>La Monica</surname><given-names>N</given-names></name><name><surname>Tuler</surname><given-names>J</given-names></name><etal></etal></person-group><article-title>The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase</article-title><source>Virology</source><year>1991</year><volume>180</volume><issue>2</issue><fpage>567</fpage><lpage>82</lpage><pub-id pub-id-type="doi">10.1016/0042-6822(91)90071-I</pub-id><pub-id pub-id-type="pmid">1846489</pub-id></element-citation></ref><ref id="CR3"><label>3.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Xu</surname><given-names>HF</given-names></name><name><surname>Wang</surname><given-names>M</given-names></name><name><surname>Zhang</surname><given-names>ZB</given-names></name><name><surname>Zou</surname><given-names>XZ</given-names></name><name><surname>Gao</surname><given-names>Y</given-names></name><name><surname>Liu</surname><given-names>XN</given-names></name><etal></etal></person-group><article-title>An epidemiologic investigation on infection with severe acute respiratory syndrome coronavirus in wild animals traders in Guangzhou</article-title><source>Zhonghua Yu Fang Yi Xue Za Zhi</source><year>2004</year><volume>38</volume><issue>2</issue><fpage>81</fpage><lpage>3</lpage><pub-id pub-id-type="pmid">15061910</pub-id></element-citation></ref><ref id="CR4"><label>4.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bredenbeek</surname><given-names>PJ</given-names></name><name><surname>Snijder</surname><given-names>EJ</given-names></name><name><surname>Noten</surname><given-names>FH</given-names></name><name><surname>den Boon</surname><given-names>JA</given-names></name><name><surname>Schaaper</surname><given-names>WM</given-names></name><name><surname>Horzinek</surname><given-names>MC</given-names></name><etal></etal></person-group><article-title>The polymerase gene of corona- and toroviruses: evidence for an evolutionary relationship</article-title><source>Adv Exp Med Biol</source><year>1990</year><volume>276</volume><fpage>307</fpage><lpage>16</lpage><pub-id pub-id-type="doi">10.1007/978-1-4684-5823-7_42</pub-id><pub-id pub-id-type="pmid">1966417</pub-id></element-citation></ref><ref id="CR5"><label>5.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Woo</surname><given-names>PC</given-names></name><name><surname>Lau</surname><given-names>SK</given-names></name><name><surname>Huang</surname><given-names>Y</given-names></name><name><surname>Yuen</surname><given-names>KY</given-names></name></person-group><article-title>Coronavirus diversity, phylogeny and interspecies jumping</article-title><source>Exp Biol Med (Maywood)</source><year>2009</year><volume>234</volume><issue>10</issue><fpage>1117</fpage><lpage>27</lpage><pub-id pub-id-type="doi">10.3181/0903-MR-94</pub-id><pub-id pub-id-type="pmid">19546349</pub-id></element-citation></ref><ref id="CR6"><label>6.</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>King</surname><given-names>AMQ</given-names></name><name><surname>Lefkowitz</surname><given-names>E</given-names></name><name><surname>Adams</surname><given-names>MJ</given-names></name><name><surname>Carstens</surname><given-names>EB</given-names></name></person-group><source>Virus Taxonomy: Ninth report of the International Committee on Taxonomy of Viruses</source><year>2011</year><publisher-loc>London</publisher-loc><publisher-name>Elsevier Inc.</publisher-name></element-citation></ref><ref id="CR7"><label>7.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van der Hoek</surname><given-names>L</given-names></name><name><surname>Pyrc</surname><given-names>K</given-names></name><name><surname>Berkhout</surname><given-names>B</given-names></name></person-group><article-title>Human coronavirus NL63, a new respiratory virus</article-title><source>FEMS Microbiol Rev</source><year>2006</year><volume>30</volume><issue>5</issue><fpage>760</fpage><lpage>73</lpage><pub-id pub-id-type="doi">10.1111/j.1574-6976.2006.00032.x</pub-id><pub-id pub-id-type="pmid">16911043</pub-id></element-citation></ref><ref id="CR8"><label>8.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zaki</surname><given-names>AM</given-names></name><name><surname>van Boheemen</surname><given-names>S</given-names></name><name><surname>Bestebroer</surname><given-names>TM</given-names></name><name><surname>Osterhaus</surname><given-names>AD</given-names></name><name><surname>Fouchier</surname><given-names>RA</given-names></name></person-group><article-title>Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia</article-title><source>N Engl J Med</source><year>2012</year><volume>367</volume><issue>19</issue><fpage>1814</fpage><lpage>20</lpage><pub-id pub-id-type="doi">10.1056/NEJMoa1211721</pub-id><pub-id pub-id-type="pmid">23075143</pub-id></element-citation></ref><ref id="CR9"><label>9.