title: Construction and characterization of a recombinant ovine lentivirus carrying the optimized green fluorescent protein gene at the dUTPase locus.
abstract: AdUTPase gene ( du) deleted ovine lentivirus (OvLV(Deltadu)) mutant, derived from Visna/maedi virus (VMV) molecular clone KV1772, was constructed. Subsequently, a copy of the optimized green fluorescent protein ( egfp) coding region was fused into the viral pol open reading frame (ORF) at the deleted du locus to generate viral mutant, OvLV(Deltadu-egfp). OvLV(Deltadu) reverse transcriptase (RT) activity and titer of infectious virus in goat synovial membrane (GSM) cell cultures were not affected compared to that of KV1772 and OvLV-85/34 strain (p < 0.05). By contrast, OvLV(Deltadu-egfp) RT activity and virus titer were lower than for KV1772 and OvLV(Deltadu) (p < 0.05). OvLV-85/34 RT in sheep monocyte-derived macrophages (SMDM) was higher than that of KV1772, OvLV(Deltadu) and OvLV(Deltadu-egfp) (p < 0.05). The ability to prevent dUTP mis-incorporation into newly synthesized DNA was disrupted in OvLV(Deltadu) and OvLV(Deltadu-egfp) (p < 0.05). Immunoprecipitation demonstrated that GFP is expressed by OvLV(Deltadu-egfp) at a low level. OvLV(Deltadu-egfp) retained egfp after 10 passages in cell culture.OvLV(Deltadu-egfp) was re-isolated in GSM cells from peripheral blood mononuclear (PBMN) cells of three of four OvLV(Deltadu-egfp)-inoculated lambs, but by contrast to the in vitro experiments OvLV(Deltadu-egfp) lost the insert. Priming with OvLV(Deltadu-egfp) did not prevent infection with pathogenic OvLV, but cell-associated viremia in a mock-infected contact control lamb was higher than in OvLV(Deltadu-egfp)-primed lambs. OvLV serum antibody titers increased steadily in OvLV(Deltadu-egfp)-inoculated lambs, but in a lamb from which OvLV(Deltadu-egfp) was not reisolated the antibody titer surpassed the negative/positive cut-off value only after challenge with OvLV-85/34. Because OvLV(Deltadu-egfp) is attenuated for pathogenicity in vitro, replicates in vivo and stimulates an antibody response, subsequent experiments need to address the likelihood of using OvLV(Deltadu-egfp) as an attenuated, live-virus vaccine to protect sheep against OvLV-induced disease when challenged with pathogenic OvLV.
authors.affiliation: Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Guo
authors.forename: Y
authors.initials: Y
authors.lastname: Ni
authors.forename: F W
authors.initials: FW
authors.lastname: Bazer
authors.forename: L
authors.initials: L
authors.lastname: Giavedoni
authors.forename: A
authors.initials: A
authors.lastname: de la Concha-Bermejillo
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002478:Cells, Cultured; D017353:Gene Deletion; D006041:Goats; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000821:virology; D009000:Monocytes / Q000821:virology; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012756:Sheep; D012757:Sheep Diseases / Q000821:virology*; D013583:Synovial Membrane / Q000821:virology; D019076:Transgenes; D014779:Virus Replication; D014790:Visna-maedi virus / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity / Q000502:physiology*
chemical_list: D000914:Antibodies, Viral; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase