{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,28]],"date-time":"2026-03-28T19:11:18Z","timestamp":1774725078413,"version":"3.50.1"},"reference-count":70,"publisher":"American Society for Microbiology","issue":"23","license":[{"start":{"date-parts":[[2008,12,1]],"date-time":"2008-12-01T00:00:00Z","timestamp":1228089600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Virol"],"published-print":{"date-parts":[[2008,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>Historically, coronaviruses were predominantly associated with mild upper respiratory disease in humans. More recently, three novel coronaviruses associated with severe human respiratory disease were found, including (i) the severe acute respiratory syndrome coronavirus, associated with a significant atypical pneumonia and 10% mortality; (ii) HKU-1, associated with chronic pulmonary disease; and (iii) NL63, associated with both upper and lower respiratory tract disease in children and adults worldwide. These discoveries establish coronaviruses as important human pathogens and underscore the need for continued research toward the development of platforms that will enable genetic manipulation of the viral genome, allowing for rapid and rational development and testing of candidate vaccines, vaccine vectors, and therapeutics. In this report, we describe a reverse genetics system for NL63, whereby five contiguous cDNAs that span the entire genome were used to generate a full-length cDNA. Recombinant NL63 viruses which contained the expected marker mutations replicated as efficiently as the wild-type NL63 virus. In addition, we engineered the heterologous green fluorescent protein gene in place of open reading frame 3 (ORF3) of the NL63 clone, simultaneously creating a unique marker for NL63 infection and demonstrating that the ORF3 protein product is nonessential for the replication of NL63 in cell culture. The availability of the NL63 and NL63gfp clones and recombinant viruses provides powerful tools that will help advance our understanding of this important human pathogen.<\/jats:p>","DOI":"10.1128\/jvi.01804-08","type":"journal-article","created":{"date-parts":[[2008,9,26]],"date-time":"2008-09-26T00:45:07Z","timestamp":1222389907000},"page":"11948-11957","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":86,"title":["Systematic Assembly of a Full-Length Infectious Clone of Human Coronavirus NL63"],"prefix":"10.1128","volume":"82","author":[{"given":"Eric F.","family":"Donaldson","sequence":"first","affiliation":[{"name":"Departments of Microbiology and Immunology"},{"name":"Epidemiology"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Boyd","family":"Yount","sequence":"additional","affiliation":[{"name":"Epidemiology"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amy C.","family":"Sims","sequence":"additional","affiliation":[{"name":"Epidemiology"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Susan","family":"Burkett","sequence":"additional","affiliation":[{"name":"Cystic Fibrosis\/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raymond J.","family":"Pickles","sequence":"additional","affiliation":[{"name":"Departments of Microbiology and Immunology"},{"name":"Cystic Fibrosis\/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ralph S.","family":"Baric","sequence":"additional","affiliation":[{"name":"Departments of Microbiology and Immunology"},{"name":"Epidemiology"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.20288"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1097\/01.inf.0000145815.70485.f7"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2006.11.022"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02263-07"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1086\/426869"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(08)60170-1"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.75.24.12359-12369.2001"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1080\/03079450500367682"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1086\/498875"},{"key":"e_1_3_2_11_2","doi-asserted-by":"publisher","DOI":"10.1086\/506350"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1086\/521308"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1186\/1743-422X-3-106"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.20289"},{"key":"e_1_3_2_15_2","first-page":"835","year":"2000","unstructured":"Enjuanes, L., D. Cavanaugh, K. Holmes, M. Lai, H. Laude, P. Masters, P. Rottier, S. Siddell, W. Spaan, F. Taguchi, and P. Talbot. 2000. Coronaviridaea, p. 835-859. In Virus taxonomy. Classification and nomenclature of viruses. Academic Press, San Diego, CA.","journal-title":"Classification and nomenclature of viruses"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01032-06"},{"key":"e_1_3_2_17_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01012-07"},{"key":"e_1_3_2_18_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-0-387-33012-9_26"},{"key":"e_1_3_2_19_2","first-page":"183","volume":"107","year":"2005","unstructured":"Fulcher, M. L., S. Gabriel, K. A. Burns, J. R. Yankaskas, and S. H. Randell. 2005. Well-differentiated human airway epithelial cell cultures. Methods Mol. Med.107:183-206.","journal-title":"Methods Mol. Med."