Barb Sherry

Evaluation of decontamination protocols and vehicle movement to mitigate the transmission risk of PED virus as a proxy for FAD

Papillomavirus (PV) infections significantly impact human health. Mucosal PVs cause virtually all cases of human cervical cancer, while cutaneous PVs cause severe skin infections and non-melanoma skin cancers in patients with immunodeficiencies resulting from e.g. HIV infection, organ transplants, or genetic disorders. Treatments for cutaneous PVs are limited and often ineffective. Thus, there is a critical need for development of novel therapeutics directed at keratinocytes, the target cell of PV infections. Keratinocytes have the potential to mount an antiviral response. Viral infection activates interferon regulator factors (IRFs), which upregulate interferons (IFN) and IFN-stimulated antiviral genes. IRFs and IFNs are also tumor-suppressing, making them particularly attractive as therapeutic targets for PV infections. However, both mucosal and cutaneous PVs can repress IRF function and IFN expression. This repression is mediated by the E6 and E7 oncogenes of mucosal PVs. While limited studies suggest that mucosal and cutaneous PVs differ in their E6 and E7 functions and that IRF regulation of the IFN response in keratinocytes differs from that in other cell types, there are many more questions than answers. Like humans, cutaneous PV infections occur in dogs and are more prevalent in immunodeficient animals. Thus, the dog offers a natural spontaneous animal model for investigation of cutaneous PV infections and for testing novel therapeutics. Indeed, our preliminary data demonstrate that canine cutaneous PV E6 and E7 differentially disrupt IFN and IFN-stimulated gene expression in canine keratinocytes. The long-range goal of our work is to develop novel therapeutics targeting IRFs and the IFN response for treatment of cutaneous PV infections in immunodeficient patients, using a canine model of PV infection. We hypothesize here that E6 and E7 from cutaneous PVs enhance viral infection by inhibiting IRF function in keratinocytes using mechanisms unique to cutaneous PVs. In Aim 1, we will inhibit expression of IRFs to determine their impact on cutaneous PV infection and on constitutive and inducible expression of IFNs and IFN-stimulated genes in keratinocytes. In Aims 2 and 3, we will determine the mechanisms for cutaneous PV E6- and E7-mediated disruption of IFNs and IFN-stimulated gene expression in keratinocytes. Specifically, we will use mass spectrometry to identify E6 and E7 binding partners that modulate IRF expression and/or function, and then identify mechanisms for E6 and E7 effects on these binding partners. Using human PV and keratinocytes will address the human disease; using canine PV and keratinocytes will advance the dog model. Results will lay the foundation for development of novel therapeutics that would by-pass E6 and E7 deleterious effects. The mentoring team includes faculty with extensive expertise in keratinocyte biology, IRF and IFN biology in differentiated cells, PV biology, and mass spectrometry proteomics and bioinformatics. In sum, the proposed research will address a critical issue in human and animal health, capitalize on the diverse skill sets of the interdisciplinary mentoring team, and enhance my maturation as an independent clinician scientist.

Our overall goal is to identify novel components of the host innate antiviral response and novel mechanisms by which viruses subvert this response to cause disease. Cardiac myocytes are essential and non-replenishable, thus the heart is unusually dependent on this first-line defense. Indeed, we have shown that the cardiac Type I interferon (IFN) response is unique, and that in reovirus-induced murine myocarditis the IFN response differs between virus strains and is critical for protection. We continue to use this powerful tool-kit of viruses to probe the innate response in a highly vulnerable organ, the heart, and have made two new discoveries. First, we found that reovirus inhibits IFN signaling by a mechanism not previously described for any virus. Specifically, reovirus protein mu2 represses IFN signaling, repression is virus strain-specific, and repression is associated with unusual nuclear accumulation of transcription factor IRF9, likely reflecting concomitant interference with its normal participation in induction of IFN-stimulated genes. We hypothesize that reovirus protein mu2 modulates IRF9 structure / function to inhibit IFN signaling, and that mu2 repression of IFN signaling in cardiac cells is critical for myocarditis. In Specific Aim 1, we will: A) determine whether mu2 alters IRF9 structure and/or binding partners to repress IFN signaling, B) identify IRF9 requirements for these events, and C) identify viral and cell variations that determine reovirus-induced myocarditis. Second, using a proteomic discovery approach, we identified a new IFN-independent protective response which can be subverted by virus. We found that Heat Shock Protein-25 (Hsp25) is phosphorylated (by non-myocarditic reoviruses) or degraded (by a myocarditic reovirus) in cardiac myocytes, and that this is cell type-specific and IFN-independent. Hsp25 is modulated by many viruses and it is protective against stress, particularly in the heart. However there have been no reports of degradation of Hsp25 by any stimulus in any cell type. The specific degradation of Hsp25 by a highly myocarditic reovirus, but no other stimuli, suggests that Hsp25 is antiviral and that it can be subverted by some viruses. We hypothesize that Hsp25 plays a cell type-specific antiviral role, that phosphorylated Hsp25 is antiviral, and that some viruses can subvert this innate response. In Specific Aim 2, we will: A) identify the antiviral effects of Hsp25, B) determine the impact of Hsp25 on virus tropism and disease, and C) determine the mechanisms by which reovirus modulates Hsp25 phosphorylation and abundance. The broader impact of our investigations is to increase the catalog of protective host factors that can be sabotaged by viruses, thereby extending the range of possible targets for antiviral therapeutics. Unique insights gained here address innate responses elicited by most viruses and provide the foundation for studies in many other systems, expanding the impact beyond myocarditis to many viral diseases.

