Journal of General Virology

In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes

Sara Alonso,¹ Isabel Sola,¹ Jens P. Teifke,² Ilona Reimann,² Ander Izeta,¹ Mónica Balasch,³ Juan Plana-Durán,³ Rob J. M. Moormann⁴ and Luis Enjuanes¹

¹ Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
² Federal Research Centre for Virus Diseases of Animals, Friedrich-Loeffler-Institutes, Insel Riems, Germany
³ Fort Dodge Veterinaria, Girona, Spain
⁴ Institute for Animal Science and Health, Lelystad, The Netherlands

A helper-dependent expression system based on transmissible gastroenteritis coronavirus (TGEV) has been developed using a minigenome of 3.9 kb (M39). Expression of the reporter gene -glucuronidase (GUS) (2–8 µg per 10⁶ cells) and the porcine respiratory and reproductive syndrome virus (PRRSV) ORF5 (1–2 µg per 10⁶ cells) has been shown using a TGEV-derived minigenome. GUS expression levels increased about eightfold with the m.o.i. and were maintained for more than eight passages in cell culture. Nevertheless, instability of the GUS and ORF5 subgenomic mRNAs was observed from passages five and four, respectively. About a quarter of the cells in culture expressing the helper virus also produced the reporter gene as determined by studying GUS mRNA production by in situ hybridization or immunodetection to visualize the protein synthesized. Expression of GUS was detected in the lungs, but not in the gut, of swine immunized with the virus vector. Around a quarter of lung cells showing replication of the helper virus were also positive for the reporter gene. Interestingly, strong humoral immune responses to both GUS and PRRSV ORF5 were induced in swine with this virus vector. The large cloning capacity and the tissue specificity of the TGEV-derived minigenomes suggest that these virus vectors are very promising for vaccine development.

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© 2002 SGM

This article is now available in the March 2002 print issue of JGV (vol. 83, 567–579). The complete issue of the journal may be seen in electronic form on JGV Online.