title: Isolation, propagation, genome analysis and epidemiology of HKU1 betacoronaviruses.
abstract: From 1 January 2009 to 31 May 2013, 15 287 respiratory specimens submitted to the Clinical Virology Laboratory at the Children's Hospital Colorado were tested for human coronavirus RNA by reverse transcription-PCR. Human coronaviruses HKU1, OC43, 229E and NL63 co-circulated during each of the respiratory seasons but with significant year-to-year variability, and cumulatively accounted for 7.4-15.6 % of all samples tested during the months of peak activity. A total of 79 (0.5 % prevalence) specimens were positive for human betacoronavirus HKU1 RNA. Genotypes HKU1 A and B were both isolated from clinical specimens and propagated on primary human tracheal-bronchial epithelial cells cultured at the air-liquid interface and were neutralized in vitro by human intravenous immunoglobulin and by polyclonal rabbit antibodies to the spike glycoprotein of HKU1. Phylogenetic analysis of the deduced amino acid sequences of seven full-length genomes of Colorado HKU1 viruses and the spike glycoproteins from four additional HKU1 viruses from Colorado and three from Brazil demonstrated remarkable conservation of these sequences with genotypes circulating in Hong Kong and France. Within genotype A, all but one of the Colorado HKU1 sequences formed a unique subclade defined by three amino acid substitutions (W197F, F613Y and S752F) in the spike glycoprotein and exhibited a unique signature in the acidic tandem repeat in the N-terminal region of the nsp3 subdomain. Elucidating the function of and mechanisms responsible for the formation of these varying tandem repeats will increase our understanding of the replication process and pathogenicity of HKU1 and potentially of other coronaviruses.
authors.affiliation: Departments of Microbiology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 E 19th Ave, Room P18-9403B, Aurora, CO 80045, USA.|Departments of Pediatrics, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 E 19th Ave, Room P18-9403B, Aurora, CO 80045, USA.
authors.forename: Samuel R
authors.initials: SR
authors.lastname: Dominguez
authors.affiliation: Department of Pathology and Clinical Medicine, Children's Hospital Colorado, 13123 E 16th Ave, Aurora, CO 80045, USA.
authors.forename: Susmita
authors.initials: S
authors.lastname: Shrivastava
authors.affiliation: Departments of Pediatrics, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 E 19th Ave, Room P18-9403B, Aurora, CO 80045, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Berglund
authors.affiliation: Departments of Pediatrics, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 E 19th Ave, Room P18-9403B, Aurora, CO 80045, USA.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Qian
authors.affiliation: Interdisciplinary Graduate Program in Biotechnology, University of São Paulo, Av Prof. Lineu Prestes, 2415, ICB-III, Cidade Universitária, CEP: 05508-900, São Paulo, SP - Brazil.|J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA.
authors.forename: Luiz Gustavo Bentim
authors.initials: LGB
authors.lastname: Góes
authors.affiliation: Department of Pathology and Clinical Medicine, Children's Hospital Colorado, 13123 E 16th Ave, Aurora, CO 80045, USA.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Halpin
authors.affiliation: Department of Pathology and Clinical Medicine, Children's Hospital Colorado, 13123 E 16th Ave, Aurora, CO 80045, USA.
authors.forename: Nadia
authors.initials: N
authors.lastname: Fedorova
authors.affiliation: Department of Pathology and Clinical Medicine, Children's Hospital Colorado, 13123 E 16th Ave, Aurora, CO 80045, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Ransier
authors.affiliation: Departments of Pediatrics, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 E 19th Ave, Room P18-9403B, Aurora, CO 80045, USA.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Weston
authors.affiliation: Interdisciplinary Graduate Program in Biotechnology, University of São Paulo, Av Prof. Lineu Prestes, 2415, ICB-III, Cidade Universitária, CEP: 05508-900, São Paulo, SP - Brazil.|J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA.
authors.forename: Edison Luiz
authors.initials: EL
authors.lastname: Durigon
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Av Prof. Lineu Prestes 1374, ICB-II, Cidade Universitária, CEP: 05580-900, São Paulo, SP - Brazil.|J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA.
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Jerez
authors.affiliation: Department of Preventive Veterinary Medicine and Animal Health, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, Cidade Universitária, CEP: 05508-270, Sao Paulo, SP - Brazil.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Robinson
authors.affiliation: Department of Pathology and Clinical Medicine, Children's Hospital Colorado, 13123 E 16th Ave, Aurora, CO 80045, USA.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Town
authors.affiliation: Departments of Microbiology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 E 19th Ave, Room P18-9403B, Aurora, CO 80045, USA.
authors.forename: Kathryn V
authors.initials: KV
authors.lastname: Holmes
mesh_terms: D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D002478:Cells, Cultured; D016000:Cluster Analysis; D003120:Colorado; D003332:Coronaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D003333:Coronaviridae Infections / Q000453:epidemiology* / Q000821:virology*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D012141:Respiratory Tract Infections / Q000453:epidemiology* / Q000821:virology*; D017422:Sequence Analysis, DNA; D064370:Spike Glycoprotein, Coronavirus / Q000235:genetics / Q000276:immunology; D014776:Virus Cultivation
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D012367:RNA, Viral; D064370:Spike Glycoprotein, Coronavirus