Our overall goal is to identify novel components of the host innate antiviral response and novel mechanisms by which viruses subvert this response to cause disease. Given that cardiac myocytes are non-replenishable, the heart is unusually dependent on this first-line defense. Indeed, we have demonstrated that the cardiac Type I interferon (IFN) response is unique. Moreover, in reovirus-induced murine myocarditis the IFN response differs between virus strains and is critical for protection. We continue to use this powerful tool-kit of viruses to probe the innate response in a highly vulnerable organ, the heart. We have made two recent discoveries towards new insights into the virus-cell tango. First, we determined that reovirus can modulate IFN signaling by a mechanism not previously described for any virus. Specifically, reovirus protein mu2 can repress IFN signaling, repression is virus strain-specific, and repression is associated with nuclear accumulation of the transcription factor IRF9, likely concomitant with obstruction of its normal role in inducing expression of interferon-stimulated genes. We hypothesize that reovirus protein mu2 modulates IRF9 structure / function to inhibit its function, and that mu2 repression of IFN signaling in cardiac cells results in myocarditis. In Specific Aim 1, we will: i) determine whether mu2 alters IRF9 structure and / or cellular binding partners; ii) identify mu2 and IRF9 domains required for IRF9 alterations, and iii) determine whether mu2 modulation of IRF9 is required for reovirus-induced myocarditis. Second, using a proteomics discovery approach, we identified a new IFN-independent protective response which can be subverted by virus. We found that reovirus induces phosphorylation or degradation of Heat Shock Protein-25 (HSP25) in cardiac myocytes, and that these effects are cell type-specific, reovirus strain-specific, and IFN-independent. Members of seven different virus families induce HSP25 expression or phosphorylation, but not degradation. Moreover, many insults to the heart result in HSP25 phosphorylation but not degradation in cardiac cells. Finally, over-expressed HSP25 is cardio-protective. We hypothesize that HSP25 plays a cell type-specific antiviral role, and that viruses have evolved mechanisms to evade this innate response. In Specific Aim 2, we will determine: i) the cell type-specificity for reovirus alteration of HSP25, ii) the mechanisms by which reovirus alters HSP25, and iii) the mechanism(s) by which HSP25 inhibits reovirus infection. The broader impact of our studies will be to increase the catalog of protective host factors that can be sabotaged by viruses, potentially providing new therapeutic targets, particularly for viral myocarditis which remains an intractable disease.

Embrex Inc. used a patented technology to develop and produce successful vaccines currently employed world-wide in the poultry industry. ImmunoBiosciences Inc. holds a patent to use the same technology for mammalian systems. Our goal is to use this ?Immune Complex Vaccine? (ICV) technology to develop vaccines against Infectious Bovine Rhinotracheitis virus, Influenza virus, and other viral pathogens that threaten veterinary and human health.

