Core 2: Virology, Molecular, and Histology Core

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ABSTRACT – Virology, Molecular, and Histology Core (Core 2) It is well known that, despite strict adherence to protocols, hidden or subtle factors that vary among laboratories can cause techniques such as those concerning virus propagation and qPCR to produce differing outcomes. Thus, to ensure cross-project consistency, minimize confounding, and maximize impact of results, a Virology, Molecular, and Histology Core is proposed. This Core will be led by Dr. Peter Barry, who brings to the Program extant and well established techniques in virus stock propagation and analyses of viral parameters of infection (including quantification of viral loads, histopathology, and antigen expression). These approaches are central to rigorous testing of the project's hypotheses addressing the immunologic (Project 1) and virologic (Project 2) determinants of congenital CMV infection (cCMV), and generating virus stocks for the NHP Core, purified DAN to assess for emergence/selection of viral variants (Core 3), and quantifiable measures of viral replication and disease for rigorous statistical analyses (Core 4). Specific outputs of the Virology, Molecular, and Histology Core are as follows: well-characterized, high titer stocks of epithelial-tropic stocks of RhCMV (Aim 1), sensitive analyses of tissues and fluids from inoculated animals to quantify RhCMV genome copy numbers by PCR (qPCR) (Aim 2), and histopathologic/immunohistochemical (IHC) analyses to characterize viral pathology and antigen expression (Aim 3). Providing singly sourced stocks of virus and sensitive and specific assays for RhCMV genome copy numbers and antigen expression will enable uniform starting material and analytic techniques—capabilities that are critical for evaluating the overall objective of this Program: to define the maternal immune responses and viral characteristics that impact cCMV transmission and the outcome of fetal infection.