Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia.
Journal: 2005/January - Journal of Virology
ISSN: 0022-538X
Abstract:
Despite extensive laboratory investigations in patients with respiratory tract infections, no microbiological cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here we report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-year-old man with pneumonia who had just returned from Shenzhen, China. Quantitative reverse transcription-PCR showed that the amount of CoV-HKU1 RNA was 8.5 to 9.6 x 10(6) copies per ml in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with immunoglobulin M (IgM) titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, respectively. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic analysis reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, negative for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an additional specimen, with a viral load of 1.13 x 10(6) copies per ml, from a 35-year-old woman with pneumonia. Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans.
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J Virol 79(2): 884-895

Characterization and Complete Genome Sequence of a Novel Coronavirus, Coronavirus HKU1, from Patients with Pneumonia

+6 authors
Department of Microbiology, State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Division of Respiratory Medicine, Department of Medicine, United Christian Hospital, Department of Microbiology, Tseung Kwan O Hospital, Hong Kong4
Corresponding author. Mailing address: Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital, Hong Kong. Phone: (852) 28554892. Fax: (852) 28551241. E-mail: kh.ukh.ccukh@orcimukh.
P. C. Y. Woo, S. K. P. Lau, and K.-y. Yuen are all principal investigators and contributed equally to the manuscript.
Received 2004 Jul 29; Accepted 2004 Sep 3.

Abstract

Despite extensive laboratory investigations in patients with respiratory tract infections, no microbiological cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here we report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-year-old man with pneumonia who had just returned from Shenzhen, China. Quantitative reverse transcription-PCR showed that the amount of CoV-HKU1 RNA was 8.5 to 9.6 × 10 copies per ml in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with immunoglobulin M (IgM) titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, respectively. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic analysis reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, negative for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an additional specimen, with a viral load of 1.13 × 10 copies per ml, from a 35-year-old woman with pneumonia. Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans.

Abstract

Since no microbiological cause can be identified for a significant proportion of patients with respiratory tract infections (18, 29), research has been conducted to identify novel agents. Of the three novel agents identified in recent 3 years, including human metapneumovirus (36), severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) (25), and human coronavirus NL63 (HCoV-NL63) (6, 37), two were coronaviruses. Coronaviruses possess the largest genomes of all RNA viruses, consisting of about 30 kb. As a result of their unique mechanism of viral replication, coronaviruses have a high frequency of recombination.

Based on genotypic and serological characterization, coronaviruses were divided into three distinct groups, with human coronavirus 229E (HCoV-229E) being a group 1 coronavirus and human coronavirus OC43 (HCoV-OC43) being a group 2 coronavirus (16). They account for 5 to 30% of human respiratory tract infections. In late 2002 and 2003, the epidemic caused by SARS-CoV affected more than 8,000 people with 750 deaths (23-25, 44, 45, 51). We have also reported the isolation of SARS-CoV-like viruses from Himalayan palm civets, which suggested that animals could be the reservoir for the ancestor of SARS-CoV (9). On the basis of genome analysis, SARS-CoV belonged to a fourth coronavirus group or alternatively was a distant relative of group 2 coronaviruses (4, 20, 28, 31, 48). Recently, a novel group 1 human coronavirus associated with respiratory tract infections, HCoV-NL63, was discovered, and its genome was sequenced (37).

In this study, we report the discovery of a novel group 2 coronavirus in the nasopharyngeal aspirates (NPAs) of patients with pneumonia. The complete genome of the coronavirus was sequenced and analyzed. Based on the findings of this study, we propose that this new virus be designated coronavirus HKU1 (CoV-HKU1).

Acknowledgments

This work was partly supported by the Research Grant Council Grant, Research Fund for the Control of Infectious Diseases, “One Mouth, One Mask” Fund, Suen Chi Sun Charitable Foundation, the Public Health Research Grant AI95357 from the National Institutes of Allergy and Infectious Diseases, and the William Benter Infectious Disease Fund.

Acknowledgments

REFERENCES

REFERENCES

References

  • 1. Apweiler, R., T. K. Attwood, A. Bairoch, A. Bateman, E. Birney, M. Biswas, P. Bucher, L. Cerutti, F. Corpet, M. D. Croning, R. Durbin, L. Falquet, W. Fleischmann, J. Gouzy, H. Hermjakob, N. Hulo, I. Jonassen, D. Kahn, A. Kanapin, Y. Karavidopoulou, R. Lopez, B. Marx, N. J. Mulder, T. M. Oinn, M. Pagni, F. Servant, C. J. Sigrist, and E. M. Zdobnov. 2001. The InterPro database, an integrated documentation resource for protein families, domains and functional sites. Nucleic Acids Res.29:37-40.
  • 2. Bateman, A., E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall, and E. L. Sonnhammer. 2002. The Pfam protein families database. Nucleic Acids Res.30:276-280.
  • 3. Combet, C., C. Blanchet, C. Geourjon, and G. Deléage. 2000. NPS@: network protein sequence analysis. Trends Biochem. Sci.25:147-150. [[PubMed]
  • 4. Eickmann, M., S. Becker, H. D. Klenk, H. W. Doerr, K. Stadler, S. Censini, S. Guidotti, V. Masignani, M. Scarselli, M. Mora, C. Donati, J. H. Han, H. C. Song, S. Abrignani, A. Covacci, and R. Rappuoli. 2003. Phylogeny of the SARS coronavirus. Science302:1504-1505. [[PubMed]
  • 5. Fischer, F., D. Peng, S. T. Hingley, S. R. Weiss, and P. S. Masters. 1997. The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication. J. Virol.71:996-1003.
  • 6. Fouchier, R. A., N. G. Hartwig, T. M. Bestebroer, B. Niemever, J. C. de Jong, J. H. Simon, and A. D. Osterhaus. 2004. A previously undescribed coronavirus associated with respiratory disease in humans. Proc. Natl. Acad. Sci. USA101:6212-6216.
  • 7. Gattiker, A., E. Gasteiger, and A. Bairoch. 2002. ScanProsite: a reference implementation of a PROSITE scanning tool. Appl. Bioinformatics1:107-108. [[PubMed]
  • 8. Goebel, S. J., B. Hsue, T. F. Dombrowski, and P. S. Masters. 2004. Characterization of the RNA components of a putative molecular switch in the 3′ untranslated region of the murine coronavirus genome. J. Virol.78:669-682.
  • 9. Guan, Y., B. J. Zheng, Y. Q. He, X. L. Liu, Z. X. Zhuang, C. L. Cheung, S. W. Luo, P. H. Li, L. J. Zhang, Y. J. Guan, K. M. Butt, K. L. Wong, K. W. Chan, W. Lim, K. F. Shortridge, K. Y. Yuen, J. S. Peiris, and L. L. Poon. 2003. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science302:276-278. [[PubMed]
  • 10. Herold, J., S. G. Siddell, and A. E. Gorbalenya. 1999. A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold. J. Biol. Chem.274:14918-14925. [[PubMed]
  • 11. Hofmann, K., and WStoffel. 1993. TMbase—a database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler374:166. [PubMed][Google Scholar]
  • 12. Jendrach, M., V. Thiel, and S. Siddell. 1999. Characterization of an internal ribosome entry site within mRNA 5 of murine hepatitis virus. Arch. Virol.144:921-933. [[PubMed]
  • 13. Kanjanahaluethai, A., D. Jukneliene, and S. C. Baker. 2003. Identification of the murine coronavirus MP1 cleavage site recognized by papain-like proteinase 2. J. Virol.77:7376-7382.
  • 14. Klausegger, A., B. Strobl, G. Regl, and A. Kaser. 1999. Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus. J. Virol.73:3737-3743.
  • 15. Krempl, C., B. Schultze, and G. Herrler. 1995. Analysis of cellular receptors for human coronavirus OC43. Adv. Exp. Med. Biol.380:371-374. [[PubMed]
  • 16. Lai, M. M., and D. Cavanagh. 1997. The molecular biology of coronaviruses. Adv. Virus Res.48:1-100. [[PubMed]
  • 17. Lai, M. M., R. S. Baric, P. R. Brayton, and S. A. Stohlman. 1984. Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus. Proc. Natl. Acad. Sci. USA81:3626-3630.
  • 18. Macfarlane, J. T., A. Colville, A. Guion, R. M. Macfarlane, and D. H. Rose. 1993. Prospective study of aetiology and outcome of adult lower-respiratory-tract infections in the community. Lancet341:511-514. [[PubMed]
  • 19. Makino, S., S. A. Stohlman, and M. M. Lai. 1986. Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription. Proc. Natl. Acad. Sci. USA83:4204-4208.
