Development of a reporter bovine viral diarrhea virus and initial evaluation of its application for high throughput antiviral drug screening.
Bovine viral diarrhea virus (BVDV) causes lethal mucosal disease of cattle and leads to severe economic loss of cattle production and reproduction worldwide. Over the past decades, vaccination was not very successful in providing prevention of BVDV infection. This reality demands that anti-BVDV drugs should be used as an alternative treatment strategy. In this study, a BAC cDNA of noncytopathic BVDV strain SD-1 is constructed to contain an enhanced green fluorescence protein (eGFP) gene between viral NS3 and NS4A coding sequences. The recombinant reporter virus is generated subsequently by transfection of MDBK cells with the transcripts produced in vitro. The rescued reporter virus is stable in MDBK cells and the eGFP protein is expressed and processed properly. Of most importance, the reporter virus shows a growth property similar to the SD-1 parent and the fluorescent signal intensity increases in parallel to the reporter virus RNA and protein replication. In addition, two known anti-BVDV drug G418 (viral assembly/release inhibitor) and ribavirin (viral RNA replication inhibitor) are identified as hits in a high-throughput format, suggesting that this system is capable of identifying BVDV inhibitors that target different steps in viral life cycle. The cell-based system developed provides a useful and versatile tool which should facilitate the identification of BVDV inhibitors on a large scale.
Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA. fanzhen@obesita-algaegen.com
Zhen-Chuan
ZC
Fan
R Curtis
RC
Bird
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000821:virology; D002417:Cattle; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D030241:Diarrhea Virus 1, Bovine Viral / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods*; D007668:Kidney / Q000166:cytology / Q000821:virology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000998:Antiviral Agents; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014764:Viral Proteins