2023 Research Symposium

P2 - Time is Brain: Stimulating Tie2 Improves Pial Collateral Growth and Reduces Tissue Damage After lschemic Stroke 

Alexandra M. Kaloss 1, Kennedie Lyles 2, Nathalie A. Groot 1, Jackie Zhu 3, John B. Matson 3, and Michelle H. Theus 1, 2 

1 Department of Biomedical Sciences and Pathabialagy
2 School of Neuroscience
3 Department of Chemistry, Virginia Tech, Blacksburg VA 

Strokes are a leading cause of death and disability, with most cases being ischemic strokes resulting from blood flow in the brain being interrupted. Immediately following this vascular obstruction, often due to a blood clot, the cells in the affected region of the brain begin to die due to lack of oxygen and nutrients, resulting in neurological impairments. Treatments for ischemic strokes currently focus on removing the clot but can fail to restore blood flow and prevent cell death. Thus, novel treatments are needed that specifically target the restoration of blood flow. Specialized pre-existing arterioles, termed pial collaterals, can remodel into larger vessels after an ischemic event allowing them to ease the loss of blood flow and prevent cell death. However, the process for pial collateral remodeling remains poorly understood. Using a permanent middle cerebral artery occlusion (pMCAO) model of ischemic stroke, our previous work has shown that loss of EphA4, a receptor tyrosine kinase, results in reduced infarct volume which correlated with increased collateral size from 4.5 to 24 hours post-pMCAO. The collateral vessels lacking EphA4 also displayed significantly higher endothelial cell proliferation and immune cell recruitment at 24 hours post-stroke. We hypothesized that EphA4 was exerting its function through the inhibition of Tie2, a protein involved in vascular stability and growth. Therefore, we employed Vasculotide (VT), an Angiopoetin-1 memetic peptide, to stimulate Tie2 signaling. In vitro, endothelial cells treated with VT displayed improved migration and wound healing. Moreover, in an in vivo mouse model, animals that received intravenous VT directly following a surgically induced stroke had significantly lower infarct volumes at 24 hours post-pMCAO compared to vehicle controls. This decreased infarct volume correlated with improved performance by VT-treated mice on sensory and motor behavior tests, including adhesive tape test, rotarod, and modified neurological severity score in the first 28 days post-pMCAO, compared to controls. VT-treated mice also displayed significantly larger pial collateral vessels 24 hours post-stroke compared to vehicle-treated control mice. Therefore, inhibiting EphA4 or stimulating Tie2 could serve as novel therapeutic strategies for improving collateral response following ischemic stroke. 

Support: NINDS R01NS112541 (MHT) and Office of Research and Graduate Studies 

P4 - Mechanical High-Intensity Focused Ultrasound (Histotripsy) in Dogs with Spontaneously Occurring Soft Tissue Sarcomas 

Ester Yang, Lauren Ruger, Jessica Gannon, Hannah Sheppard, Sheryl Coutermarsh-Ott, Timothy J. Ziemlewicz, Nikolaos Dervisis, Irving C. Allen, Gregory B. Daniel, Joanne Tuohy, Eli Vlaisavljevich, Shawna Klahn

Department of Small Animal Clinical Sciences, Virginia-Maryland College of Veterinary Medicine 
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine
Department of Biomedical Engineering and Applied Mechanics, Virginia Tech 

High-intensity focused ultrasound (HIFU) is a non-invasive technique able to ablate tissue by either thermal or mechanical means. Histotripsy is a non-thermal HIFU ablative therapy that causes mechanical disruption of tissue. Prior rodent studies have demonstrated that HIFU is immunogenic. Soft tissue sarcomas (STS) are a common form of cancer in dogs with biological behavior similar to humans. Adequate local tumor control in veterinary medicine may require extensive surgical resection. There is need for alternative therapies. Our objective was to investigate the in vivo feasibility of ablating STS in client-owned dogs with histotripsy and to characterize the impact of acute immunologic response. Methods: CT of the chest/abdomen/tumor was performed for staging and treatment­planning. Partial tumor ablation was performed using a prototype histotripsy system. Anatomical ablation zones were evaluated with CT at 1- and 4-days post-treatment, with tumor resection at 4-days post-treatment. Safety was monitored with exams, owner reports, and bloodwork. TME gene expression was evaluated with the Nanostring Canine IO panel. Multiplex serum cytokine levels were used to evaluate systemic immune response. TME was evaluated by characterizing changes in the infiltration of T AMs and TILs using mIHC panels. Results: Ten dogs were recruited and treated. Tumor histologies included all STS of various subtypes and grades. Differential gene expression analysis identified 79 genes with at least 2-fold upregulation between treated and untreated groups. Genes associated with inflammation, stress, immune cell migration and interactions were the highest upregulated. Myeloid compartment, NK cell functions, and interleukin gene sets obtained the highest significance score. There were no statistically significant differences in plasma cytokine concentrations between groups. In more than half of all examined samples, mIHC investigating macrophage populations showed mild to marked increases in IBA-1 and CD206 double-positive cells, representing M2-polarized macrophages. There were no significant differences in lymphocyte staining between groups. Conclusions: Histotripsy can achieve safe and effective tumor ablation in dogs with STS. Changes in the tumor microenvironment reflect increases in the expression of genes associated with inflammation, matrix remodeling, and immune cell interactions. Histotripsy as an immunotherapeutic treatment option needs to be further investigated. 

Support: Focused Ultrasound Foundation  

P6 - Non-essential role for Cx3cr1-expressing EPH receptor Alt in a murine model of TBI 

Jatia Mills, Eman Soliman, Jing Ju, Alexandra M. Kaloss, Erwin Kristobal Gudenschwager Basso, Nathalie Groot, Colin Kelly, Elizabeth A. Kowalski, Mohamed Elhassanny, Michael Chen, Xia Wang, and Michelle H. Theus

Department of Biomedical Sciences and Pathabialagy, Virginia Tech, Blacksburg, VA, United States, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt, Schaal of Neuroscience, Virginia Tech, Blacksburg, VA, United States, Center far Engineered Health, Virginia Tech, Blacksburg, VA, United States 

Erythropoietin-producing human hepatocellular (Eph) receptors contribute significantly to central nervous system injury. Our findings demonstrated that Cx3cr1-expressing cells within the perilesional cortex showed increased levels of EphA4 after induction of controlled cortical impact (CCI) injury in mice. Cx3cr1 is a fractalkine receptor, commonly expressed on resident microglial and peripheral-derived macrophage (PDM) cells. The aim of this study is to identify the role of microglial-specific EphA4 in CCI-induced damage. Cx3cr1 CreER/EYFP knock-in/knock-out mice expressing EYFP in Cx3cr1 + cells were used to evaluate microglia in EphA4-deficient mice following 1-month tamoxifen injections. CCI-Injured wild-type (WT) Cx3cr1CreER/EYFP/EphA4+/+ mice displayed increased EphA4 expression on the EYFP-positive cx3crl cells within the peri-lesion. Immunohistochemical applications were further used to differentiate between the peripheral-derived macrophage and resident microglia using anti-Ccr2, which selectively labeled PDMs and not microglia. We then exploited GFP bone marrow chimeric mice to discriminate EphA4 expression on microglia (TMEM 119 +/ G FP-) versus PD Ms ( G FP+) following CCI. Finally, the use of Cx3cr1CreER/EYFP/EphA4f/f (KO) mice, which show no detectable transcript for EphA4 in microglia only, demonstrated no discernible difference in lesion volume or blood brain barrier (BBB) disruption when compared to the WT mice. These findings illustrate that although EphA4 is upregulated on cortical microglia after TBI, it plays a nonessential role in acute response following TBI.

Support: We recognize The Center for Engineered Health for grant support, and Mellissa Markus for flow cytometry support. This work was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health, NS096281, NS119540, and NS121103 (MHT).   