},{"key":"e_1_3_2_20_2","first-page":"547","volume":"46","year":"2006","unstructured":"Ge, J. Y., Z. Y. Wen, Y. Wang, E. D. Bao, and Z. G. Bu. 2006. Rescue of a recombinant Newcastle disease virus expressing the green fluorescent protein. Wei Sheng Wu Xue Bao46:547-551. (In Chinese.)","journal-title":"Wei Sheng Wu Xue Bao"},{"key":"e_1_3_2_21_2","doi-asserted-by":"publisher","DOI":"10.1002\/path.2162"},{"key":"e_1_3_2_22_2","doi-asserted-by":"publisher","DOI":"10.1002\/path.1570"},{"key":"e_1_3_2_23_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-006-0746-7"},{"key":"e_1_3_2_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcv.2006.10.009"},{"key":"e_1_3_2_25_2","doi-asserted-by":"publisher","DOI":"10.1093\/clinids\/18.5.770"},{"key":"e_1_3_2_26_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0409465102"},{"key":"e_1_3_2_27_2","doi-asserted-by":"publisher","DOI":"10.1084\/jem.115.5.903"},{"key":"e_1_3_2_28_2","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.21067"},{"key":"e_1_3_2_29_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1469-0691.2006.01506.x"},{"key":"e_1_3_2_30_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01782-06"},{"key":"e_1_3_2_31_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0065-3527(08)60286-9"},{"key":"e_1_3_2_32_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2004.09.031"},{"key":"e_1_3_2_33_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.02614-05"},{"key":"e_1_3_2_34_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2008.05.025"},{"key":"e_1_3_2_35_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2007.04.035"},{"key":"e_1_3_2_36_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-007-0961-x"},{"key":"e_1_3_2_37_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0065-3527(06)66005-3"},{"key":"e_1_3_2_38_2","first-page":"85","volume":"63","year":"1974","unstructured":"McIntosh, K. 1974. Coronaviruses: a comparative review. Curr. Top. Microbiol. Immunol.63:85-129.","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"e_1_3_2_39_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.78.14.7400-7409.2004"},{"key":"e_1_3_2_40_2","unstructured":"Studies on human pathogenic coronaviruses: survey for coronaviruses in African bat species and characterization of the novel human coronavirus NL63 (HCoV-NL63) open reading frame 3 (ORF3) 2007"},{"key":"e_1_3_2_41_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.76.22.11518-11529.2002"},{"key":"e_1_3_2_42_2","doi-asserted-by":"publisher","DOI":"10.1155\/2006\/152612"},{"key":"e_1_3_2_43_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4615-5331-1_65"},{"key":"e_1_3_2_44_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.72.7.6014-6023.1998"},{"key":"e_1_3_2_45_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-0-387-33012-9_47"},{"key":"e_1_3_2_46_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pmed.0020274"},{"key":"e_1_3_2_47_2","first-page":"25","volume":"3","year":"2005","unstructured":"Pyrc, K., B. Burkhout, and L. van der Hoek. 2005. Molecular characterization of human coronavirus NL63. Recent Res. Dev. Infect. Immun.3:25-48.","journal-title":"Recent Res. Dev. Infect. Immun."},{"key":"e_1_3_2_48_2","doi-asserted-by":"publisher","DOI":"10.1186\/1743-422X-1-7"},{"key":"e_1_3_2_49_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.0030005"},{"key":"e_1_3_2_50_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02041-07"},{"key":"e_1_3_2_51_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.79.24.15511-15524.2005"},{"key":"e_1_3_2_52_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2007.03.013"},{"key":"e_1_3_2_53_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-0-387-33012-9_48"},{"key":"e_1_3_2_54_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro775"},{"key":"e_1_3_2_55_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.micinf.2004.01.011"},{"key":"e_1_3_2_56_2","doi-asserted-by":"publisher","DOI":"10.1099\/0022-1317-82-6-1273"},{"key":"e_1_3_2_57_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00384-06"},{"key":"e_1_3_2_58_2","first-page":"176","volume":"44","year":"2007","unstructured":"Vabret, A., J. Dina, S. Gouarin, J. Petitjean, V. Tripey, J. Brouard, and F. Freymuth. 2007. Human (non-severe acute respiratory syndrome) coronavirus infections in hospitalised children in France. J. Paediatr. Child Health.44:176-181.","journal-title":"J. Paediatr. Child Health."},{"key":"e_1_3_2_59_2","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.82590-0"},{"key":"e_1_3_2_60_2","doi-asserted-by":"publisher","DOI":"10.1038\/nm1024"},{"key":"e_1_3_2_61_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pmed.0020240"},{"key":"e_1_3_2_62_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-0-387-33012-9_86"},{"key":"e_1_3_2_63_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00431-007-0429-8"},{"key":"e_1_3_2_64_2","first-page":"245","volume":"23","year":"2007","unstructured":"Xing, J. F., R. N. Zhu, Y. Qian, L. Q. Zhao, J. Deng, F. Wang, and Y. Sun. 2007. Sequence analysis for genes encoding nucleoprotein and envelope protein of a new human coronavirus NL63 identified from a pediatric patient in Beijing by bioinformatics. Bing Du Xue Bao23:245-251. (In Chinese.)","journal-title":"Bing Du Xue Bao"},{"key":"e_1_3_2_65_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2004.10.045"},{"key":"e_1_3_2_66_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.74.22.10600-10611.2000"},{"key":"e_1_3_2_67_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1735582100"},{"key":"e_1_3_2_68_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.76.21.11065-11078.2002"},{"key":"e_1_3_2_69_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.79.23.14909-14922.2005"},{"key":"e_1_3_2_70_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.79.2.1113-1124.2005"},{"key":"e_1_3_2_71_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.76.11.5654-5666.2002"}],"container-title":["Journal of Virology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/JVI.01804-08","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/JVI.01804-08","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,3,5]],"date-time":"2022-03-05T12:08:27Z","timestamp":1646482107000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/JVI.01804-08"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2008,12]]},"references-count":70,"journal-issue":{"issue":"23","published-print":{"date-parts":[[2008,12]]}},"alternative-id":["10.1128\/JVI.01804-08"],"URL":"https:\/\/doi.org\/10.1128\/jvi.01804-08","relation":{},"ISSN":["0022-538X","1098-5514"],"issn-type":[{"value":"0022-538X","type":"print"},{"value":"1098-5514","type":"electronic"}],"subject":[],"published":{"date-parts":[[2008,12]]},"assertion":[{"value":"2008-08-27","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2008-09-19","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2008-12-01","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}