In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes.
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 beta-glucuronidase (GUS) (2-8 microg per 10(6) cells) and the porcine respiratory and reproductive syndrome virus (PRRSV) ORF5 (1-2 microg per 10(6) 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.
Alonso
Sara
S
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain1.
Sola
Isabel
I
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain1.
Teifke
Jens P
JP
Federal Research Centre for Virus Diseases of Animals, Friedrich-Loeffler-Institutes, Insel Riems, Germany2.
Reimann
Ilona
I
Federal Research Centre for Virus Diseases of Animals, Friedrich-Loeffler-Institutes, Insel Riems, Germany2.
Izeta
Ander
A
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain1.
Balasch
Mónica
M
Fort Dodge Veterinaria, Girona, Spain3.
Plana-Durán
Juan
J
Fort Dodge Veterinaria, Girona, Spain3.
Moormann
Rob J M
RJM
Institute for Animal Science and Health, Lelystad, The Netherlands4.
Enjuanes
Luis
L
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain1.
D000818:Animals; D000831:Animals, Newborn / Q000276:immunology / Q000821:virology; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D006378:Helper Viruses / Q000235:genetics / Q000502:physiology; D017403:In Situ Hybridization; D007422:Intestines / Q000821:virology; D008168:Lung / Q000821:virology; D016366:Open Reading Frames / Q000235:genetics; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012692:Serial Passage; D013552:Swine / Q000276:immunology / Q000821:virology; D019076:Transgenes / Q000235:genetics*; D005760:Transmissible gastroenteritis virus / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology; D014779:Virus Replication
D012333:RNA, Messenger; D014764:Viral Proteins; D014765:Viral Vaccines; D005966:Glucuronidase