Characterization of HIV Reservoirs in the Brain

National Institute Of Neurological Disorders And Stroke

Investigators

Abstract

To achieve the goals of this project, the following is the summary of what we have done: 1) We have collected autopsy brain and peripheral lymphoid tissue from 4 persons with HIV(PWH). Additional tissues are being obtained from NNTC. 2) We have synthesized digital droplet PCR primers for the detection of HIV reservoir in tissue, and performed confirmative PCR run to validate the primers. 3) We cultured 4 different cell lines (including Jurkat cell as negative control, 8E5 cell with single copy of integrated HIV, and two more cell lines with multiple integrated HIV copies) for the extraction of genomic DNA and verification of integrated HIV genome with our synthesized primers. In addition, this genomic DNA will be used as positive control for our long read sequencing of HIV reservoir from HIV patient tissue. 4) We designed hybridization capture probe panel for the enrichment of HIV genomic fragment from the total genomic DNA extraction. 5) We use the extracted cell line genomic DNA to perform DNA fragmentation using MegaRuptor from Diagenode . The targeted DNA fragment size is around 7 kb. 5) We used the cell line genomic DNA for genomic HIV fragment enrichment with the HIV hybridization capture probe panel and made DNA library for nanopore sequencing. 6) We are currently using the newly purchased Oxford nanopore sequencing device (GridION) for establishing sequencing and data analysis protocol. We had trouble with the purchase and installation of the GridION device because of the delayed shipment and replacement of defective device. We are still troubleshooting one of the flow cell holder in the GridION device. 7) We are now preparing genomic DNA library from PWH. Once we successfully run the cell line genomic DNA library on the GridION, we will start sequencing PWH genomic DNA for integrated HIV.