Infection of neurons by varicella-zoster virus (VZV) is not well understood, although it is a serious medical problem. VZV infects and becomes latent in dorsal root ganglia (DRG) during varicella. Subsequent reactivation of VZV gives rise to zoster (shingles). It has previously been difficult to study VZV latency and reactivation because of the virus' limited host range and the consequent lack of suitable animal or in vitro neuronal models. Identification of viral genes expressed during latency has been complicated by the potential problem of distinguishing latent from reactivating virus in ganglia obtained at autopsy. They propose to take advantage of an in situ hybridization -based method that permits human DRG neurons harboring latent or reactivating VZV to be distinguished. To identify viral genes which may be involved in latency, they will analyze viral gene expression in individual human DRG cells which are determined in serial section to contain latent or reactivating VZV. A multiplex method of in situ hybridization will be employed to identify every VZV open reading frame (ORF) expressed during latent infection. The abundance of these transcripts will be quantified by RT-PCR. Purified antibodies to representative proteins of the three major kinetic classes of VZV genes have been prepared and will also be used to identify protein expression during latent infection in human ganglia. They will attempt also to confirm the recent demonstration that VZV can infect rat DRG. To validate the use of rat DRG as a model of VZV latency, they will compare VZV gene expression during latency in rat DRG neurons with those expressed in their human counterparts in latent infection. The relative contributions made by viremia and retrograde transport in enabling VZV to infect rat DRG cells will be studied. Finally, the relative abilities of wild-type VZV and vaccine type (Oka) VZV to infect rat DRG will be determined. VZV latency will also be examined in cultured hNT neurons (postmitotic human neurons that differentiate in vitro from proliferating neuronal precursors). They will investigate gene expression during putative latency, including the comparative virulence of wild type and vaccine type VZV.
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