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Journal of Virology, February 2005, p. 1409-1416, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1409-1416.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Reverse Genetics System for Porcine Enteric Calicivirus, a Prototype Sapovirus in the Caliciviridae

Kyeong-Ok Chang,1* Stanislav S. Sosnovtsev,1 Gaël Belliot,1 Qiuhong Wang,2 Linda J. Saif,2 and Kim Y. Green1

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland,1 Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio2

Received 9 July 2004/ Accepted 10 September 2004

A porcine enteric calicivirus (PEC), strain Cowden in the genus Sapovirus of the Caliciviridae family, can be propagated in a porcine kidney continuous cell line (LLC-PK) in the presence of bile acids in the cell culture medium. A full-length cDNA copy of the Cowden PEC genome was cloned into a plasmid vector directly downstream from the T7 RNA polymerase promoter, and capped RNA transcripts derived from this clone were infectious when transfected into LLC-PK cells. The recovery of PEC after transfection of RNA transcripts was dependent on the presence of bile acids, consistent with our recent identification of a bile acid-mediated signaling pathway required for PEC replication (Chang et al., Proc. Natl. Acad. Sci. USA 101:8733-8788, 2004). Recovery of virus was verified by detection of PEC antigen in transfected cells by immunofluorescence and enzyme-linked immunosorbent assays, direct observation of recovered viral particles by electron microscopy, and partial sequence analysis of their genomes (first 1,070 nucleotides) to differentiate them from tissue culture-adapted parental virus. The recovered virus retained its ability to infect piglets when administered by the oral route and showed an attenuated phenotype similar to that of the tissue culture-adapted parental virus. This reverse genetics system for PEC provides a new tool to study the molecular basis of replication and pathogenesis for caliciviruses associated with diarrheal disease.

* Corresponding author. Mailing address: National Institutes of Health/DHHS, NIAID/LID, Bldg. 50, Room 6316, 9000 Rockville Pike, Bethesda, MD 20892-8026. Phone: (301) 496-5130. Fax: (301) 480-5031. E-mail: kchang{at}niaid.nih.gov.

Journal of Virology, February 2005, p. 1409-1416, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1409-1416.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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