P8 - The T2-FLAIR mismatch sign as an imaging biomarker for canine oligodendrogliomas 

Josefa Garcia-Mora 1, 2, Rell L. Parker 1, Thomas Gecere 3, John L. Robertson 2, 4, 5, John H. Ross meisl 1,2,4, 5 

1 Department of Small Animal Clinical Sciences and Animal Cancer Care and Research Center, Virginia­Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, United States of America 
2 Veterinary and Comparative Neuro-oncology Laboratory, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, United States of America 
3 Department of Biomedical Sciences & Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, United States of America 
4 School of Biomedical Engineering and Sciences, Virginia Tech-Wake Forest University, Blacksburg, Virginia, United States of America 
5 Comprehensive Cancer Center and Brain Tumor Center of Excellence, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America 

Background: The T2-FLAIR mismatch sign (T2FMM) is an MRI imaging biomarker of human ID H 1-m utated, 1 p 19q non-codeleted low-grade astrocytomas. It is characterized by the presence of a homogeneously hyperintense tumor signal on T2W images and a hypointense signal with a hyperintense peripheral rim on FLAIR images. On histopathology, the T2FMM is correlated with the presence of microcysts in the mismatch regions. In canine gliomas, the T2FMM has not been described Hypotheses/Objectives: The T2FMM in canine brain MRI studies will be associated with the low-grade astrocytoma phenotype and the presence of microcysts on histopathology. Interobserver agreement will be high for the qualitative assessments of the T2FMM. Animals: 186 dogs with brain MRI evidence of focal intra-axial brain lesions;146 histologically confirmed intracranial gliomas ( 90 oligodendrogliomas, 4 7 astrocytomas, 9 undefined gliomas), 3 3 cerebrovascular accidents, and 7 with inflammatory lesions Methods: Two blinded raters evaluated all sequences from 186 canine MRI brain studies to identify cases with the T2FMM. Interobserver agreement was determined for qualitative MRI features defining the T2FMM by calculating intraclass correlation coefficients (ICC). Fisher exact tests were used to compare the proportions of binary MRI features present in dogs with and without the T2FMM. Immunohistochemistry for the IDH1 (R132H) mutation and gene expression analyses (Illumina) of oligodendrogliomas with and without T2FMM were performed to examine genotypes associated with this sign. Results: T2FMM was identified in 14/ 186 (8%) canine brain MRI, and was exclusively detected in oligodendrogliomas (14/14; 100%). Interobserver agreement for detecting T2FMM was good (ICC>0. 81). The presence of the T2FMM was significantly associated with the low-grade oligodendroglioma (LGO) phenotype (p < 0.001), absence of tumor enhancement on post-contrast images (p=0.002), and microcysts or myxoid lakes on histopathology (p <0.00001). In oligodendrogliomas with the T2FMM, IDH1-mutations or specific differentially expressed genes were not identified. Conclusion and clinical importance: T2FMM can be readily identified on routinely obtained MRI sequences. T2FMM is a specific biomarker for canine oligodendroglioma, and was significantly associated with non-enhancing LGO.

P18 - Transcriptomic and Proteomic Analysis of Venezuelan and Eastern Equine Encephalitis Virus Infected Astrocytes 

Rafaela Flor 1,2, Amy Smith 1, Alan Baer 3, Kimberley Alex Hodge 4, Emanuel F. Petricoin 4, Jon D. Dinman 5, Jonathan Jacobs 6, Kehn-Hall 1,2

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA 
2 Genter for Emerging, Zoonotic, and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA 
3 National Genter for Biodefense and Infectious Diseases, George Mason, Manassas, VA 
4 Genter for Applied Proteomics and Molecular Medicine, School of Systems Biology, George Mason University, Manassas, Virginia, United States of America 
5 University of Maryland, College Park, MD 
6 American Tissue Type Culture, Manassas, VA 

The mosquito-borne equine encephalitic alphaviruses, Venezuelan and eastern and equine encephalitis viruses (VEEV and EEEV), are known to cause disease in both humans and horses in sporadic outbreaks across the Americas. These viruses vary in severity of pathogenesis and sequelae, but both can lead to significant brain inflammation with long lasting debilitating neurological effects in humans. However, there is a lack of effective countermeasures available to treat these infections and knowledge regarding host processes impacted by these viruses, especially for EEEV, is lacking. Two complementary "omics" approaches, RNAseq and reverse phase protein array (RPPA), were utilized to determined host factors and pathways altered in VEEV and EEEV infected U87MG astrocytoma cells. RNAseq identified 407 and 1235 differentially expressed genes in VEEV and EEEV infected cells, respectively. EIF2 signaling, Regulation of elF4 and p70S6K Signaling, mTOR Signaling, Protein Ubiquitination Pathway, Mitochondrial Dysfunction, Huntington's Disease Signaling, and Caveolar-mediated Endocytosis Signaling were amongst the top dysregulated pathways in both VEEV and EEEV infected cells. RPPA analysis was performed to detect alterations in the phosphorylation status or overall protein levels of 113 proteins involved in a wide array of cellular pathways including cell cycle, apoptosis, growth, inflammation, transcription, translation, autophagy, and innate immune responses. Twenty-three and 31 proteins were found to be statistically significantly altered in VEEV and EEEV infected cells, respectively. Phosphorylation of HSP27, p38 MAPK, SMAD 1/5/9, and S6 Ribosomal protein was significantly increased with both infections. In contrast, phosphorylation of Histone H3, pRB, and Aurora A were commonly downregulated by VEEV and EEEV. Currently our studies are confirmed these alterations using orthogonal assays and testing inhibitors of these pathways to determine their influence on alphavirus replication.

Support: Defense Threat Reduction Agency (DTRA) / HDTRA1-21-1-0008   

P20 - Transcriptomic Profiling of Astrocyte Development  

Xiaoran Wei, Leanne M Holt, Natasha L Pacheco, Boyu Lyu, Raymundo Hernandez, Guoqiang Yu and Michelle L Olsen 

School of Neuroscience, College of Science, Virginia-Polytechnic Institute and State University 

Astrocytes are the most abundant glial cell type in the central nervous system (CNS) and are responsible for many crucial roles in CNS homeostasis and synaptic transmission. Surprisingly little is understood regarding the gene expression changes occurring during early postnatal astrocytic development, when astrocytes undergo complex morphological and maturational changes. Understanding these developmental gene expression patterns may provide much needed insight into neurodevelopmental diseases such as Rett syndrome, autism spectrum disorder and fragile X syndrome. In the present study, we examined gene expression in wild type (WT) astrocytes at five early developmental timepoints spanning the period from astrocyte birth to maturation. Unsurprisingly, we found the highest number of differentially expressed genes (DEGs) between postnatal days (P) 14 and P28 in WT animals, a critical time in astrocyte morphological maturation. This period of time has been linked to changes in gene and protein expression associated with astrocyte maturation, as well as active astrocytic morphological maturation processes. Focusing on this developmental time period, pathway analysis of these DEGs help us identified that development, ion transporters and channels, G-protein-coupled receptors and kinase signaling cascades, cellular metabolism, and several pathways implicating cellular structure, growth, and morphology are upregulated in WT astrocytes between P14 and P28. In contrast, WT astrocytes downregulated biological pathways associated with gene and protein expression, the cell cycle, cellular metabolism (including fatty acid metabolism), and regulation of cell size at the same time point comparison. During time period, multiple pathways associated with cellular morphology and maturation functions are enriched in WT astrocytes. 