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vabret</surname><given-names>A</given-names></name><name><surname>Mourez</surname><given-names>T</given-names></name><name><surname>Dina</surname><given-names>J</given-names></name><name><surname>van der Hoek</surname><given-names>L</given-names></name><name><surname>Gouarin</surname><given-names>S</given-names></name><name><surname>Petitjean</surname><given-names>J</given-names></name><etal></etal></person-group><article-title>Human coronavirus NL63, France</article-title><source>Emerg Infect Dis</source><year>2005</year><volume>11</volume><issue>8</issue><fpage>1225</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.3201/eid1108.050110</pub-id><pub-id pub-id-type="pmid">16102311</pub-id></element-citation></ref><ref id="CR10"><label>10.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hammitt</surname><given-names>LL</given-names></name><name><surname>Kazungu</surname><given-names>S</given-names></name><name><surname>Welch</surname><given-names>S</given-names></name><name><surname>Bett</surname><given-names>A</given-names></name><name><surname>Onyango</surname><given-names>CO</given-names></name><name><surname>Gunson</surname><given-names>RN</given-names></name><etal></etal></person-group><article-title>Added value of an oropharyngeal swab in detection of viruses in children hospitalized with lower respiratory tract infection</article-title><source>J Clin Microbiol</source><year>2011</year><volume>49</volume><issue>6</issue><fpage>2318</fpage><lpage>20</lpage><pub-id pub-id-type="doi">10.1128/JCM.02605-10</pub-id><pub-id pub-id-type="pmid">21490188</pub-id></element-citation></ref><ref id="CR11"><label>11.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chiu</surname><given-names>SS</given-names></name><name><surname>Chan</surname><given-names>KH</given-names></name><name><surname>Chu</surname><given-names>KW</given-names></name><name><surname>Kwan</surname><given-names>SW</given-names></name><name><surname>Guan</surname><given-names>Y</given-names></name><name><surname>Poon</surname><given-names>LL</given-names></name><etal></etal></person-group><article-title>Human coronavirus NL63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in Hong Kong, China</article-title><source>Clin Infect Dis</source><year>2005</year><volume>40</volume><issue>12</issue><fpage>1721</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1086/430301</pub-id><pub-id pub-id-type="pmid">15909257</pub-id></element-citation></ref><ref id="CR12"><label>12.</label><mixed-citation publication-type="other">WHO. Middle East respiratory syndrome coronavirus (MERS-CoV): Summary of Current Situation, Literature Update and Risk Assessment–as of 5 February 2015. Global Alert and Response (GAR) 2015 [cited 2015 17 March 2015]; Available from: <ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/disease/coronavirus_infections/mers-5-february-2015.pdf?ua=1">http://www.who.int/csr/disease/coronavirus_infections/mers-5-february-2015.pdf?ua=1</ext-link>. Accessed 27 Jan 2016.</mixed-citation></ref><ref id="CR13"><label>13.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bulimo</surname><given-names>WD</given-names></name><name><surname>Achilla</surname><given-names>RA</given-names></name><name><surname>Majanja</surname><given-names>J</given-names></name><name><surname>Mukunzi</surname><given-names>S</given-names></name><name><surname>Wadegu</surname><given-names>M</given-names></name><name><surname>Osunna</surname><given-names>F</given-names></name><etal></etal></person-group><article-title>Molecular characterization and phylogenetic analysis of the hemagglutinin 1 protein of human influenza A virus subtype H1N1 circulating in Kenya during 2007–2008</article-title><source>J Infect Dis</source><year>2012</year><volume>206</volume><issue>Suppl 1</issue><fpage>S46</fpage><lpage>52</lpage><pub-id pub-id-type="doi">10.1093/infdis/jis586</pub-id><pub-id pub-id-type="pmid">23169971</pub-id></element-citation></ref><ref id="CR14"><label>14.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sueker</surname><given-names>J</given-names></name><name><surname>Blazes</surname><given-names>DL</given-names></name><name><surname>Johns</surname><given-names>MC</given-names></name><name><surname>Blair</surname><given-names>PJ</given-names></name><name><surname>Sjoberg</surname><given-names>PA</given-names></name><name><surname>Tjaden</surname><given-names>JA</given-names></name><etal></etal></person-group><article-title>Influenza and respiratory disease surveillance: the US military’s global laboratory-based network</article-title><source>Influenza Other Respi Viruses</source><year>2010</year><volume>4</volume><issue>3</issue><fpage>155</fpage><lpage>61</lpage><pub-id pub-id-type="doi">10.1111/j.1750-2659.2010.00129.x</pub-id></element-citation></ref><ref id="CR15"><label>15.