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Journal of Virology, March 2005, p. 3097-3106, Vol. 79, No. 5
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.5.3097-3106.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Recombinant Mouse Hepatitis Virus Strain A59 from Cloned, Full-Length cDNA Replicates to High Titers In Vitro and Is Fully Pathogenic In Vivo

Scott E. Coley ,1,{dagger},{ddagger} Ehud Lavi,2,{dagger},§ Stanley G. Sawicki,3 Li Fu,2 Barbara Schelle,4 Nadja Karl,4,§,¶ Stuart G. Siddell,1* and Volker Thiel5*

Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom,1 Division of Neuropathology, Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania,2 Department of Medical Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio,3 Institute of Virology and Immunology, University of Würzburg, Würzburg, Germany,4 Research Department, Cantonal Hospital St. Gallen, St. Gallen, Switzerland5

Received 3 August 2004/ Accepted 13 October 2004

Mouse hepatitis virus (MHV) is the prototype of group II coronaviruses and one of the most extensively studied coronaviruses. Here, we describe a reverse genetic system for MHV (strain A59) based upon the cloning of a full-length genomic cDNA in vaccinia virus. We show that the recombinant virus generated from cloned cDNA replicates to the same titers as the parental virus in cell culture (~109 PFU/ml), has the same plaque morphology, and produces the same amounts and proportions of genomic and subgenomic mRNAs in virus-infected cells. In a mouse model of neurological infection, the recombinant and parental viruses are equally virulent, they replicate to the same titers in brain and liver, and they induce similar patterns of acute hepatitis, acute meningoencephalitis, and chronic demyelination. We also describe improvements in the use of the coronavirus reverse genetic system based on vaccinia virus cloning vectors. These modifications facilitate (i) the mutagenesis of cloned cDNA by using vaccinia virus-mediated homologous recombination and (ii) the rescue of recombinant coronaviruses by using a stable nucleocapsid protein-expressing cell line for the electroporation of infectious full-length genomes. Thus, our system represents a versatile and universal tool to study all aspects of MHV molecular biology and pathogenesis. We expect this system to provide valuable insights into the replication of group II coronaviruses that may lead to the development of novel strategies against coronavirus infections, including the related severe acute respiratory syndrome coronavirus.

* Corresponding author. Mailing address for V. Thiel: Research Department, Cantonal Hospital St. Gallen, 9007 St. Gallen, Switzerland. Phone: 41-71-4941074. Fax: 41-71-4946321. E-mail: volker.thiel{at}kssg.ch. Mailing address for S. G. Siddell: Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom. Phone: 44-117-9287889. Fax: 44-117-9287896. E-mail: Stuart.Siddell{at}bristol.ac.uk.

{dagger} S.E.C. and E.L. contributed equally to this work.

{ddagger} Present address: Department of Medicine, University of Massachusetts, Worcester, MA 01605.

§ Department of Pathology, Weill Medical College of Cornell University, New York, NY 10021.

Present address: Rudolf-Virchow Center for Experimental Biomedicine, 97078 Würzburg, Germany.

Journal of Virology, March 2005, p. 3097-3106, Vol. 79, No. 5
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.5.3097-3106.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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