Support: NIH RO1 NS120746, Virginia Tech Start Up Funds    

P22 - Attenuation mechanism of an interferon gamma expressing chikungunya vaccine  

Christina Chuong, Chelsea Cereghino, James Weger-Lucarelli

Biomedical and Veterinary Sciences; Biomedical and Pathobiology; Center for Emerging, Zoonotic, and Arthopod-borne Pathogens  

Chikungunya virus (CHIKV) is an emerging pathogen that has caused millions of infections globally within the last 15 years and has the potential to become endemic in the US. CHIK disease is characterized by debilitating chronic arthritis which causes a significant reduction in quality of life. The current standard of care is based on treating symptoms and is often ineffective, and no vaccine is available to prevent disease. Furthermore, current live-attenuated CHIKV vaccine candidates in development have safety concerns. Previously, our group developed a mouse interferon-gamma expressing CHIKV-Semliki Forest virus (SFV) chimera (CHIKV-SFV /IFNy) vaccine that was extremely safe, resulting in reduced footpad swelling and limited capacity to replicate in immunocompromised mice. Towards understanding the increased attenuation mechanism of CHIKV-SFV / IFNy, we have identified a subset of IFNy-regulated antiviral genes in the guanylate binding protein (GBP) family that are highly upregulated in CHIKV-SFV/DomC-IFNy infected fibroblasts and mice. We hypothesized that vaccine-driven IFNy-expression stimulates these genes to restrict viral replication and thus self-contain the virulence of the vaccine. To scrutinize these genes' role in attenuation, we used siRNA to knockdown GBP gene expression or GBP overexpression plasmids prior to infection with CHI KV -SFV /IFN y. We also created a human IFNy-expressing vaccine to understand if these identified mechanisms could be directly applicable for human health. Lastly, we also explored the promise of IFNy as a broadly effective antiviral against several other viruses and thus demonstrate further use of our vaccine platform. This project will define a clear role for IFNy in modulating viral replication of CHIKV, particularly in regard to the enhancement of vaccine safety, and may identify novel strategies to improve vaccination or therapeutic development. Furthermore, the data obtained from these studies will establish a foundation to investigate the use of IFNy against other pathogens threatening human health. 

Support: Virginia-Maryland College of Veterinary Medicine     

P24 - Regulation of developmental cell death in hypothalamic corticotrophin-releasing hormone neurons through DSCAML 1 and cortisol signaling   

Michael Brooks, Yueh in Albert Pan

Department of Biomedical Sciences and Pathbiology and Fralin Biomedical Research Institute  

The neuroendocrine stress axis, known as the hypothalamic-pituitary-adrenal (HP A) axis, is mediated by the hormones CRH, ACTH, and cortisol. CRH is produced in the neurosecretory area of the hypothalamus and regulates the release of ACTH, which then regulates the release of cortisol. The number of CRH-expressing neuron is precisely regulated during development, but it remains unknown what molecular mechanisms determine cell number and how cell number contributes to HPA axis function. In previous work, we found that Down syndrome cell adhesion molecule like-1 (DSCAMLl), a cell adhesion molecule with links to neurodevelopmental disorders, promotes CRH neuron cell death and reduces overall CRH neuron number. In addition, DSCAMLl deficiency also results in abnormal function of the stress axis, notably the overproduction of cortisol at baseline. As cortisol has been shown to affect cell death during development, we hypothesized that DSCAMLl may affect CRH neuron cell death via the regulation of baseline cortisol. To test this hypothesis, we sought to modulate cortisol independently of dscamll and determine the effects on developmental CRH neuron cell death. We will use zebrafish as the model organism, as they boast a homologous hypothalamic-pituitary­interrenal (HPI) axis and the ability to easily visualize the brain in early development. To control cortisol levels, CRISPR-mediated genome engineering will be used to delete the Steroidogenic acute regulatory protein (STaR) gene, which is produced in the interrenal glands and is necessary for the syn thesis of cortisol. This gene will be targeted using three (3) short-guide RNAs (sgRNAs) to introduce frameshift mutations, inhibiting STaR and consequently cortisol production. Fluorescence in-situ hybridization (FISH) will be used to label CRH neurons and immunostaining with anti-activated caspase 3 will be used to label apoptotic cells. Group-wise comparisons of cell death and CRH neuron cell number will first be made between STaR deficient animals versus sibling controls in the wild-type background. Then, we will examine STaR deficiency in the dscamll mutant background to determine whether dscamll deficiency affects CRH neuron cell death in the absence of cortisol signaling. These experiments will advance our understanding of stress axis development as well as the role of cortisol in cell death, and provide insights into stress axis-linked human neurodevelopmental and psychiatric disorders.

Support: NIH T32 (T32OD028239-03)     

P34 - A potent antibacterial activity against Clostridioides difficile is exhibited by antifungal drugs  

Ahmed A. Abouelkhair, Nader S. Abutaleb, Mohamed N. Seleem

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University.   

Clostridioides difficile is a prominent source of healthcare-associated infections and is regarded as an urgent public health problem globally. The only FDA-approved antibiotics for the treatment of C. difficile infection (CDI) are vancomycin and fidaxomicin. The high rate of treatment failure and recurrence linked to these antibiotics, as well as the rising number of infections, make CDI treatment extremely difficult. Therefore, it is imperative to find new, powerful anti-C. difficile drugs. When compared to de nova drug innovation, drug repurposing is a potential technique for reducing costs and time and decreasing risks associated with de nova drug discovery. Utilizing this approach, we screened 3200 FDA-approved drugs against C. difficile, and the results showed that miconazole is a powerful inhibitor of the bacterium with a minimum inhibitory concentration (MIC) of 1 µg/ml. On the strength of this, we tested a library of 24 azoles against a diverse range of pathogenic C. difficile strains. Miconazole, econazole, and tioconazole displayed the most potent activity against C. difficile inhibiting the growth of 50% of tested isolates (MIC50) at concentrations of 1 µg/ml, 2 µg/ml, and 2 µg/ml, respectively. Miconazole was selected for further investigation since it demonstrated the most potent anti-C. difficile activity, and it is orally bioavailable. In a time-kill kinetics study, miconazole showed a fast bactericidal activity outperforming vancomycin, where it decreased a high bacterial inoculum by more than 3 logl0 within 2-4 hours and completely cleared the bacterial burden after 4 hours. Physicochemical properties of miconazole including, the effects of pH, pre-exposure to simulated gastric fluid (SGF), and simulated intestinal fluid (SIF), were also examined. High pH values did not affect the miconazole's antibacterial action, and it retained the same potency after being exposed to SGF and SIF. Furthermore, miconazole did not show inhibitory activity against key species that compose the host intestinal microbiota and showed a prolonged post-antibiotic effect (PAE) (>6 hours) exceeding that of the drug of choice, vancomycin. Overall, these results indicate that miconazole merits further investigation as a potent and selective anti-clostridial agent to replenish the dry pipeline of new anti-C. difficile therapeutics.

Support: NIH   

P36 - Construction of novel antibiotic-resistant mutants of Neisseria gonorrhoeae strains suitable for mouse vaginal infection model  

Babatomiwa Kikiowo, Atoka B. Bandura, Nader S. Abutaleb, and Mohamed N. Seleem 

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.  Center for One Health Research, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, is the second most common sexually transmitted bacterial infection in the United States. The increasing prevalence of N. gonorrhoeae infections has been due to the emergence of antimicrobial­resistant strains. Most seriously, this uprising resistance to all classes of antibiotics could lead to a future with untreatable gonorrhea. Thus, the development of novel anti-N. gonorrhoeae drugs is urgently needed. N. gonorrhoeae FA1090 is the only strain reported to be used for in vivo mouse models because of its natural resistance to streptomycin. Streptomycin is a necessary antibiotic utilized in the mouse model to inhibit the commensal flora in the lower genital tract of mice to enhance N. gonorrhoeae colonization. However, this strain is susceptible to all antibiotics used to treat gonorrhea, and therefore, it is not suitable for drug discovery. To test the efficacy of new therapeutics against clinically important N. gonorrhoeae isolates, such as ceftriaxone-resistant and azithromycin-resistant strains in vivo, streptomycin resistance is a required phenotype for performing the in vivo mouse model. Thus, there is a requirement to develop N. gonorrhoeae strains that are simultaneously resistant to streptomycin as well as standard-of-care antibiotics, azithromycin and ceftriaxone. In this study, using allelic-exchange procedures, we constructed a N. gonorrhoeae mutant that is resistant to both streptomycin and azithromycin, and another N. gonorrhoeae mutant that is resistant to both streptomycin and ceftriaxone. The minimum inhibitory concentrations of standard antibiotics were determined against the newly constructed strains compared to their wild-type strains. When used in N. gonorrhoeae genital tract infection mouse model, mice were colonized with the new mutants for 14 days like N. gonorrhoeae FA1090. Overall, our results indicate that the newly constructed mutants proved to be suitable to be utilized in the N. gonorrhoeae infection mouse model for drug discovery studies.