</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>FISCHERS</surname><given-names>A</given-names></name><name><surname>ANDREW</surname><given-names>A</given-names></name><name><surname>TOWNSEND</surname><given-names>W</given-names></name></person-group><source>Handbook for family planning operations research designs</source><year>1998</year><publisher-loc>USA</publisher-loc><publisher-name>Population Council</publisher-name></element-citation></ref><ref id="CR16"><label>16.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kuypers</surname><given-names>J</given-names></name><name><surname>Martin</surname><given-names>ET</given-names></name><name><surname>Heugel</surname><given-names>J</given-names></name><name><surname>Wright</surname><given-names>N</given-names></name><name><surname>Morrow</surname><given-names>R</given-names></name><name><surname>Englund</surname><given-names>JA</given-names></name></person-group><article-title>Clinical disease in children associated with newly described coronavirus subtypes</article-title><source>Pediatrics</source><year>2007</year><volume>119</volume><issue>1</issue><fpage>e70</fpage><lpage>6</lpage><pub-id pub-id-type="doi">10.1542/peds.2006-1406</pub-id><pub-id pub-id-type="pmid">17130280</pub-id></element-citation></ref><ref id="CR17"><label>17.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Woo</surname><given-names>PC</given-names></name><name><surname>Lau</surname><given-names>SK</given-names></name><name><surname>Chu</surname><given-names>CM</given-names></name><name><surname>Chan</surname><given-names>KH</given-names></name><name><surname>Tsoi</surname><given-names>HW</given-names></name><name><surname>Huang</surname><given-names>Y</given-names></name><etal></etal></person-group><article-title>Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia</article-title><source>J Virol</source><year>2005</year><volume>79</volume><issue>2</issue><fpage>884</fpage><lpage>95</lpage><pub-id pub-id-type="doi">10.1128/JVI.79.2.884-895.2005</pub-id><pub-id pub-id-type="pmid">15613317</pub-id></element-citation></ref><ref id="CR18"><label>18.</label><mixed-citation publication-type="other">H.B.S.R.A. DNA Baser Sequence Assembler v3.x. 2012 [cited 2014; Available from: <ext-link ext-link-type="uri" xlink:href="http://www.DnaBaser.com">http://www.DnaBaser.com</ext-link>]. Accessed 27 Jan 2016.</mixed-citation></ref><ref id="CR19"><label>19.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Altschul</surname><given-names>SF</given-names></name><name><surname>Gish</surname><given-names>W</given-names></name><name><surname>Miller</surname><given-names>W</given-names></name><name><surname>Myers</surname><given-names>EW</given-names></name><name><surname>Lipman</surname><given-names>DJ</given-names></name></person-group><article-title>Basic local alignment search tool</article-title><source>J Mol Biol</source><year>1990</year><volume>215</volume><issue>3</issue><fpage>403</fpage><lpage>10</lpage><pub-id pub-id-type="doi">10.1016/S0022-2836(05)80360-2</pub-id><pub-id pub-id-type="pmid">2231712</pub-id></element-citation></ref><ref id="CR20"><label>20.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Edgar</surname><given-names>RC</given-names></name></person-group><article-title>MUSCLE: multiple sequence alignment with high accuracy and high throughput</article-title><source>Nucleic Acids Res</source><year>2004</year><volume>32</volume><issue>5</issue><fpage>1792</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1093/nar/gkh340</pub-id><pub-id pub-id-type="pmid">15034147</pub-id></element-citation></ref><ref id="CR21"><label>21.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ronquist</surname><given-names>F</given-names></name><name><surname>Teslenko</surname><given-names>M</given-names></name><name><surname>van der Mark</surname><given-names>P</given-names></name><name><surname>Ayres</surname><given-names>DL</given-names></name><name><surname>Darling</surname><given-names>A</given-names></name><name><surname>Hohna</surname><given-names>S</given-names></name><etal></etal></person-group><article-title>MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space</article-title><source>Syst Biol</source><year>2012</year><volume>61</volume><issue>3</issue><fpage>539</fpage><lpage>42</lpage><pub-id pub-id-type="doi">10.1093/sysbio/sys029</pub-id><pub-id pub-id-type="pmid">22357727</pub-id></element-citation></ref><ref id="CR22"><label>22.