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Journal of Virology, November 2008, p. 11429-11436, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.00696-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Recovery of Infectious Virus by Transfection of In Vitro-Generated RNA from Tulane Calicivirus cDNA{triangledown}

Chao Wei,1 Tibor Farkas,1,2* Karol Sestak,3,4 and Xi Jiang1,2*

Cincinnati Children's Hospital Medical Center,1 University of Cincinnati College of Medicine, Cincinnati, Ohio,2 Tulane National Primate Research Center,3 Tulane University School of Medicine, New Orleans, Louisiana4

Received 28 March 2008/ Accepted 29 August 2008

Tulane virus (TV) is a newly reported calicivirus that was isolated from stool samples of captive rhesus macaques from the Tulane National Primate Research Center (TNPRC). The virus has been cultivated successfully in LLC-MK2 rhesus monkey kidney cells. Its complete genomic sequence suggests that TV represents a new genus and is evolutionarily more closely related to Norovirus than to any other genus of Caliciviridae. In this study, we demonstrated that RNA transcripts made in vitro from the full-length genomic cDNA of TV were infectious upon transfection into permissive LLC-MK2 cells. The recombinant virus exhibited plaque morphologies and growth kinetics similar to those of the wild-type virus in this cell line. Capping was required for TV RNA infectivity. Although a subgenomic RNA has been detected in TV-transfected cells, a separate subgenomic RNA transcript was not required for the initial transfection to establish the replication. Transfection of truncated RNA lacking open reading frame 2 (ORF2) and ORF3 or TV-norovirus chimeric RNA resulted in abortive replication without the production of infectious progeny viruses, indicating that both ORFs are essential for the replication of TV. A heterologous insertion at the 5' end of the genome also hampered viral replication, suggesting that an authentic 5' end of the genome is critical for replication. The availability of the complete genomic sequence and the reverse genetics system described herein make TV a valuable model for studying calicivirus pathogenesis and replication.

* Corresponding author. Mailing address: Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. Phone: (513) 636-0119. Fax: (513) 636-7655. E-mail for Xi Jiang: jason.jiang{at}cchmc.org. E-mail for Tibor Farkas: tibor.farkas{at}cchmc.org

{triangledown} Published ahead of print on 10 September 2008.

Journal of Virology, November 2008, p. 11429-11436, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.00696-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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