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Journal of Virology, November 2002, p. 11518-11529, Vol. 76, No. 22
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.22.11518-11529.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Generation of a Replication-Competent, Propagation-Deficient Virus Vector Based on the Transmissible Gastroenteritis Coronavirus Genome

Javier Ortego,1 David Escors,1 Hubert Laude,2 and Luis Enjuanes1*

Centro Nacional de Biotecnología, CSIC, Department of Molecular and Cell Biology, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain,1 Unité de Virologie Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France2

Received 8 May 2002/ Accepted 6 August 2002

Replication-competent propagation-deficient virus vectors based on the transmissible gastroenteritis coronavirus (TGEV) genome that are deficient in the essential E gene have been developed by complementation within E+ packaging cell lines. Cell lines expressing the TGEV E protein were established using the noncytopathic Sindbis virus replicon pSINrep21. In addition, cell lines stably expressing the E gene under the CMV promoter have been developed. The Sindbis replicon vector and the ectopic TGEV E protein did not interfere with the rescue of infectious TGEV from full-length cDNA. Recombinant TGEV deficient in the nonessential 3a and 3b genes and the essential E gene (rTGEV-{Delta}3ab{Delta}E) was successfully rescued in these cell lines. rTGEV-{Delta}3ab{Delta}E reached high titers (107 PFU/ml) in baby hamster kidney cells expressing porcine aminopeptidase N (BHK-pAPN), the cellular receptor for TGEV, using Sindbis replicon and reached titers up to 5 x 105 PFU/ml in cells stably expressing E protein under the control of the CMV promoter. The virus titers were proportional to the E protein expression level. The rTGEV-{Delta}3ab{Delta}E virions produced in the packaging cell line showed the same morphology and stability under different pHs and temperatures as virus derived from the full-length rTGEV genome, although a delay in virus assembly was observed by electron microscopy and virus titration in the complementation system in relation to the wild-type virus. These viruses were stably grown for >10 passages in the E+ packaging cell lines. The availability of packaging cell lines will significantly facilitate the production of safe TGEV-derived vectors for vaccination and possibly gene therapy.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco, 28029 Madrid, Spain. Phone: 34-91-585 4555. Fax: 34-91-585 4915. E-mail: L.Enjuanes{at}cnb.uam.es.

Journal of Virology, November 2002, p. 11518-11529, Vol. 76, No. 22
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.22.11518-11529.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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