Varicella zoster virus (VZV) causes chickenpox, after which the virus becomes latent in cranial nerve, dorsal root and autonomic nervous system ganglia along the entire neuraxis. Decades later, virus reactivation produces zoster (pain and rash restricted to 1-3 dermatomes) in about 1 million Americans annually. Zoster is often followed by postherpetic neuralgia (PHN, chronic pain), as well as retinitis, myelitis and vasculopathy, which are significant causes of blindness, paralysis and death. Although zoster vaccine reduces the incidence of zoster by 50% over a 3-year period, even if everyone over age 60 received it, there would still be 500,000 cases of zoster and 200,000 of PHN annually. Thus, as the elderly population increases in number, the incidence of zoster and its attendant neurological complications is still likely to increase. In 1983, we first proved that VZV is latent in human ganglia. Our subsequent analyses of >6,000 human ganglia from >700 randomly autopsied subjects showed that the entire virus genome is present, maintained as a histone-coated circular episome with a variable abundance of VZV DNA, and that VZV gene expression is restricted and regulated epigenetically without transcription of VZV micro-RNA. Nonetheless, mechanism(s) underlying VZV gene expression during latency have been hampered by the lack of a model that faithfully recapitulates the molecular events in latently infected human neurons. We have now developed an in vitro model in which human neural stem cells were induced to differentiate to >90% neurons, as evidenced by immunohistochemical staining. After infection with cell-free VZV, the neurons can be maintained for weeks in the absence of a cytopathic effect. We hypothesize that these VZV-infected neurons can be used to study VZV latency, thus obviating the need for thousands of human ganglia, and enable analyses not currently possible in human ganglia. Our goal is to prove that the in vitro differentiated neurons are latently infected with VZV after which we will use them to further study VZV latency. Specifically, we will isolate VZV-infected differentiated neuronal cells by optimized laser capture microdissection (LCM) and determine the purity of LCM-captured infected neurons by RT-PCR with neuron- and glial-specific primers (Aim 1). We will verify that these neurons are latently infected by showing that the physical state of VZV DNA and VZV gene transcription in LCM-captured neurons is the same as in latently infected human ganglia (Aim 2);we will then analyze epigenetics of the VZV DNA;and use GenomeLab Genetic Analysis System to analyze the entire VZV transcriptome (Aim 3). An in-depth understanding of the mechanism(s) of VZV gene expression in latently infected human neurons will lead to experiments designed to prevent the cascade of events leading to VZV reactivation, a cause of serious neurologic disease and blindness, particularly in the rapidly increasing elderly and immunocompromised populations.

Public Health Relevance

An in-depth understanding of how varicella zoster virus lives in latently infected human nerve cells is prerequisite to developing strategies that prevent the cascade of events leading to virus reactivation, a cause of serious neurologic disease and blindness, particularly in the rapidly increasing elderly and immunocompromised populations.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG006127-27
Application #
8522096
Study Section
Special Emphasis Panel (ZRG1-BDCN-W (03))
Program Officer
Mackiewicz, Miroslaw
Project Start
1987-07-01
Project End
2015-05-31
Budget Start
2013-09-01
Budget End
2014-05-31
Support Year
27
Fiscal Year
2013
Total Cost
$366,218
Indirect Cost
$126,860
Name
University of Colorado Denver
Department
Neurology
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
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