Support: NIH   

P38 - Discovering Antiviral Treatments for Alphaviruses Through Proteomic Analysis of the VEEV E1 Glycoprotein 

Lauren Panny 1,2, Ivan Akrhymuk 1, Nicole Bracci 1, Caitlin Woodson 1, Rafaela Flor 1,2, Weidong Zhou 3, Aarthi Narayanan 4, Catherine Campbell 5, Kylene Kehn-Hall 1,2 

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24060, USA
2 Genter for Emerging, Zoonotic, and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24060, USA
3 Genter for Applied Proteomics and Molecular Medicine, School of Systems Biology, George Mason University, Manassas, VA, 20110, USA
4 Department of Biology, George Mason University, Fairfax, VA, 22030, USA
5 DGE Consulting, Vienna, VA, 2218, USA

Venezuelan Equine Encephalitis virus (VEEV) is an alphavirus that can cause febrile disease, encephalitis and sometimes death in humans and equines. This emerging infectious disease currently has no FDA approved treatments or vaccines. Host protein interactions with the El fusion glycoprotein of VEEV remain mostly unknown, presenting an untapped source for potential antiviral targets. As a means to effectively isolate the El protein and characterize host interactors, we designed a VEEV TC83 molecular clone with a tag inserted in the C-terminal of the El protein. Utilizing this tagged virus (VEEV TC83 El­VS) and mass spectrometry, we have discovered 182 host interactors of El after normalization. Many of these proteins are involved in pathways known to be modulated during viral infection, including the unfolded protein response and a utophagy. E 1 interaction with protein disulfide isomerase family a member 6 (PDia6), valosin containing protein (VCP) and ER lipid raft associated 2 (ERLIN2) was further verified through co­imm unoprecipitation. Inhibition of PDia6 with the POI inhibitor Loc14 resulted in decreased viral titers and viral RNA levels. These data suggest that El interacts with VCP, PDia6 and ERLIN2 to facilitate viral replication. Mechanism of action studies are ongoing to further elucidate the role of these host interactors in the VEEV replication cycle. 

Support: DTRA    

P40 - Pre- and Post-Phacoemulsification Morphologic lridocorneal Angle Analysis by Anterior Segment Gonioscopic Imaging as a Predictor of Glaucoma in Dogs  

SR Hanes, RV Ramos, IP Herring 

Department of Small Animal Clinic Sciences 

Purpose. To serially assess iridocorneal angle morphology following phacoemulsification rn dogs to determine risk of post-operative glaucoma development. Methods. Client-owned dogs presenting for surgical removal of cataract by phacoemulsification were included. Each eye underwent serial anterior segment imaging using a retinal imaging camera 

(RetCamTM). In addition to standard-of-care post-phacoemulsification management, images of the complete iridocorneal angle were obtained in four quadrants both pre­operatively and at follow-up up to one year post-operatively. Each image was evaluated to measure both angle width and pectinate ligament dysplasia (PLD) severity. Together, angle width and PLD are interpreted per a previously described equation (Zibura 2020) to determine an overall ZibWest Angle Index score for each eye at each timepoint. Results. 40 patients have been enrolled, and five patients (10 eyes) have been measured through the 6-9 month timepoint and statistically evaluated, all of which have not been diagnosed with postoperative glaucoma. A statistically significant difference in Zib West Angle Index was observed between the following: pre-operative & 3-4 months post-operative (p=0.0027); pre-operative & 6-9 months post-operative (p=0.0009); 1-2 weeks post-operative & 3-4 months post-operative (p=0.0328); 1-2 weeks post-operative & 6-9 months post-operative (p=0.0117); 3-6 weeks & 6-9 months post-operative (p=0.0453). Though not always statistically significant, a decrease in ZibWest Angle Index was observed at each subsequent timepoint. Conclusion. Serial imaging of the iridocorneal angle post­phacoemulsification reveals a decrease in ZibWest score even in non-glaucomatous eyes. Supported by the Virginia-Maryland College of Veterinary Medicine Veterinary Memorial Fund. None. 

Support: Veterinary Memorial Fund   

P10 - Comparison of semi-automated and automated diffusion tensor imaging scalar value acquisition in dogs

Michael Edwards, John Rossmeisl, Gregory Daniel, Richard Shinn

Department of Small Animal Clinical Sciences

Accurate assessment of spinal cord injury (SCI) severity improves the ability of veterinarians to develop an appropriate clinical treatment plan and discuss prognosis with clients. Quantitative imaging surrogates of myelin and axonal integrity not routinely performed provide more detailed information of SCI than routine imaging and neurologic exam. Quantitative magnetic resonance imaging metrics (qMRI) previously explored for use in veterinary medicine include fractional anisotropy, apparent diffusion coefficient, mean, axial, and radial diffusivity, and magnetization transfer ratio. A limitation for including qMRI metrics in routine imaging is the wide range of variability in reported normal values. Factors involved in data acquisition of qMRI include acquisition parameters, analytic methods, and region of interest selection variability. The implementation of automated data acquisition through open-source software, such as Spinal Cord Toolbox (SCT), will aid in reducing variability of qMRI metrics due to reduced variability in region of interest placement and wide-spread software access by various institutions and clinical practices. Due to anatomic differences between human and canine vertebral columns and spinal cords, a canine atlas was created for use with SCT to improve the accuracy of automated qMRI data acquisition. Five medium size healthy dogs had spinal MRI performed from the foramen magnum caudally to the con us medullaris. The MRI data and Digital Imaging and Communications in Medicine (DICOM) images were shared with collaborators at the University of Montreal, and an atlas was created for use with SCT. qMRI ranges of fractional anisotropy, mean diffusivity, axial diffusivity, radial diffusivity, magnetic transfer ratio, and grey matter to white matter intensity ratio will be processed using SCT in the normal dogs. Data acquisition from automated region of interest (ROI) placement and automated region of interest placement with manual adjustment (semi-automated) values will be compared, and intra-operator and inter-operator variability will be assessed. 

Support: Veterinary Memorial Fund Grant 

P12 - HDM2 inhibitor NVP-CGM097 significantly reduces alphavirus replication in microglia 

Morgen VanderGiessen 1,2, Victoria Callahan 3, Brian Carey 3, Kylene Kehn-Hall 1,2 

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
2 Center for Emerging, Zoonotic, and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
3 National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA, USA

Eastern and Venezuelan equine encephalitis virus (EEEV and VEEV) are new world alphaviruses that can cause mild flu-like illness which can progress into severe neurological deficits in both humans and equines. The primary virulence factor of these viruses, the capsid protein has a variety of functions including binding to membrane glycoproteins, inhibiting transcription of host cells, and blocking nucleocytoplasmic transport. Interestingly, the capsid protein of some other encephalitic viruses, including West Nile virus, has been demonstrated to mediate apoptosis through the p53/HDM2 cell apoptosis pathway. The role of HDM2 specifically has only recently been highlighted as a contributing factor impacting viral proliferation in non-oncogenic viruses. We hypothesize that HDM2 is a pro-viral factor which is activated during EEEV and VEEV infection with the goal of establishing HDM2 as a promising antiviral target against encephalitic alpha virus replication. Here, we investigated the antiviral potential of an HDM2 inhibitor, NVP-CGM097. Treatment of microglia cells (HMC3s) with NVP-CGM097 at nontoxic concentrations (>80% cell viability) resulted in up to 4 log 10 reduction in viral titers of VEEV TC-83. Additional studies revealed that treatment of EEEV (FL93-939) infected microglia with NVP-CGM097 at nontoxic concentrations resulted in a> lloglO reduction of viral titers. LC-MS/MS analysis of capsid immunoprecipitated samples from VEEV infected cells identified p53 as a potential host interactor of VEEV capsid. Furthermore, Co- imm unoprecipitation with a VEEV TC-83 V5 tagged capsid verified that there is an interaction between HDM2 and VEEV capsid protein as well as p53 and VEEV capsid protein. Further studies are aimed at determining the impact of VEEV capsid on HDM2 expression and the importance of HDM2 to viral proliferation. Ultimately, this study highlights the potential for HDM2 inhibition as an antiviral against encephalitic viruses.