  • 20. Marra, M. A., S. J. Jones, C. R. Astell, R. A. Holt, A. Brooks-Wilson, Y. S. Butterfield, J. Khattra, J. K. Asano, S. A. Barber, S. Y. Chan, A. Cloutier, S. M. Coughlin, D. Freeman, N. Girn, O. L. Griffith, S. R. Leach, M. Mayo, H. McDonald, S. B. Montgomery, P. K. Pandoh, A. S. Petrescu, A. G. Robertson, J. E. Schein, A. Siddiqui, D. E. Smailus, J. M. Stott, G. S. Yang, F. Plummer, A. Andonov, H. Artsob, N. Bastien, K. Bernard, T. F. Booth, D. Bowness, M. Czub, M. Drebot, L. Fernando, R. Flick, M. Garbutt, M. Gray, A. Grolla, S. Jones, H. Feldmann, A. Meyers, A. Kabani, Y. Li, S. Normand, U. Stroher, G. A. Tipples, S. Tyler, R. Vogrig, D. Ward, B. Watson, R. C. Brunham, M. Krajden, M. Petric, D. M. Skowronski, C. Upton, and R. L. Roper. 2003. The genome sequence of the SARS-associated coronavirus. Science300:1399-1404. [[PubMed]
  • 21. Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng.10:1-6. [[PubMed]
  • 22. Nussenzweig, V., and R. S. Nussenzweig. 1985. Circumsporozoite proteins of malaria parasites. Cell42:401-403. [[PubMed]
  • 23. Peiris, J. S., C. M. Chu, V. C. Cheng, K. S. Chan, I. F. Hung, L. L. Poon, K. I. Law, B. S. Tang, T. Y. Hon, C. S. Chan, K. H. Chan, J. S. Ng, B. J. Zheng, W. L. Ng, R. W. Lai, Y. Guan, K. Y. Yuen, and the HKU/UCH SARS Study Group. 2003. Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet361:1767-1772. [[PubMed]
  • 24. Peiris, J. S., K. Y. Yuen, A. D. Osterhaus, and K. Stohr. 2003. The severe acute respiratory syndrome. N. Engl. J. Med.349:2431-2441. [[PubMed]
  • 25. Peiris, J. S., S. T. Lai, L. L. Poon, Y. Guan, L. Y. Yam, W. Lim, J. Nicholls, W. K. Yee, W. W. Yan, M. T. Cheung, V. C. Cheng, K. H. Chan, D. N. Tsang, R. W. Yung, T. K. Ng, K. Y. Yuen, and the SARS Study Group. 2003. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet361:1319-1325. [[PubMed]
  • 26. Popova, R., and XZhang. 2002. The spike but not the hemagglutinin/esterase protein of bovine coronavirus is necessary and sufficient for viral infection. Virology294:222-236. [[PubMed][Google Scholar]
  • 27. Riski, H., and THovi. 1980. Coronavirus infections of man associated with diseases other than the common cold. J. Med. Virol.6:259-265. [[PubMed][Google Scholar]
  • 28. Rota, P. A., M. S. Oberste, S. S. Monroe, W. A. Nix, R. Campagnoli, J. P. Icenogle, S. Penaranda, B. Bankamp, K. Maher, M. H. Chen, S. Tong, A. Tamin, L. Lowe, M. Frace, J. L. DeRisi, Q. Chen, D. Wang, D. D. Erdman, T. C. Peret, C. Burns, T. G. Ksiazek, P. E. Rollin, A. Sanchez, S. Liffick, B. Holloway, J. Limor, K. McCaustland, M. Olsen-Rasmussen, R. Fouchier, S. Gunther, A. D. Osterhaus, C. Drosten, M. A. Pallansch, L. J. Anderson, and W. J. Bellini. 2003. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science300:1394-1399. [[PubMed]
  • 29. Ruiz, M., S. Ewig, M. A. Marcos, J. A. Martinez, F. Arancibia, J. Mensa, and A. Torres. 1990. Etiology of community-acquired pneumonia: impact of age, comorbidity, and severity. Am. J. Respir. Crit. Care Med.160:397-405. [[PubMed]
  • 30. Schwarz, B., E. Routledge, and S. G. Siddell. 1990. Murine coronavirus nonstructural protein ns2 is not essential for virus replication in transformed cells. J. Virol.64:4784-4791.
  • 31. Snijder, E. J., P. J. Bredenbeek, J. C. Dobbe, V. Thiel, J. Ziebuhr, L. L. Poon, Y. Guan, M. Rozanov, W. J. Spaan, and A. E. Gorbalenya. 2003. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J. Mol. Biol.331:991-1004. [[PubMed]
  • 32. Sonnhammer, E. L., G. von Heijne, and A. Krogh. 1998. A hidden Markov model for predicting transmembrane helices in protein sequences. Proc. Int. Conf. Intell. Syst. Mol. Biol.6:175-182. [[PubMed]
  • 33. Suzuki, H., and FTaguchi. 1996. Analysis of the receptor-binding site of murine coronavirus spike protein. J. Virol.70:2632-2636. [Google Scholar]
  • 34. Thiel, V., and S. G. Siddell. 1994. Internal ribosome entry in the coding region of murine hepatitis virus mRNA 5. J. Gen. Virol.75:3041-3046. [[PubMed]