</label><mixed-citation publication-type="other">Rambaut A. 2009 [cited 2012; Available from: <ext-link ext-link-type="uri" xlink:href="http://tree.bio.ed.ac.uk/software/figtree/">http://tree.bio.ed.ac.uk/software/figtree/</ext-link>. Accessed 27 Jan 2016.</mixed-citation></ref><ref id="CR23"><label>23.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lau</surname><given-names>SK</given-names></name><name><surname>Woo</surname><given-names>PC</given-names></name><name><surname>Yip</surname><given-names>CC</given-names></name><name><surname>Tse</surname><given-names>H</given-names></name><name><surname>Tsoi</surname><given-names>HW</given-names></name><name><surname>Cheng</surname><given-names>VC</given-names></name><etal></etal></person-group><article-title>Coronavirus HKU1 and other coronavirus infections in Hong Kong</article-title><source>J Clin Microbiol</source><year>2006</year><volume>44</volume><issue>6</issue><fpage>2063</fpage><lpage>71</lpage><pub-id pub-id-type="doi">10.1128/JCM.02614-05</pub-id><pub-id pub-id-type="pmid">16757599</pub-id></element-citation></ref><ref id="CR24"><label>24.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Owusu</surname><given-names>M</given-names></name><name><surname>Annan</surname><given-names>A</given-names></name><name><surname>Corman</surname><given-names>VM</given-names></name><name><surname>Larbi</surname><given-names>R</given-names></name><name><surname>Anti</surname><given-names>P</given-names></name><name><surname>Drexler</surname><given-names>JF</given-names></name><etal></etal></person-group><article-title>Human coronaviruses associated with upper respiratory tract infections in three rural areas of Ghana</article-title><source>PLoS One</source><year>2014</year><volume>9</volume><issue>7</issue><fpage>e99782</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0099782</pub-id><pub-id pub-id-type="pmid">25080241</pub-id></element-citation></ref><ref id="CR25"><label>25.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kuypers</surname><given-names>J</given-names></name><name><surname>Wright</surname><given-names>N</given-names></name><name><surname>Ferrenberg</surname><given-names>J</given-names></name><name><surname>Huang</surname><given-names>ML</given-names></name><name><surname>Cent</surname><given-names>A</given-names></name><name><surname>Corey</surname><given-names>L</given-names></name><etal></etal></person-group><article-title>Comparison of real-time PCR assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children</article-title><source>J Clin Microbiol</source><year>2006</year><volume>44</volume><issue>7</issue><fpage>2382</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1128/JCM.00216-06</pub-id><pub-id pub-id-type="pmid">16825353</pub-id></element-citation></ref><ref id="CR26"><label>26.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Berkley</surname><given-names>JA</given-names></name><name><surname>Munywoki</surname><given-names>P</given-names></name><name><surname>Ngama</surname><given-names>M</given-names></name><name><surname>Kazungu</surname><given-names>S</given-names></name><name><surname>Abwao</surname><given-names>J</given-names></name><name><surname>Bett</surname><given-names>A</given-names></name><etal></etal></person-group><article-title>Viral etiology of severe pneumonia among Kenyan infants and children</article-title><source>JAMA</source><year>2010</year><volume>303</volume><issue>20</issue><fpage>2051</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1001/jama.2010.675</pub-id><pub-id pub-id-type="pmid">20501927</pub-id></element-citation></ref><ref id="CR27"><label>27.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dijkman</surname><given-names>R</given-names></name><name><surname>Jebbink</surname><given-names>MF</given-names></name><name><surname>Gaunt</surname><given-names>E</given-names></name><name><surname>Rossen</surname><given-names>JW</given-names></name><name><surname>Templeton</surname><given-names>KE</given-names></name><name><surname>Kuijpers</surname><given-names>TW</given-names></name><etal></etal></person-group><article-title>The dominance of human coronavirus OC43 and NL63 infections in infants</article-title><source>J Clin Virol</source><year>2012</year><volume>53</volume><issue>2</issue><fpage>135</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1016/j.jcv.2011.11.011</pub-id><pub-id pub-id-type="pmid">22188723</pub-id></element-citation></ref><ref id="CR28"><label>28.