The Viral Evolution Core (VEC) provides expertise and innovative sequencing techniques, molecular cloning, as well as viral evolution analyses to support extramural and intramural investigators in order to increase the overall understanding of HIV biology with a major focus on exploiting the unique advantages afforded by utilizing non-human primates and SIV infection. Currently the VEC utilizes single genome amplification (SGA) and deep sequencing approaches (including Illumina, and SMRT sequencing technologies) to identify genetic changes in viral populations.
The Viral Evolution Core (VEC) provides expertise and innovative sequencing techniques, molecular cloning, as well as viral evolution analyses to support extramural and intramural investigators in order to increase the overall understanding of HIV biology with a major focus on exploiting the unique advantages afforded by utilizing non-human primates and SIV infection. Currently the VEC utilizes single genome amplification (SGA) and deep sequencing approaches (including Illumina, and SMRT sequencing technologies) to identify genetic changes in viral populations. This service includes viral RNA/DNA extraction from cell free or cell associated samples. cDNA will then be generated and using a limiting dilution method, will be diluted to a single copy prior to nested PCR spanning the viral gene(s) of interest. To assure only a single molecule is present in positive reactions, PCR is performed with many replicates and using the Poisson distribution, the proportion of positive to negative reactions indicates the probability of a single template. Confirmation of single molecule PCR is obtained by directly sequencing PCR products and excluding amplicons with evidence of more than one template. Finally, under the guidance of Dr. Keele, the VEC will provide comprehensive sequence analysis including enumerating transmitted/founder genomes, phylogenetic tree construction, and genetic comparisons of diversity. Relevant data will be provided electronically and a report with interpretation/conclusions will be provided. Consultation with the customer will be provided as needed. In addition to SGA and sanger sequencing, we have the ability to perform bulk PCR and MiSeq sequencing. This assay is particularly helpful when using molecularly tagged, or genetically barcoded virus model systems. Both of these approaches have been useful in mucosal transmission studies including testing preventative measures with vaccines, microbicides, passive administered neutralizing antibodies, and to characterize the viral reservoir in HIV and SIV infection. Finally, we have pioneered an innovative approach to isolate and sequence viral RNA and DNA directly from Formalin-Fixed, Paraffin-Embedded (FFPE) tissues that can be specifically isolated by laser-capture microdissection.
Quantitative Molecular Diagnostics Core (QMDC) provides state-of-the-art quantitative molecular analyses to measure specific nucleic acid sequences in specimens provided by laboratories within the ACVP, intramural NIH laboratories, and federally supported studies in extramural laboratories. In particular, the QMDC performs testing for simian immunodeficiency virus and related viruses, with emphasis on determination of viral loads in macaque models of AIDS, including plasma viral loads, cell-associated viral loads and tissue associated viral loads using quantitative PCR and RT PCR methods.
Over the years, the Cellular Immunity Core (CIC) has developed novel NHP/SIV reagents, methods and skills in support of ACVP and collaborative research endeavors. The CIC offers some of its more useful and qualified reagents and methods to the AIDS research community. The CIC also offers consultation on NHP immunology, NHP-reactive reagents and developing custom NHP-specific, polychromatic multi-parameter flow cytometry antibody panels.
- SIV ENV monoclonal antibody (Clone 7D3) labeled with Alexa Fluor 647
- SIV GAG monoclonal antibody (Clone 55-2F12) labeled with FITC/Alexa Fluor 647/Alexa Fluor 532
Flow cytometry panels and protocols:
- Polychromatic multi-parameter surface and intracellular flow cytometry assays and antibody panels to measure: cellular immune responses via intracellular cytokine staining (ICS); HIV and SIV Infection; transcription factors; cell viability, apoptosis and death; enzymes and enzymatic activity; and protein phosphorylation
- NHP cell flow cytometry phenotyping assays and panels to monitor detailed cell subsets and activation of: memory T cell populations; monocytes and macrophages; granulocytes and eosinophils; B cell populations; regulatory T cells (Treg); natural killer (NK) cell populations; dendritic cell (DC) populations; and T-follicular helper cells (Tfh and GC-Tfh).
- NHP whole blood absolute cell counting assays and flow cytometry panels to detect: memory/naïve T cells; NK cell subpopulations (CD8a/CD16/CD56); activated B cells and subpopulations; macrophage and monocyte subpopulations; granulocyte populations; and myeloid and plasmacytoid DCs (mDC/pDC)
- Polychromatic multi-parameter flow cytometry detection of acetylated-histones in NHP leukocyte subpopulations
- Flow cytometry SIV RNA detection assay (RNAflow)
- Flow cytometry single-HIV/SIV-infected-cell phenotyping assay
This website catalogs the cellular proteins found in the HIV-1 virions and contains recent protocols for subtilisin digestion and CD45 immunoaffinity depletion.