Support: Office of Research and Graduate Studies, Defense Threat Reduction Agency (DTRA) grant HDTRA1-18-1-0040

P14 - Rift Valley fever virus NSs protein interacts with LC3 family members to modulate autophagy   

Kaylee Petraccione 1,2, Mohamed Ali3, Nicole Bracci 1,2, Normand Gyr 3, Haytham M. Wahba 3, Andrew Silberfarb 4, Danuta Sastre 5, Paul 0'Maille 5, James 0michinski 3, Kylene Kehn-Hall 1, 2

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
2 Center for Emerging, Zoonotic, and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
3 Department of Biochemistry and Molecular Medicine, Universite de Montreal, Montreal, QC, Ganado
4 Artificial Intelligence Center, SRI International, Menlo Park, CA, USA
5 Biosciences Division, SRI International, Menlo Pork, CA, USA 

Rift Valley fever virus (RVFV) is a viral zoonosis that causes severe disease in ruminants and humans. Impregnated ruminant infections are characterized by 'abortion storms' in which abortions occur in nearly 100% of cases. In humans, RVFV causes a variance of clinical symptoms ranging from a flu-like illness to hemorrhagic fever, neurological deficits, and encephalitis. RVFV is mainly transmitted via mosquito bite and concerns are rising regarding the introduction of RVFV to the U.S. as competent mosquito vectors were identified. Despite its pathogenic potential, there are no FDA-approved therapeutics or vaccines to challenge the global spread of this infectious organism. The nonstructural small (NSs) protein is the main virulence factor of RVFV, making it an attractive antiviral target. Bioinformatic and structural analysis identified four potential LC3-interacting region (LIR) motifs in the RVFV NSs protein, suggesting that NSs forms polyvalent interactions with LC3, the host key autophagy protein. Autophagy is a homeostatic process in which cellular materials are degraded and it can act in either a proviral or antiviral manner. To determine whether NSs interacts with LC3-family proteins, isothermal titration calorimetry (ITC) experiments were performed with peptides corresponding to the predicted LIRs. ITC demonstrated that LIR4 interacts with high affinity with all six LC3 proteins, whereas weak or no binding was observed with LIRl-3. To confirm the NSs-LC3 interaction, plasmids encoding LC3-family members were utilized, and co-immunoprecipitation confirmed that NSs interacts with all six LC3-family members in RVFV-infected cells. NSs also co-immunoprecipitated with endogenous LC3A and LC3B in RVFV-infected cells. Substitution of key amino acids in LIR4 of NSs resulted in significant loss of binding to LC3B in infected cells, further indicating a crucial role for LIR4. Cellular fractionation followed by co-immunoprecipitation demonstrated the NSs-LC3 interaction occurred predominantly in the nucleus. Confocal microscopy demonstrated that NSs colocalized with LC3A in perinuclear and filamentous regions, suggesting NSs is sequestering LC3A in the nucleus to prevent antiviral autophagy. This is supported by experiments demonstrating that NSs downregulates autophagy. These results demonstrate that RVFV NSs modulates autophagy through interaction with LC3-family proteins, providing another mechanism that RVFV NSs dampens the host antiviral response.

Support: Defense Threat Reduction Agency (DTRA), grant HDTRA1-22-1-0009 

P16 - NAK associated protein 1/NAP1 is required for mitosis and cytokinesis through TBK1 activation   

Swagatika Paul 1, Shireen A. Sarra/2, Ki Hong Nam3, Leila Zavar4, Sahitya Ranjan Biswas5, Lauren E. Fritsch5, Nicole DeFoor4, Tomer M. Yaron6J, Jared L. Johnson6J, Emily M. Huntsman6J, Lewis C. Cantley6,7, Alban 0rdureau3, and Alicia M. Pickrel/4, *  

1 Graduate Program in Biomedical and Veterinary Sciences, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA 24061 USA
2 Biochemistry Section, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 USA
3 Cell Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065 USA
4 School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 USA
5 Translational Biology, Medicine, and Health Graduate Program, Virginia Tech, Roanoke, VA 24016 USA
6 Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10065 USA
7 Department of Medicine, Weill Cornell Medicine, New York, NY 10065 USA;

Successful cell division is dependent on precise and timely transition between different cell cycle phases, which is regulated by dynamic changes in protein phosphorylation status. Thus, protein kinases play a vital role to orchestrate almost every step of cell division. Impaired kinase activity often leads to abnormal cell cycle events which consequently become the underlying cause for developmental defects or abnormal cell proliferation leading to cancer. Tank Binding Kinase 1 (TBK1) is one such kinase which is often over­expressed in certain cancer types and also regulates the process of cell division. TBK1 is activated on the centrosomes during mitosis, and its loss impairs cell division resulting in growth defects and the accumulation of multi-nucleated cells. Therefore, both proper activation and subcellular localization of TBK1 are essential for mitotic progression. Yet, the upstream regulation of TBK1 during mitosis is unknown, and the function of activated TBK1 on the centrosomes is understudied. Activation of TBK1 depends on its binding to an adaptor protein which induces a conformational change leading to transautophoshorylation on serine 172 of TBK1. Our study objective is to identify the unknown upstream adaptor(s) and downstream substrates of TBK1 during mitosis. Using a combination of shRNA mediated knockdown cell lines, editing of several TBK1-associated genes via CRISPR, conditional protein degradation, and co-immunoprecipitation experiments, we identified the adaptor protein for TBK1 activation during mitosis is NAK Associated Protein 1(NAP1/AZI2). Characterization of mitotic and cytokinetic defects suggests that loss of either NAP1 or TBK1 results in the accumulation of binucleated and multinucleated cells, possibly due to several mitotic and cytokinetic defects seen in these knockout cells. We establish NAP1 as a cell cycle protein which colocalizes with activated TBK1 on the centrosomes. Interestingly, NAP1 levels during mitosis are tightly regulated by TBK1, where activated TBK1 phosphorylates NAP1 on serine 318 flagging it for ubiquitin proteasomal degradation. Further, by an unbiased quantitative phosphoproteomics analysis during mitosis, the substrates discovered reveal that TBK1 also regulates essential cell cycle kinases such as Aurora A and Aurora B. In conclusion, our work has uncovered a novel function for the NAP1-TBK1 complex during mitosis, which is distinctive from its previously known role in innate immune signaling.

