JCM Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martinez-Picado, J.
Right arrow Articles by D'Aquila, R. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martinez-Picado, J.
Right arrow Articles by D'Aquila, R. T.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, September 1999, p. 2943-2951, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Human Immunodeficiency Virus Type 1 Cloning Vectors for Antiretroviral Resistance Testing

Javier Martinez-Picado,1,2,3 Lorraine Sutton,1 Maria Pia De Pasquale,1,2 Anu V. Savara,1 and Richard T. D'Aquila1,2,*

Massachusetts General Hospital1 and Harvard Medical School,2 Boston, Massachusetts 02129, and Foundation irsi-Caixa, Barcelona, Spain3

Received 15 March 1999/Returned for modification 26 April 1999/Accepted 7 June 1999

Better detection of minority human immunodeficiency virus type 1 (HIV-1) populations containing gene mutations may improve the usefulness of antiretroviral resistance testing for clinical management. Molecular cloning of HIV-1 PCR products which might improve minority detection can be slow and difficult, and commercially available recombinant virus assays test drug susceptibility of virus pools. We describe novel plasmids and simple methods for rapid cloning of HIV-1 PCR products from patient specimens and their application to generate infectious recombinant virus clones for virus phenotyping and genotyping. Eight plasmids with differing deletions of sequences encoding HIV-1 protease, reverse transcriptase, or Gag p7/p1 and Gag p1/p6 cleavage sites were constructed for cloning HIV-1 PCR products. A simple HIV-1 sequence-specific uracil deglycosylase-mediated cloning method with the vectors and primers designed here was more rapid than standard ligase-mediated cloning. Pooled and molecularly cloned infectious recombinant viruses were generated with these vectors. Replicative viral fitness and drug susceptibility phenotypes of cloned infectious viruses containing patient specimen-derived sequences were measured. Clonal resistance genotyping analyses were also performed from virus isolates, plasma HIV-1 RNA, and infected cell DNA. Sequencing of a limited number of molecular clones detected minorities of resistant virus not identified in the pooled population PCR product sequence and linkage of minority mutations.

* Corresponding author. Mailing address: Infectious Disease Division and AIDS Research Center, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129. Phone: (617) 726-5776. Fax: (617) 726-5411. E-mail: daquila{at}helix.mgh.harvard.edu.

Journal of Clinical Microbiology, September 1999, p. 2943-2951, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:



Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Antimicrob. Agents Chemother. Clin. Microbiol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS

Copyright © 1999 by the American Society for Microbiology. All rights reserved.