We have studied the mechanisms that permit and regulate latency and reactivation of herpes simplex viruses (HSV) 1 and 2 and the varicella-zoster virus (VZV). We have been determining the level of latent infection with HSV and VZV in human neurons. The HSV latency associated transcripts (LATs) are the only viral RNAs consistently expressed during latency. While earlier work from our section examined latent viral loads and LATs in extracts from whole ganglia, we have recently developed and standardized assays using laser capture microdissection (LCM) in which single cells or groups of cells can be removed from a histologic section and subjected to quantitative PCR. ? ? We showed that HSV-1 persists in 2 to 10.5% of human trigeminal neurons with a median of 11.3 copies per infected cell, very much concordant with our projections from bulk PCR studies. HSV-1 genome copies vary widely within individual neurons ranging from 5 to 3,955 copies per neuron; most neurons have <20 copies of HSV-1 DNA. Analysis of neurons using in situ hybridization for LAT RNA showed that 0.2 to 1.5% of neurons are positive for LAT. Combining the techniques of in situ hybridization with LCM, we found that most neurons with latently infected DNA (based on LCM), do not express LAT RNA (assayed by in situ hybridization). The percentages of HSV-1 DNA positive neurons were 5-fold to 41-fold higher than those positive for LAT. These data indicate that HSV-1 infects more neurons than previously estimated. HSV-1 DNA was detected very rarely in non-neuronal cells; of 5,200 satellite cells tested, 21 copies of HSV DNA-1 were detected. These findings provide evidence that the lack of viral transcripts detected in non-neuronal cells is due to the absence of latent infection, rather than a lack of transcription from latent viral genomes in these cells.? ? We have also used LCM to quantify the level of VZV latent infection in single neurons. VZV DNA was detected in 1.0 to 6.9% of neurons with a mean of 4.1% neurons containing viral DNA. The viral DNA load in the infected neurons ranged from 2.6 to 5,773 copies per cell. 70% of the VZV DNA positive neurons contained 20 or fewer copies of viral DNA, with a median of 6.9 copies per infected cell. VZV DNA was rarely detected in non-neuronal cells; of 14,200 non-neuronal cells tested, 9 copies of VZV DNA were detected.
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