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

Loading next page...
 
/lp/springer_journal/construction-and-characterization-of-a-recombinant-ovine-lentivirus-Poh4oI48x3
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

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 12 million articles from more than
10,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Unlimited reading

Read as many articles as you need. Full articles with original layout, charts and figures. Read online, from anywhere.

Stay up to date

Keep up with your field with Personalized Recommendations and Follow Journals to get automatic updates.

Organize your research

It’s easy to organize your research with our built-in tools.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve Freelancer

DeepDyve Pro

Price
FREE
$49/month

$360/year
Save searches from
Google Scholar,
PubMed
Create lists to
organize your research
Export lists, citations
Read DeepDyve articles
Abstract access only
Unlimited access to over
18 million full-text articles
Print
20 pages/month
PDF Discount
20% off