Construction and characterization of a recombinant ovine lentivirus carrying the optimized green fluorescent protein gene at the dUTPase locus

Construction and characterization of a recombinant ovine lentivirus carrying the optimized green... A dUTPase gene ( du ) deleted ovine lentivirus (OvLV Δdu ) 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 Δdu-egfp . OvLV Δdu 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 Δdu-egfp RT activity and virus titer were lower than for KV1772 and OvLV Δdu (p < 0.05). OvLV-85/34 RT in sheep monocyte-derived macrophages (SMDM) was higher than that of KV1772, OvLV Δdu and OvLV Δdu-egfp (p < 0.05). The ability to prevent dUTP mis-incorporation into newly synthesized DNA was disrupted in OvLV Δdu and OvLV Δdu-egfp (p < 0.05). Immunoprecipitation demonstrated that GFP is expressed by OvLV Δdu-egfp at a low level. OvLV Δdu-egfp retained egfp after 10 passages in cell culture. OvLV Δdu-egfp was re-isolated in GSM cells from peripheral blood mononuclear (PBMN) cells of three of four OvLV Δdu-egfp -inoculated lambs, but by contrast to the in vitro experiments OvLV Δdu-egfp lost the insert. Priming with OvLV Δdu-egfp did not prevent infection with pathogenic OvLV, but cell-associated viremia in a mock-infected contact control lamb was higher than in OvLV Δdu-egfp -primed lambs. OvLV serum antibody titers increased steadily in OvLV Δdu-egfp -inoculated lambs, but in a lamb from which OvLV Δdu-egfp was not reisolated the antibody titer surpassed the negative/positive cut-off value only after challenge with OvLV-85/34. Because OvLV Δdu-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 Δdu-egfp as an attenuated, live-virus vaccine to protect sheep against OvLV-induced disease when challenged with pathogenic OvLV. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Construction and characterization of a recombinant ovine lentivirus carrying the optimized green fluorescent protein gene at the dUTPase locus

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Publisher
Springer-Verlag
Copyright
Copyright © 2003 by Springer-Verlag/Wien
Subject
LifeSciences
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s00705-003-0123-8
Publisher site
See Article on Publisher Site

Abstract

A dUTPase gene ( du ) deleted ovine lentivirus (OvLV Δdu ) 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 Δdu-egfp . OvLV Δdu 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 Δdu-egfp RT activity and virus titer were lower than for KV1772 and OvLV Δdu (p < 0.05). OvLV-85/34 RT in sheep monocyte-derived macrophages (SMDM) was higher than that of KV1772, OvLV Δdu and OvLV Δdu-egfp (p < 0.05). The ability to prevent dUTP mis-incorporation into newly synthesized DNA was disrupted in OvLV Δdu and OvLV Δdu-egfp (p < 0.05). Immunoprecipitation demonstrated that GFP is expressed by OvLV Δdu-egfp at a low level. OvLV Δdu-egfp retained egfp after 10 passages in cell culture. OvLV Δdu-egfp was re-isolated in GSM cells from peripheral blood mononuclear (PBMN) cells of three of four OvLV Δdu-egfp -inoculated lambs, but by contrast to the in vitro experiments OvLV Δdu-egfp lost the insert. Priming with OvLV Δdu-egfp did not prevent infection with pathogenic OvLV, but cell-associated viremia in a mock-infected contact control lamb was higher than in OvLV Δdu-egfp -primed lambs. OvLV serum antibody titers increased steadily in OvLV Δdu-egfp -inoculated lambs, but in a lamb from which OvLV Δdu-egfp was not reisolated the antibody titer surpassed the negative/positive cut-off value only after challenge with OvLV-85/34. Because OvLV Δdu-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 Δdu-egfp as an attenuated, live-virus vaccine to protect sheep against OvLV-induced disease when challenged with pathogenic OvLV.

Journal

Archives of VirologySpringer Journals

Published: Jul 1, 2003

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