Support: National Institutes of Health Grants GM142368 (AMP) & Departmental startup fund (AMP)

P26 - Mounting a mitochondrial defense: Innate immune receptor NLRX1 regulates cell metabolism and death for protection against Lyme disease  

Juselyn D. Tupik 1, Mecaila E. McGlune 2, Hailey W. Camp 1, Julia A. Gregory 1, Jules M. Dressler 2, Justin W. Markov Madanick 1, Margaret A. Nagai-Singer 1, Brandon L. Jutras 2, Irving C. Allen 1,3 

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, VA
2 Department of Biochemistry, Virginia Tech, Blacksburg, VA
3 Department of Basic Science Education, Virginia Tech Carilion School of Medicine, Roanoke, VA 

Lyme disease, caused by the bacterium Borrelia burgdorferi, is an emerging infectious disease of global concern. Roughly 60% of untreated patients will develop inflammation of the joints termed Lyme arthritis. This persistent phenotype results from sustained inflammatory signaling by the innate immune system. Currently, there are limited treatments for antibiotic-refractory Lyme arthritis, warranting our investigation into how the innate immune system can mitigate this inflammation. Here, we studied how the anti­inflammatory innate immune receptor NLRX1 regulates host-pathogen interactions in response to Borrelia burgdorferi. Expressed almost ubiquitously in mammalian cells, NLRX1 senses conserved genetic motifs of pathogens to regulate innate immune pathways. Specifically, NLRX1 associates with the mitochondria, playing unique roles in regulating cell metabolism and function. Because NLRX1 modulates cell function, including inflammatory signaling, cell death, autophagy, and cell metabolism, we hypothesized that NLRX1 could have unique antimicrobial defenses against Lyme arthritis. Through 30-day models of infection in novel Nlrx1-/- mice, we found that NLRX1 significantly decreased arthritis severity in wildtype mice when compared with knockouts, modulating bacterial load in vivo. We next determined in Nlrx1 -/- murine macrophages that NLRX1 may control B. burgdorferi persistence by promoting Reactive Oxygen Species (ROS)-mediated oxidative stress, subsequently promoting mitochondrial dysfunction and inflammatory cell death. Finally, by infecting novel NLRX1 overexpression human monocytes, we found that elevated NLRX1 significantly decreased pro-inflammatory NF-xB cytokine secretion. These results indicate that NLRX1 plays a protective role in mitigating Lyme arthritis in both murine and human models. Further, this protection could occur through NLRX1 's promotion of oxidative stress, which may initiate pore formation and inflammatory cell death in tandem with suppression of pro-inflammatory NF-xB signaling. Therefore, we emphasize the importance of NLRX1 host sensing of B. burgdorferi and encourage its further exploration for new treatments for Lyme arthritis. 

Support: NIH/NIAID (R21AI159800), Cohen Foundation 

P28 - Regulation of and by Quorum Sensing Proteins in the Zoonotic Pathogen Brucella abortus 

Mitchell T. Caudill and Clayton C. Caswell 

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, VA

The zoonotic pathogen Brucella abortus is a causative agent of brucellosis, a potentially debilitating, persistent bacterial disease characterized by fatigue, arthritis, and periodic fever. Brucella is able to disseminate throughout the mammalian host via replication and survival in mesenchymal cells, particularly macrophages, while eliciting only a minimal immune response. How Brucella is adapted to this stealthy lifestyle, and how it regulates its genetic expression to achieve persistence in the host are major questions regarding Brucella pathogenesis. One set of virulence factors utilized by Brucella are the two quorum sensing proteins VjbR and BabR, which are master transcriptional regulators that are major determinants of Brucella's metabolic and virulence state. While strains lacking one of these two proteins have been explored, including as potential vaccines, we have created the first strain of Brucella lacking both proteins. Our data show that after removing both proteins, mice clear the resultant strain of Brucella by 4 weeks post-infection, and that the lack of both proteins has physiological consequences for how Brucella responds to host-derived stresses encountered during its infection cycle. Current work is being pursued to better define in vitro stress responses in this Brucella strain, as well as to better determine how the two proteins regulate one another in natural Brucella infections. 

Support: Animal Model Research for Veterinarians Program (T32OD028239)  

P30 - Proliferation, Plasticity, and Migration: Exploring the synergistic relationship between eosinophils and TH2 cells in development of HES-like syndrome 

Brie Trusiano, I.C. Allen 

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, VA

Eosinophils play an important therapeutic role in combating fungal and helminthic pathogens. However, aberrancies in eosinophil proliferation, activation, and chemotaxis can also lead to acute eosinophilic leukemia, paraneoplastic eosinophilia associated with T cell lymphoma and carcinomas, systemic and local hypersensitivity reactions, and Hypereosinophilic Syndrome (HES), respectively. HES is an idiopathic disease affecting humans and veterinary patients characterized by persistent eosinophilia (>1,500 cells/uL) and eosinophilic infiltration of solid organs and tissues leading to increased morbidity and mortality. Previous studies have demonstrated the role of NF-kB inducing kinase (NIK) an upstream regulator of the noncanonical NF-KB signaling pathway, in development of a HES-like syndrome due to aberrant TH2 polarization in the Nik-/- murine model. However, the intricacies of eosinophilic granulopoiesis, education of eosinophilic precursors in different hematopoietic compartments, eosinophil/neutrophil plasticity, and chemotactic ability has not been assessed during development of HES-like syndrome in Nik-/- mice. Here we show that loss of NIK potentially enhances eosinophilic chemotactic potential to eotaxin (CCL1 1), enhances eosinophilic/neutrophilic plasticity, and affects granulopoiesis in the bone marrow and spleen beginning at the level of eosinophilic metamyelocytes, despite potentially decreasing the proliferative potential of these cells. Taken together, these results suggest that NIK and the noncanonical NF-KB signaling pathway plays a role in dictating the hematopoietic and chemotactic potential of eosinophils and highlights a synergistic relationship with TH2 cells and their respective cytokines during development of HES-like syndrome in Nik-/- mice. Overall, these findings warrant further study to better characterize the synergistic relationship described above to better understand development of HES and HES-like diseases. Furthermore, these results may also suggest a role for NIK and the noncanonical NF-KB signaling pathway when attempting to better characterize, diagnose, and treat eosinophilic leukemias or paraneoplastic eosinophilia associated with T cell lymphoma. 

Support: ICTAS, Office of Research and Graduate Studies 

P32 - LAMPore sequencing as a novel diagnostic for equine herpesvirus 1 using equine nasal swabs

Nadia Saklou 1, Brandy Burgess 2, Kevin Lahmers 1

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, VA 2 University of Georgia

Equine herpesvirus type 1 (EHV-1) is an alphaherpes virus that is ubiquitous in the equine population, affecting horses of every breed and discipline. While the majority of horses have been exposed by one year of age, and typically experience mild respiratory disease, infection is also associated with late term abortion, neonatal foal death, and neurologic disease- Equine Herpes Myeloencephalopathy or EHM. Test results for current detection methods, such as PCR and virus isolation, can take multiple days before they become available. A novel DNA sequencing technology, the MinION nanopore sequencer, offers a new tool to rapidly detect and differentiate EHV-1 (i.e., neurotropic and non-neurotropic strains) as a stall-side diagnostic and in field applications (e.g., naturally occurring disease and outbreaks). DNA is extracted from clinical samples, such as nasal swabs, in preparation for sequencing. Extraction of EHV-1 DNA from nasal swabs is often low yield, thus viral amplification prior to sequencing is ideal. Loop-mediated isothermal amplification (LAMP) allows for simple and rapid viral amplification. This method does not require a thermocycler nor nucleic acid purification, thus it can be done quickly with minimal equipment and will be less effected by inhibitors found in clinical samples. The purpose of our study is to compare the efficacy of LAMP and DNA sequencing using the Minion nanopore sequencer to the current gold standard qPCR for diagnosing equine herpesvirus type-1 in equine nasal swabs. Nasal swabs from Six EHV-1 positive and six EHV-1 negative horses were processed using LAMP followed by Minion sequencing. DNA from each sample was also analyzed by qPCR. Our data currently shows that LAMP and DNA sequencing with the Minion successfully identify EHV-1 in nasal swabs from horses. 

Support: Veterinary Memorial Fund and American Quarter Horse Foundation  

P42 - Evaluation of a feline optimized TSH assay in cats with hyperthyroidism and with non-thyroidal illness 

Camille Brassard, Stephanie DeMonaco 

Department of Small Animal Clinical Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech Blacksburg, VA, USA

About 10% of hyperthyroid cats have a normal total T4 requiring further testing to make the diagnosis. In people, thyroid stimulating hormone (TSH) can be used to make the diagnosis. As a specific feline assay is not currently available, TSH is measured using the canine assay (cTSH). However, this assay cannot differentiate between subnormal and low-­normal TSH concentrations in cats, making it a poor test for diagnosing feline hyperthyroidism. A novel feline optimized TSH assay (fTSH, Truforma by Zomedica) was recently developed. It differentiates better between euthyroid and hyperthyroid cats compared to cTSH. However, the effect of non-thyroidal illness (NTI) on fTSH has not been evaluated. Objectives: Comparison of fTSH and cTSH concentrations among hyperthyroid cats, cats with non-thyroidal illness (NTI), and healthy cats. Evaluation of sensitivity and specificity of fTSH to diagnose hyperthyroidism. Hypotheses: Hyperthyroid cats have lower fTSH compared to healthy and ill cats. The fTSH detects more hyperthyroid cats than cTSH. Cats with NTI have similar fTSH and cTSH concentrations as compared to healthy cats. Prospective study consisting of hyperthyroid cats, healthy cat, and cats with NTI. Serum was collected to measure T4 and cTSH (Immulite 2000) and fTSH (Truforma platform) concentrations in all cats. Hyperthyroid cats had a thyroid scintigraphy performed to confirm hyperthyroidism. Healthy and NTI cats had T4 repeated 3 months from enrollment if available for follow up to rule out subclinical hyperthyroidism. The fTSH was compared among groups using Kruskal Wallis followed by Wilcoxon pairwise method. Significance was set at P <0.05. Preliminary results of 29 hyperthyroid cats, 29 healthy cats and 30 cats with NTI. The mean age of healthy, hyperthyroid, and NTI cats were 8. 7 yrs, 11.6 yrs, and 12.1 yrs, respectively, with healthy cats being significantly lower in age compared to the other groups. There is no difference between mean fTSH and cTSH concentrations between healthy and NTI cats. TSH concentrations from both assays are significantly lower in hyperthyroid cats than in healthy and NTI cats (P<0.001 ). Five ( 17%) hyperthyroid cats had a normal fTSH but undetectable cT SH. One cat has a normal cTSH ( 0. 035), but undetectable fTSH ( <0 .008). Conclusions: The fTSH identifies normal TSH in healthy cats more often than cTSH and appears to not be affected by NTI. The fTSH can be a useful tool for the diagnosis of feline hyperthyroidism. 

Support: Zomedica 

P44 - Combined live oral priming and intramuscular boosting regimen with Rotarix and a nanoparticle-based multivalent rotavirus vaccine confers significant protection against both G4P[6] and G1P[8] human rotavirus infection in gnotobiotic pigs  

Casey Hensley, Charlotte Nyblade, Peng Zhou, Viviana Parreno, Annie Frazier, Maggie Frazier, Ariana Fantasia-Davis, Sarah Garrison, Ruiqing Cai, Ming Xia, Ming Tan, Lijuan Yuan 

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA.
Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA 

Human rotavirus (HRV) is the causative agent of severe dehydrating diarrhea in children under the age of five, resulting in up to 215,000 deaths yearly. With the introduction of live, oral attenuated vaccines nearly two decades ago, these deaths now almost exclusively occur in low and middle-income countries where vaccine efficacy is the lowest. Reasons for decreased efficacy in these areas include gut dysbiosis, concurrent use of other live oral vaccines, enteric viral infection, and malnutrition. These factors make parenteral vaccines for HRV particularly attractive, as they avoid many of the issues associated with oral vaccines. Our collaborators at Cincinnati Children's Hospital Medical Center developed a nanoparticle-based nonreplicating, parenteral HRV vaccine, utilizing the shell (S) domain of the capsid of norovirus (NoV). The S domain of NoV self-assembles into a nanoparticle with exposed C-termini, which are amenable to the insertion of foreign proteins. In this vaccine, HRV's VP8"' from the three most predominant P types were fused to the exposed C-termini, forming the S60-VP8* nanoparticle cocktail vaccine. The safety, immunogenicity and protective efficacy of this cocktail vaccine as a two-dose intramuscularly administrated regimen, as well as a booster using one dose of the commercial oral Rotarix vaccine as a priming immunization was evaluated in the gnotobiotic pig models of P[6] and P[8] HRV infection and diarrhea. The prime/boost regimen provided partial protection from diarrhea and significantly delayed the onset of virus shedding in pigs challenged orally with the virulent Wa (G1P[8]) HRV. This regimen also significantly shortened mean duration, mean peak titer and AUC of virus shedding after challenge with Arg (G4P[6]) HRV. Both vaccine regimens were highly imm unogenic and induced significantly higher titers of HRV-specific serum IgG, IgA and virus neutralizing antibodies in both challenge groups. Prime/boost-vaccinated pigs challenged with Wa HRV had significantly higher P[8]-specific IgG antibody secreting cells (ASCs) rn the spleen post-challenge. Prime/boost-vaccinated pigs challenged with P[6] HRV had significantly higher numbers of P[8]-specific IgA ASCs in the spleen, as well as significantly higher numbers of P[6] and P[8]-specific IgG ASCs in the ileum post­challenge. These results suggest the promise and warrant further investigation in to the oral priming and parenteral boosting strategy for future HRV vaccines.

Support: NIAID, VMCVM, CCHMC  

P46 - Exploiting virus-host interactions to develop novel inhibitors against Venezuelan equine encephalitis virus

Abdullahi Jamiu 1,2, Ivan Akhrymuk 1, Kenneth Foreman 3, Dmitri Klimov 4, Mikell Paige 3, Kylene Kehn-Hall  1,2 

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
2 Center for Emerging, Zoonotic, and Arthropod-Borne Pathogens (CeZAP), Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
3 Department of Chemistry and Biochemistry, George Mason University, Manassas, VA 20110, USA
4 School of Systems Biology, George Mason University, Manassas, VA 20110, USA 

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne, positive sense, single­stranded RNA virus that belongs to the genus Alphavirus. VEEV can infect both equines and humans, with associated neurological complications in ~14% of human cases. Due to its low infectious dose, ease of aerosolization and manipulation, this virus is classified as a select agent by both the CDC and USDA. However, there are currently no FDA-approved therapeutics or licensed vaccines against VEEV infection in humans. The VEEV capsid protein is an essential virulence factor of VEEV. The capsid protein can simultaneously bind to the host's nuclear import receptors, importin a/b1, and the host export receptor, CRMl to form a tetrameric complex. This complex accumulates at the nuclear pore channel, halting nucleocytoplasmic trafficking, downregulating host transcription and inhibiting cellular antiviral response. Moreover, VEEV TC83 Cm, with a mutated non­functional nuclear localization sequence within the capsid, failed to downregulate cellular transcription and antiviral response. This suggests that the nuclear import of VEEV capsid is pertinent for pathogenesis and could be exploited as an attractive target for therapeutic development. We hypothesized that chemical inhibitors capable of disrupting the interaction of capsid with importin al􀀃 1 should increase cellular antiviral response, resulting in reduced viral titers and rescue of cells from VEEV-induced cell death. Two small molecule inhibitors, I2 and 1564, were designed to disrupt the interaction between capsid and importin a. These inhibitors were well tolerated by HMC3 microglia cells with CC50 of >250 µM and >500 µM for 12 and 1564, respectively. These compounds impacted VEEV TC83 titer with> 1 logl0 decrease at 9 hpi. Furthermore, 12 displayed an EC50 of 2.96 µM and 1564 an EC50 of 5.38 µM against VEEV. Both compounds also rescued infected cells from VEEV-induced cell death. In order to evaluate the impact of these compounds on the capsid-importin a interaction, we cloned two viruses that contain a VS tag at the N­terminus of the capsid, TC83 VS-C and TC83 VS-Cm. The replication kinetics of these new viruses were similar to that of parental TC83 and TC83 Cm. Future studies will involve evaluating the impact of these compounds on capsid-importin interaction and capsid localization using co-immunoprecipitation and confocal microscopy. 

