Development of a Recombinant Noncytopathic Bovine Viral Diarrhea Virus Stably Expressing Enhanced Green Fluorescent Protein
Type of DegreeThesis
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Noncytopathic (NCP) type-I bovine viral diarrhea virus (BVDV) strain SD1 was originally isolated from a persistently infected heifer with fatal mucosal disease (MD) and represents the only reported nucleotide sequence of a NCP BVDV isolate determined without extensive cell culture passage in the laboratory. In this study, a full length infectious cDNA clone of SD1 was constructed in a plasmid backbone, pACYC1180, a low-copy-number plasmid, by RT-PCR and standard molecular techniques. In vitro transcripts from this template directed the generation of infectious BVDV upon transfection of MDBK cells. The rescued virus, designated as ASD1, contained all of the artificially introduced genetic markers and had similar growth properties as wild type SD1 (wt SD1). Based on pASD1, a recombinant BVDV stably expressing enhanced green fluorescent protein (eGFP) was successfully generated and designated as ASD1-eGFP2A3. ASD1-eGFP2A3 was stable upon passage in tissue culture and eGFP accumulated to high levels in vivo. eGFP expression was maintained stably over time following the passage of persistently-infected MDBK cells. Furthermore, ASD1-eGFP2A3 was similar to wt SD1 in viral growth properties, viral RNA replication, and viral protein expression. Thus, this marker virus may provide a tool for studying the viral transplacental infection in animal test as a real-time monitoring system and a backbone for construction of recombinant marker BVDV vaccine.