Lab data entry
Co-investigators: Deenan Pillay, Dermot Maher, Anatoli Kamali, Judith Vandepitte, Heiner Grosskurth, Josephine Birungi, David Yirrell, Jim Mullins, George Shaw and Beatrice Hahn
Funding: MRC; European and Developing Countries Clinical Trials Partnership ,(Collaboration on AIDS Vaccine Development)
Objectives: The purpose of this work is to provide information on the HIV-1 subtypes circulating in Uganda especially in our study populations, on the frequency of HIV-1 dual infection in our cohorts and on sequences for epitope mapping for various immunology studies.
Methods: Specimens from the various cohorts that the MRC Uganda Unit is maintaining in Uganda are examined, using a variety of techniques. For DNA sequencing of specimens from the various cohort, viral RNA is extracted from cryopreserved plasma using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany); PCR is performed in the pol-integrase and env-gp41genes. The amplified products are cleaned and directly sequenced using a Beckman Coulter CEQ8800 sequencer. Phylogenetic trees are constructed using the neighbour-joining method, and the reliability of topologies is estimated through the bootstrap approach. For other cohorts, viral DNA samples are amplified by PCR using an in-house assay already.....
..established and optimized at UVRI. For investigations of viral resistance, HIV-1 RNA is extracted from plasma using RNA extraction columns (Qiagen, Hilden, Germany), amplified using the pol region and then sequenced by means of a Beckman CEQ 8000/8800 automated capillary sequencer and an ABI 3130 genetic sequencer platform. Sequence data are analysed using MEGA 4.3, ClustalX 1.7, ChromasPro, R.2.5 package and Sequencher 4.6 software programmes. After submission to the Stanford database, phylogenetic trees are reconstructed using distance-based, maximum likelihood and Bayesian inference techniques and the robustness of the clustering evaluated using statistical techniques included in the PHYLIP package. For dual infection stuidies, we use methods that allow high throughput, including the Multiple Hybridization Assay (MHA) and gag HMA. more
HIV Drug resistance reference activities
The MRC/UVRI Basic Sciences laboratory is currently a WHO accredited laboratory for HIV drug resistance testing. It functions as the National and Regional reference laboratory.