VZV Foundation - A Decade Of Leadership In The Fight Against VZV Infections

	Oral Presentation Abstracts: 64 [64] FRACTIONATION OF NEURONS AND SATELLITE CELLS FROM HUMAN SENSORY GANGLIA IN ORDER TO STUDY HERPESVIRUS LATENCY Guang-Yun Cai, Ph.D. (1), Lewis Pizer, Ph.D. (1,2), & *Myron J. Levin, M.D. (1) (1) Section of Pediatric Infectious Disease & (2) Department of Microbiology, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, USA Herpes simplex virus (HSV) and varicella zoster virus (VZV) establish latency in sensory ganglia. Animal models indicate that certain types of neurons are preferred targets for HSV latency, but whether VZV has a similar neuronal tropism and can also establish latency in non-neuronal cells remain as open questions. The availability of isolated neurons and non-neuronal satellite cells would offer an approach to answering these questions. We developed a method of fractionating cells from human ganglia that results in highly purified populations of neurons and satellite cells suitable for single-cell PCR studies. This method avoids the use of fixatives and proteases that would alter cell surfaces. Trigeminal ganglia, removed at autopsy, were minced, passed through a 60 uM mesh filter, and the cell suspension sedimented (1xG) for 6 to 12 hours. Neurons and satellite cells were selected on the basis of morphology, with a micromanipulator connected to a Hamilton syringe and washed. Photomicrographs demonstrate the purity of the separated cells. These cells were subjected to PCR analysis to verify the fractionation procedure. Cells from eleven trigeminal ganglia were analyzed for HSV DNA. A total of 103 tubes containing neurons and 75 tubes containing clumps of satellite cells (50-60 cells per clump) were analyzed. Of the 965 neurons analyzed, 31 were HSV-positive, indicating that about 3% of the neurons contained latent HSV genomes. A total of 390 clumps of satellite cells, approximately 20,000 cells, were analyzed without detecting an HSV-positive tube. These data on the frequency and cellular location of latent HSV, which are consistent with previous publications, indicate that mechanical fractionation of cell types results in low levels of cross-contamination. The isolated neurons and satellite cells are being analyzed for VZV DNA and proteins. Corresponding Author: Myron J. Levin, M.D., University of Colorado Health Sciences Center, 4200 East Ninth Avenue, C-227, Denver, CO 80262, USA

Oral Presentation Abstracts: 64

[64]

FRACTIONATION OF NEURONS AND SATELLITE CELLS FROM HUMAN SENSORY GANGLIA IN ORDER TO STUDY HERPESVIRUS LATENCY

Guang-Yun Cai, Ph.D. (1), Lewis Pizer, Ph.D. (1,2), & *Myron J. Levin, M.D. (1)
(1) Section of Pediatric Infectious Disease & (2) Department of Microbiology, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, USA

Herpes simplex virus (HSV) and varicella zoster virus (VZV) establish latency in sensory ganglia. Animal models indicate that certain types of neurons are preferred targets for HSV latency, but whether VZV has a similar neuronal tropism and can also establish latency in non-neuronal cells remain as open questions. The availability of isolated neurons and non-neuronal satellite cells would offer an approach to answering these questions. We developed a method of fractionating cells from human ganglia that results in highly purified populations of neurons and satellite cells suitable for single-cell PCR studies. This method avoids the use of fixatives and proteases that would alter cell surfaces. Trigeminal ganglia, removed at autopsy, were minced, passed through a 60 uM mesh filter, and the cell suspension sedimented (1xG) for 6 to 12 hours. Neurons and satellite cells were selected on the basis of morphology, with a micromanipulator connected to a Hamilton syringe and washed. Photomicrographs demonstrate the purity of the separated cells. These cells were subjected to PCR analysis to verify the fractionation procedure. Cells from eleven trigeminal ganglia were analyzed for HSV DNA. A total of 103 tubes containing neurons and 75 tubes containing clumps of satellite cells (50-60 cells per clump) were analyzed. Of the 965 neurons analyzed, 31 were HSV-positive, indicating that about 3% of the neurons contained latent HSV genomes. A total of 390 clumps of satellite cells, approximately 20,000 cells, were analyzed without detecting an HSV-positive tube. These data on the frequency and cellular location of latent HSV, which are consistent with previous publications, indicate that mechanical fractionation of cell types results in low levels of cross-contamination. The isolated neurons and satellite cells are being analyzed for VZV DNA and proteins.

Corresponding Author: Myron J. Levin, M.D., University of Colorado Health Sciences Center, 4200 East Ninth Avenue, C-227, Denver, CO 80262, USA