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Smuts</surname><given-names>HE</given-names></name><name><surname>Workman</surname><given-names>LJ</given-names></name><name><surname>Zar</surname><given-names>HJ</given-names></name></person-group><article-title>Human rhinovirus infection in young African children with acute wheezing</article-title><source>BMC Infect Dis</source><year>2011</year><volume>11</volume><fpage>65</fpage><pub-id pub-id-type="doi">10.1186/1471-2334-11-65</pub-id><pub-id pub-id-type="pmid">21401965</pub-id></element-citation></ref><ref id="CR29"><label>29.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lu</surname><given-names>R</given-names></name><name><surname>Yu</surname><given-names>X</given-names></name><name><surname>Wang</surname><given-names>W</given-names></name><name><surname>Duan</surname><given-names>X</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Zhou</surname><given-names>W</given-names></name><etal></etal></person-group><article-title>Characterization of human coronavirus etiology in Chinese adults with acute upper respiratory tract infection by real-time RT-PCR assays</article-title><source>PLoS One</source><year>2012</year><volume>7</volume><issue>6</issue><fpage>e38638</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0038638</pub-id><pub-id pub-id-type="pmid">22719912</pub-id></element-citation></ref><ref id="CR30"><label>30.</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Mclntosh</surname><given-names>K</given-names></name><name><surname>Englund</surname><given-names>JA</given-names></name><etal></etal></person-group><person-group person-group-type="editor"><name><surname>Cherry</surname><given-names>J</given-names></name><name><surname>Harrison</surname><given-names>G</given-names></name><name><surname>Kaplan</surname><given-names>S</given-names></name><etal></etal></person-group><article-title>Coronaviruses and Toroviruses, Including Severe Acute Respiratory Syndrome</article-title><source>Feigin and Cherry’s Textbook of Pediatric infectious diseases. Vol. 7</source><year>2014</year><edition>7</edition><publisher-loc>Philadelphia</publisher-loc><publisher-name>Elsevier Saunders</publisher-name></element-citation></ref><ref id="CR31"><label>31.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Elden</surname><given-names>LJ</given-names></name><name><surname>van Loon</surname><given-names>AM</given-names></name><name><surname>van Alphen</surname><given-names>F</given-names></name><name><surname>Hendriksen</surname><given-names>KA</given-names></name><name><surname>Hoepelman</surname><given-names>AI</given-names></name><name><surname>van Kraaij</surname><given-names>MG</given-names></name><etal></etal></person-group><article-title>Frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction</article-title><source>J Infect Dis</source><year>2004</year><volume>189</volume><issue>4</issue><fpage>652</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1086/381207</pub-id><pub-id pub-id-type="pmid">14767819</pub-id></element-citation></ref><ref id="CR32"><label>32.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Kraaij</surname><given-names>MG</given-names></name><name><surname>van Elden</surname><given-names>LJ</given-names></name><name><surname>van Loon</surname><given-names>AM</given-names></name><name><surname>Hendriksen</surname><given-names>KA</given-names></name><name><surname>Laterveer</surname><given-names>L</given-names></name><name><surname>Dekker</surname><given-names>AW</given-names></name><etal></etal></person-group><article-title>Frequent detection of respiratory viruses in adult recipients of stem cell transplants with the use of real-time polymerase chain reaction, compared with viral culture</article-title><source>Clin Infect Dis</source><year>2005</year><volume>40</volume><issue>5</issue><fpage>662</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1086/427801</pub-id><pub-id pub-id-type="pmid">15714410</pub-id></element-citation></ref><ref id="CR33"><label>33.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pyrc</surname><given-names>K</given-names></name><name><surname>Sims</surname><given-names>AC</given-names></name><name><surname>Dijkman</surname><given-names>R</given-names></name><name><surname>Jebbink</surname><given-names>M</given-names></name><name><surname>Long</surname><given-names>C</given-names></name><name><surname>Deming</surname><given-names>D</given-names></name><etal></etal></person-group><article-title>Culturing the unculturable: human coronavirus HKU1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures</article-title><source>J Virol</source><year>2010</year><volume>84</volume><issue>21</issue><fpage>11255</fpage><lpage>63</lpage><pub-id pub-id-type="doi">10.1128/JVI.00947-10</pub-id><pub-id pub-id-type="pmid">20719951</pub-id></element-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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