Many viruses infect the heart, and >5% of the human population has experienced some form of viral myocarditis. Unfortunately, cardiac myocytes are not replenished. This cardiac vulnerability likely necessitates a uniquely effective cardiac response, to limit virus spread through the heart until immune defenses can be deployed. Interferon-?Ã’ (IFN-?Ã’) can provide this critical first line of defense. Viruses induce / activate interferon regulatory factors (IRFs) which induce IFN-?Ã’ expression. Secreted IFN-?Ã’ then induces a large number of interferon-stimulated genes (ISGs). Some ISGs have antiviral function and some are IRFs, which can both further induce IFN-?Ã’ and induce ISGs directly. Previously, we demonstrated that variations in cardiac damage induced by a panel of reoviruses in mice correlate with both viral induction of and sensitivity to IFN-?Ã’ in primary cardiac myocyte cultures (PCMCs). We found, however, that IFN-?Ã’ protection varied significantly between PCMCs, primary cardiac fibroblast cultures (PCFCs), and skeletal muscle cells, indicating cell type-specific differences in the IFN-?Ã’ response. Moreover, these differences were determinants of cell type-specific variations in viral replication and cytopathogenic effect. Importantly, multiple lines of evidence suggest that IRFs, IFN-?Ã’, and ISGs function uniquely in cardiac cells. Therefore, we hypothesize that cell type-specific responses to viral infection relating to IFN-?Ã’ determine viral replication and damage in cardiac cells and the heart. In our first Aim, we will identify cell type-specific differences in expression of IFN-?Ã’ and ISGs, and determine the molecular basis for these variations. Results will identify cardiac-specific, muscle-specific, and other differences in constitutive and induced IFN-?Ã’ and ISG expression; and will identify cell type-specific variations in underlying regulatory factors. In our second Aim, we will identify cell type-specific differences in the role of IFN-?Ã’ in protection against viral replication and cell damage, and determine the molecular basis for these variations. Results will identify the role of components of the IFN-?Ã’-response in cell type-specific differences in viral replication, cytopathogenic effect, and cardiac cell damage. In our third Aim, we will determine the role of factors that regulate IFN-?Ã’ in protection against myocarditis. In sum, results will identify cell type-specific IFN-?Ã’-related responses critical for protection against myocarditis, potentially providing new avenues for intervention against viral infections of the heart.

Project Summary/Abstract and Relevance This application is submitted for the purchase of a microscope that offers a unique opportunity to capture detailed images of macro as well as microscopic subjects. The Nikon AZ100 Macro/Micro Zoom Microscope was introduced to the U.S. market earlier this year and offers the advantages of a stereoscope (e.g., wide field of view with a long working distance) combined with the advantages of a compound microscope featuring high-resolution images. The AZ100 images specimens using white light as well as UV illumination over a broad range of magnifications (5-400x) using a novel vertical optical system that eliminates distortion introduced by stereoscopic inclination. This novel optical configuration facilitates the continuous switching of magnified images of a single specimen from macro to micro ranges. As a consequence of the elimination of inclination distortion, the AZ100 also facilitates real time deconvolution of macro subjects resulting in unique representations of three-dimensional subjects. We propose to purchase the AZ100 and install this device in a facility proximal to seven laboratories that will be major users. The research conducted within each of these laboratories requires imaging of macro (e.g., developing zebrafish, frog, mouse, and pig embryos, fetal mouse hearts, canine lenses) as well as micro (e.g., tissue sections) subjects using white light and UV illumination. This equipment will also be available to other interested laboratories at North Carolina State University and the broader Research Triangle research community as required. The long term objective and specific aims of this proposal are to utilize this versatile microscope to capture detailed images of a variety of macro and microscopic subjects for research and teaching purposes. We anticipate that these images will greatly enhance our capacity to analyze and document our experimental results as well as communicate with students and colleagues. The relevance of this equipment proposal is the each of the proposed major users is currently using a bevy of animal models to reveal mechanisms underlying normal human development, human developmental defects, human cancers, and virus-induced human diseases.

Genomic science has revolutionized the biological sciences by providing the power to characterize systems in their entirety and the ability to ask biological questions that were previously intractable. Universities must keep pace with the demand for scientists knowledgeable in this interdisciplinary area. Building on strengths in agriculture, forestry, biotechnology, statistical modeling, and veterinary medicine, NCSU has made a major commitment to graduate training in genomics, emphasizing both the inter-dependence of Functional Genomics and Bioinformatics as well as the importance of training depth in each area. Functional Genomics (which includes structural genomics and proteomics, in our program) applies high-throughput molecular biology and robotic technologies to the study of gene structure, expression, and function. Bioinformatics uses statistical and computational approaches to develop new methods to store, manage, and analyze these massive datasets. Progress in genomics requires effective collaboration between these scientists, which in turn, requires effective communication. Our program brings all students together for foundation classes but progresses to a student-driven curriculum that provides advanced training in a choice of specialized areas. Here, we propose to train 5 graduate students each year seeking their PhD in either Bioinformatics or Functional Genomics. Students select faculty advisors from 35 trainers spanning ten departments in five colleges at NCSU, and include a faculty member with complementary expertise on their advisory committee to ensure cross-disciplinary interactions. This proposal includes a new computational track in Bioinformatics to complement our current statistical strengths, strengthening our program in this increasingly competitive environment. Funds from the UNC-Office of the President will provide compelling evidence of strong UNC system support for our planned submission of an NIH training grant. With 80 current genomics graduate students, 32 Master?s and PhD degrees already awarded, and several hundred applications each year, the program serves as a model for successful interdisciplinary graduate education, providing the new interdisciplinary research workforce genomics requires.

