Varicella-zoster virus, an ?-herpesvirus, causes varicella and zoster. Infection of T cells allows VZV transfer from the respiratory tract to skin ad the typical zoster rash results from axonal transport after VZV reactivation in neurons. Our strategy uses human tonsil T cells and skin xenografts in the SCID mouse model to investigate molecular mechanisms that mediate VZV tropism for these differentiated human cells, which are critical during pathogenesis, and the counterbalancing innate immune responses. Since VZV must infect primary host cells exhibiting their inherently stochastic states, and alter conditions o support infection within each cell, our strategy emphasizes multi-parametric single cell analyses. Our aims related to T cell tropism (Aim 1) address the hypothesis that VZV modulation of tonsil T cell mircoRNAs (miRs) regulates cell signaling networks, enhancing infection and skin homing independently of the usual TCR- mediated processes that promote T cell trafficking for antiviral immune functions, as well as other host cell factors that support replication in T cells. Single cel mass cytometry (CyTOF) will be used to relate the expression of selected miRs to changes in T cell surface and intracellular proteins and virus production in VZV-infected T cells. Contributions of VZV and host cell transactivators to miR expression will be determined. Our aims about skin tropism (Aim 2) focus on hair follicle cells as an initial site of VZV infection. Our hypothesis is that infection of follicle stem cells triggers activation and accelerates VZV delivery to interfollicular epidermal cells where lesions form, and conversely, that multifactorial IFN-related and cytokine responses of uninfected epidermal cells control the process. It will be addressed primarily by quantifying combinatorial expression of 30 intracellular and cell surface proteins in infected and uninfected skin with CyTOF. Mass spectroscopy combined with tissue section imaging for high dimensional immunohistochemistry (MIBI-TOF) using these markers will also be developed to map lesion formation in skin xenografts. These methods will be applied to determine whether age-related changes in human skin impair local innate control, contributing to severe cutaneous zoster in the elderly. Studying VZV pathogenesis is relevant for public health because new antiviral drug targets are needed, given the limited benefit of available agents. In addition, live attenuated VZV vaccines are unsafe for immunocompromised patients and can cause zoster; whether new subunit vaccines will prevent varicella is uncertain. Single cell methods also have utility for screening drug candidates for antiviral activity while revealing their potential cell toxicities. For basic research, developing approaches to assess the complex, multi-factorial effects of viruses on host cell protein networks and other regulatory elements, lik miRs, has broad applications for studying viral pathogenesis. Finally, multi-parametric single cell analyses of uninfected primary human cells should reveal aspects of T cell and skin cell biology pointing to new research directions for many purposes.