CFAR Virology Core Facility
Resources and Services
Virus & Cells
The VC facility will provide phenotypically and genotypically characterized viral stocks of laboratory-adapted HIV-1 and SIV. In addition, the facility will co-culture primary isolates from HIV-1 infected adult and pediatric individuals with either a progressive infection or a long-term non-progressive infection. Viral stocks of primary isolates and functional, biological clones derived from these isolates will be characterized and provided to the investigators by the facility. In conjunction with the Molecular Biology Core facility, the VC facility will also serve as a resource for genotypically characterized molecular clones of primary isolates. The facility will separate PBMC from normal human donor blood and will supply activated or non-activated primary PBMC for viral infectivity studies. T-cell lines and T-cells enriched for CD4 or CD8 cells and macrophages free of T-cell contamination using immunobeads or other improved cell-separation techniques will be supplied to investigators for infectivity and tropism studies.
Assays
Phenotypic characterizations of the virus will include: 
- titering of virus in appropriate host cells;
- characterizing the non-syncytium-inducing (NSI) and SI phenotype in MT-2 cells;
- determining the infectivity of the virus in CD4 T-cells;
- determining the replication kinetics and receptor-tropism in primary T-cells, T-cell lines, and cell lines co-expressing CD4 and chemokine receptors;
- characterizing the cytopathic and non-cytopathic phenotype of the virus in CD4 T-cells (envelope-mediated syncytial death) and in CD4-single cell killing or CD4-SCK assay (non-syncytial death);
- determining the in vivo cytopathic and non-cytopathic phenotype of the virus in mice reconstituted with human T-cells (in association with the Animal Core facility).
The facility provides resources to perform molecular detection/diagnostic assays including qualitative and quantitative RNA and DNA-PCR for viral genomes. This year the VC facility has expanded its resources by providing Taqman-based PCR assays for real-time detection and quantitation of viral DNA. Using this state of the art, cutting edge technique, investigators can detect and quantify low levels of active viral replication (LTR-circle assay). These studies will be very helpful to understand viral latency and viral pathogenesis.
The facility will function as a site to identify and/or to monitor the efficacy of potential anti-HIV compounds/drugs targeted to structural or regulatory gene functions using cell-culture systems or small animal models. The facility will also perform antibody neutralization assays to determine antibody titer in serum of infected individuals enrolled in viral-intervention (vaccine, immunotherapy) protocols/studies.
Repository
The VC facility will maintain a repository of cell lines routinely used for viral culture for immediate availability to investigators. The facility will isolate and titer viral stocks in primary CD4 T cells, monocyte-derived macrophages or T-cell lines. These viruses will be genotypically analyzed and will be archived in aliquots at -80C or liquid nitrogen.
Additional Resources
The participating CFAR investigators can avail themselves of the services of the Facscan and Facsort flow cytometers located in the VC facility. Analysis of T-cell surface markers (CD3, CD4, CD8), monocyte surface markers (CD14), mouse cell surface markers (CD19, CD45) and differentiation of activated (HLADR) and non-activated T-cells are routinely performed cytofluorographically using Facscan flow cytometer. The CD4/CD8 T-cell counts of infected individuals can be determined and the efficacy of anti-viral therapy on the profile of CD4 T-cell counts can be monitored flow cytometrically. The facility will also have the capability to analyze multiple cell-surface markers by 2-color or 3-color fluorescence (FITC, PE, PERCP or CYCHROME) staining technique using the flow cytometer. The flow cytometer can be used to detect and analyze virus-infected cells, to detect apoptotic cells, and to measure mitochondrial viability using fluorescent dyes. The FACsort flow cytometer in the facility is available to enrich and asceptically sort specific cell-populations for infectivity and cell-tropism studies. The FACsort flow cytometer is also available to asceptically sort virus-infected cells from in vivo (patient) and in vitro (cell-culture) under biocontainment to determine the intracellular events post-infection.