Genomic science has revolutionized the biological sciences by providing the power to characterize systems in their entirety and the ability to ask biological questions that were previously intractable. Universities must keep pace with the demand for scientists knowledgeable in this interdisciplinary area. NC State University was one of the first to offer graduate degree programs in Functional Genomics and Bioinformatics. Building on strengths in agriculture, forestry, biotechnology, statistical modeling, and veterinary medicine, NC State University made a major commitment to graduate training in genomics, emphasizing both the inter-dependence of Functional Genomics and Bioinformatics as well as the importance of training depth in each area. Functional Genomics (which includes structural genomics and proteomics, in our program) applies high-throughput molecular biology and robotic technologies to the study of gene structure, expression, and function, promising insights into the way organisms work. Bioinformatics uses statistical and computer science approaches to develop new methods to store, manage, and analyze these massive datasets. Progress in genomic science requires effective collaboration between these scientists, which in turn, requires effective communication. Our program brings all students together for foundation classes but progresses to a student-driven curriculum that provides advanced training in a choice of specialized areas. Here, we propose to train 26 graduate students seeking their PhD in either Bioinformatics or Functional Genomics. Students select faculty advisors from a group of 35 trainers spanning ten departments in five colleges at NC State University, and include a faculty member with complementary expertise on their advisory committee to ensure cross-disciplinary interactions. With 80 current genomics graduate students, 32 Master?s and PhD degrees already awarded, and several hundred applications each year, the program serves as a model for successful interdisciplinary graduate education, providing the new interdisciplinary research workforce genomics requires.

In response to concerns that bioterrorists may release smallpox virus in the United States, the government has implemented a multi-stage vaccination program. However, the observation of cardiac adverse events following vaccination has resulted in the Centers for Disease Control recommending that people at risk for heart disease should be excluded from the vaccination program. Notably, myocarditis has been diagnosed in 18 recent smallpox vaccinees in the USA. Despite this recent temporal association between myocarditis and smallpox vaccination as well as a similar historic association, there have been no studies of the effects of poxviruses on cardiac cells, nor the development of any animal models to study poxvirus-induced myocarditis. For other viruses, myocarditis can reflect a direct cytopathogenic effect of the virus on cardiac myocytes, or can be mediated by immune cells, cytokines, or other soluble factors. Poxviruses can be directly cytopathogenic, but also encode a number of modulators of cytokine responses. Cowpox virus contains the full complement of known orthopoxvirus accessory genes that affect immune responses, while DryVax (the current vaccine strain of vaccinia virus) encodes fewer accessory genes, and modified vaccinia Ankara virus (MVA, which is replication-defective in human cells) contains even fewer accessory genes. Our preliminary data demonstrate that cowpox virus and DryVax replicate in murine primary cardiac myocyte cultures (PCMCs) whereas MVA does not. Moreover, cowpox, DryVax, and MVA are each cytopathogenic to these cultures, though the type of cytopathogenicity is virus strain-specific. We hypothesize that poxviruses have virus strain-specific adverse effects on cardiac cells, either directly or indirectly, resulting in damage to cardiac myocytes and the heart. In Specific Aim 1, we will determine the effect of these three poxviruses (cowpox, DryVax, and MVA) on murine primary cardiac myocyte cultures (PCMCs) and control cultures, analyzing viral infection and gene expression, and induction of cytopathogenicity. In Specific Aim 2, we will determine the association between cytokines and poxvirus infection on cytopathogenicity to PCMCs and control cultures. In Specific Aim 3, we will determine whether these poxviruses induce myocarditis in a variety of mouse strains. Studies proposed in this R21 application will provided the foundation for future in-depth studies with the goal of improving current and future smallpox vaccines.