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Journal of Virology, December 2008, p. 11948-11957, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01804-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Systematic Assembly of a Full-Length Infectious Clone of Human Coronavirus NL63{triangledown} ,{dagger}

Eric F. Donaldson,1,2,§ Boyd Yount,2,§ Amy C. Sims,2 Susan Burkett,3 Raymond J. Pickles,1,3 and Ralph S. Baric1,2*

Departments of Microbiology and Immunology,1 Epidemiology,2 Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina3

Received 27 August 2008/ Accepted 19 September 2008

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.

* Corresponding author. Mailing address: 2107 McGavran-Greenberg, CB# 7435, Chapel Hill, NC 27599-7435. Phone: (919) 966-3895. Fax: (919) 966-0584. E-mail: rbaric{at}email.unc.edu

{triangledown} Published ahead of print on 25 September 2008.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

§ These authors contributed equally to this work.

Journal of Virology, December 2008, p. 11948-11957, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01804-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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