  • 35. Vabret, A., T. Mourez, S. Gouarin, J. Petitiean, and F. Freymuth. 2003. An outbreak of coronavirus OC43 respiratory infection in Normandy, France. Clin. Infect. Dis.36:985-989. [[PubMed]
  • 36. van den Hoogen, B. G., J. C. de Jong, J. Groen, T. Kuiken, R. de Groot, R. A. Fouchier, and A. D. Osterhaus. 2001. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat. Med.7:719-724. [[PubMed]
  • 37. van der Hoek, L., K. Pyrc, M. F. Jebbink, W. Vermeulen-Oost, R. J. Berkhout, K. C. Wolthers, P. M. Wertheim-Van Dillen, J. Kaandorp, J. Spaargaren, and B. Berkhout. 2004. Identification of a new human coronavirus. Nat. Med.10:368-373. [[PubMed]
  • 38. Vlasak, R., W. Luytjes, J. Leider, W. Spaan, and P. Palese. 1988. The E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity. J. Virol.62:4686-4690.
  • 39. Vlasak, R., W. Luytjes, W. Spaan, and P. Palese. 1988. Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses. Proc. Natl. Acad. Sci. USA85:4526-4529.
  • 40. Wenzel, R. P., J. O. Hendley, J. A. Davies, and J. M. Gwaltney, Jr. 1974. Coronavirus infections in military recruits. Three-year study with coronavirus strains OC43 and 229E. Am. Rev. Respir. Dis.109:621-624. [[PubMed]
  • 41. Wessner, D. R., P. C. Shick, J. H. Lu, C. B. Cardellichio, S. E. Gagneten, N. Beauchemin, K. V. Holmes, and G. S. Dveksler. 1998. Mutational analysis of the virus and monoclonal antibody binding sites in MHVR, the cellular receptor of the murine coronavirus mouse hepatitis virus strain A59. J. Virol.72:1941-1948.
  • 42. Williams, G. D., R. Y. Chang, and D. A. Brian. 1999. A phylogenetically conserved hairpin-type 3′ untranslated region pseudoknot functions in coronavirus RNA replication. J. Virol.73:8349-8355.
  • 43. Williams, R. K., G. S. Jiang, and K. V. Holmes. 1991. Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins. Proc. Natl. Acad. Sci. USA88:5533-5536.
  • 44. Woo, P. C., S. K. Lau, B. H. Wong, H. W. Tsoi, A. M. Fung, K. H. Chan, V. K. Tam, J. S. Peiris, and K. Y. Yuen. 2004. Detection of specific antibodies to severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein for serodiagnosis of SARS coronavirus pneumonia. J. Clin. Microbiol.42:2306-2309.
  • 45. Woo, P. C., S. K. Lau, H. W. Tsoi, K. H. Chan, B. H. Wong, X. Y. Che, V. K. Tam, S. C. Tam, V. C. Cheng, I. F. Hung, S. S. Wong, B. J. Zheng, Y. Guan, and K. Y. Yuen. 2004. Relative rates of non-pneumonic SARS coronavirus infection and SARS coronavirus pneumonia. Lancet363:841-845. [[PubMed]
  • 46. Woo, P. C., S. S. Chiu, W. H. Seto, and M. Peiris. 1997. Cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients. J. Clin. Microbiol.35:1579-1581.
  • 47. Yang, H. J., S. J. Park, K. I. Im, and T. S. Yong. 2000. Identification of a Clonorchis sinensis gene encoding a vitellaria antigenic protein containing repetitive sequences. Mol. Biochem. Parasitol.111:213-216. [[PubMed]
  • 48. Yeh, S. H., H. Y. Wang, C. Y. Tsai, C. L. Kao, J. Y. Yang, H. W. Liu, I. J. Su, S. F. Tsai, D. S. Chen, P. J. Chen, and the National Taiwan University SARS Research Team. 2004. Characterization of severe acute respiratory syndrome coronavirus genomes in Taiwan: molecular epidemiology and genome evolution. Proc. Natl. Acad. Sci. USA101:2542-2547.
  • 49. Yokomori, K., L. R. Banner, and M. M. Lai. 1991. Heterogeneity of gene expression of the hemagglutinin-esterase (HE) protein of murine coronaviruses. Virology183:647-657. [[PubMed]
  • 50. Yokomori, K., M. Asanaka, S. A. Stohlman, S. Makino, R. A. Shubin, W. Gilmore, L. P. Weiner, F. I. Wang, and M. M. Lai. 1995. Neuropathogenicity of mouse hepatitis virus JHM isolates differing in hemagglutinin-esterase protein expression. J. Neurovirol.1:330-339. [[PubMed]
  • 51. Zhong, N. S., B. J. Zheng, Y. M. Li, L. L. Poon, Z. H. Xie, K. H. Chan, P. H. Li, S. Y. Tan, Q. Chang, J. P. Xie, X. Q. Liu, J. Xu, D. X. Li, K. Y. Yuen, J. S. Peiris, and Y. Guan. 2003. Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China, in February, 2003. Lancet362:1353-1358. [[PubMed]
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