Support: National Institute of Health, VMCVM Office of Research and Graduate Studies  

P48 - Optimizing centrifugation parameters for cooled canine semen processing

Nicole Sugai, Stephen Werre, Julie Cecere and Orsolya Balogh 

Department of Small Animal Clinical Sciences, Virginia-Maryland College of Veterinary Medicine 

Semen processing is critical during preparation for cooled shipments or sperm cryopreservation in dogs. The process involves centrifugation and re-suspension of the sperm pellet to appropriate volumes using semen extender. Our hypothesis was that longer centrifugation times at higher speed will lead to increased sperm recovery rates (RR) but reduced sperm viability, motility, and percent normal morphology. Semen was collected from 14 healthy client-owned stud dogs of medium to large breeds and mean age of 3.3 years. Inclusion criteria were negative Brucella canis serology, ejaculate with ≥70x106/ml sperm concentration, ≥70% total motility and ≥40% normal morphology. Ejaculates were divided into six equal volume aliquots, subjected to one of six centrifugation treatments (400g, 720g, and 900g, each for 5 or 10 minutes; denoted as A-F, respectively). Sperm pellet was re-extended to original aliquot volume with CaniPlus Chill LT (Minitube) and cooled at 4°C for 48 hrs. Sperm viability was evaluated by mixed model two-way ANOVA using percentage change from baseline, and sperm RR, subjective total motility (TM ) and computer-assisted sperm analysis (CASA) total (TM) and progressive motility (PM),% normal morphology were analyzed by a linear generalized estimating equations model using change from baseline, comparing treatment groups and time points (T0: initial raw as baseline, T1: post-centrifugation, T2: 24 hrs, and T3: 48 hrs). P<0.05 was considered significant. 

Sperm RR was similar among the six treatment groups (p≥0.06 2; 9 8. 8-102 .1 %) . The viability significantly declined at T2 and T3 compared to T1 (p≤0.001) in all treatment groups. Subjective TM decreased over time in all groups (T1 to T2 p≤0.041, T1 to T3 p≤0.0002, T2 to T3 p≤0.016). CASA TM showed significant decline by T3 (T1 to T3 p≤0.023, T2 to T3 p≤0.031) for all groups. CASA PM decreased from T1 to T3 (p≤0.004) for all groups; A, C, D, E, and F from T2 to T 3 (p≤0.0 13) and T 1 to T2 for C and E (p≤0.044). Percent normal spermatozoa decreased already by T2, independent of treatment groups (T1 to T2 p≤0.0001, T1 to T3 p≤0.0001). For healthy, fertile stud dogs, semen parameters after centrifugation and standard shipping conditions were not significantly affected by centrifugation speed or time, but all treatment groups showed declines over the 48 hrs period of cooled storage. The centrifugation speeds of 400-900g for 5-10 min duration are appropriate for canine semen processing. 

Support: Dr. JoAnne S O'Brien Endowment Fund at Virginia Tech 

P50 - Development of a novel approach to broadly-protective anti-enterovirus vaccines

Anna Zimina 

Department af Veterinary Medicine, University af Maryland 

Enteroviruses are arguably the most numerous human viral pathogens. Due to high antigenic diversity, the development of traditional capsid-targeting vaccines is feasible for only few enteroviruses. Yet, their replication proteins are much more conserved and are essentially interchangeable, as evidenced by the extensive recombination of enterovirus genomes. We observed that upon immunization with live polioviruses of mice and non­human primates, the animals develop antibodies not only against the capsid proteins but also against a number of non-structural proteins, indicating that antigens from conserved non-structural proteins are presented to and recognized by the immune system. We set to investigate if a vaccine based on expression of conserved replication proteins only could be protective, and if that protection could be extended to antigenically diverse enteroviruses. We trans-packaged the poliovirus replicon RNA coding for P2P3 proteins using our previously developed Newcastle Disease Virus (NOV) vectored vaccine expressing poliovirus virus-like particles. This trans-packaging system based on efficiently replicating poliovirus P2P3 RNA and NOV allows cost-effective production and purification of packaged replicons. These replicons can be administered similarly to live poliovirus vaccine, but the capsid proteins are present only in the original inoculum and are not produced upon replicon RNA replication. Preliminary experiments in transgenic mice demonstrate that replicon immunization indeed promote a much higher presentation of non-structural proteins to the immune system, however a significant development of antibodies against capsid proteins was also observed, indicating that an alternative replicon RNA delivery system is required to completely remove the input of immunodominant capsid proteins. 

P52 - Development of a novel approach to broadly-protective anti-enterovirus vaccines

Urmil Dave

Department af Veterinary Medicine, University af Maryland 

Streptococcus agalactiae and Staphylococcus aureus are the leading pathogens responsible for bovine mastitis and an array of human diseases. The global burden is only aggravated by the ascend of drug resistant strains such as methicillin resistant Staphylococcus aureus (MRSA). An alternative to antibiotic resistance comes from bacteriophages, the viruses of the bacteria. Some bacteriophage cell wall hydrolases, termed endolysins, responsible for lysis the bacterial cell from within during the phage life cycle, also demonstrate the ability to break down the peptidoglycan of a bacterial cell wall when applied externally. This peculiar characteristic of endolysins along with their immunity to efflux pumps, penicillin binding proteins, alterations of metabolic pathways, or other mechanisms of traditional antibiotic resistance make them excellent candidates to combat these drug resistant bacteria. A typical endolysin targeting gram-positive bacteria employ a modular structure, with an N-terminal enzymatically active domain (EAD) which cleaves the peptidoglycan, linked to a C-terminal cell wall binding domain ( CBD) which binds to the surface proteins/ carbohydrates in the cell wall. Harvesting the knowledge garnered over the years and employing the tools generated over a decades of research on these endolysins, the aim is to create a chimeric endolysin that combats both S. agalactiae and S. aureus including the drug resistant strains.

P1 - ADAMTS13 activity in dogs with chronic enteropathies

SI Barth, AR Wilkinson, SM DeMonaco, KM Boes, and BJ Conner

Department of Small Animal Clinical Sciences

Background: Chronic enteropathies (CE) predispose dogs to thromboembolic disease, but the underlying mechanisms are poorly understood. Humans with CE have decreased activity of ADAMTS 13, a von Wille brand factor (vWF) cleaving enzyme, and increased circulating vWF. The primary aim of this study is to determine whether dogs with CE have reduced ADAMTS 13 act1v1ty,  increased  plasma  vWF  antigen  concentration  (vWF:Ag),  and increased vWF:collagen binding activity (vWF:CBA) compared to healthy control dogs. Hypothesis: Dogs with CE have reduced ADAMTS 13 activity, increased vWF:Ag, and increased vWF:CBA compared to healthy control dogs. Methods:In this prospective study, ADAMTS 13 activity, vWF:Ag, and vWF:CBA will be assessed in 20 client-owned dogs with CE. Forty healthy dogs will serve as controls for ADAM TS 13 activity. Twenty healthy dogs will serve as controls for vWF:Ag and vWF:CBA. The mean ADAMTS 13 activity, vWF:Ag, and vWF:CBA of the control group will be compared with the affected group using a two­ sample t-test, or if the data is not normally disturbed, a Wilcoxon  rank sum test. Preliminary results: Sixteen dogs with CE have been enrolled at this time. The mean age of this population is 5.9 years (standard deviation (SD) of 3.7). Mean ADAMTS13 activity is 74.5% (SD 22.6) in affected dogs (n = 9) and 83.9% (SD 14.2) in healthy dogs (n = 36). Mean vWF:Ag is 109.8% (SD 41.4) in affected dogs (n = 7) and  159.8% (SD 33.4) in healthy dogs (n = 17). Mean vWF:CBA is 97.9% (SD 42.2) in affected dogs (n = 7) and  129.8% (SD 25.9) in healthy dogs (n = 17). Preliminary analysis revealed that ADAMTS 13 activity is not significantly different between dogs with CE and healthy dogs (P = 0.12). However, vWF:Ag and vWF:CBA are significantly decreased in dogs with CE compared to healthy control dogs (P = 0.005 and P = 0.032, respectively).

Support: Internally funded

P3 - Historical redlining and contemporary racial disparities in sports and recreation­related injury hospitalizations in the United States 

Tosin Ogunmayowa 1, Alicia Lozano 2, Alexandra Hanlon 2, Natalie Cook 1, Freddy Paige 3, Charlotte Baker 1 

1 Department of Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech
2 Center far Biostatistics and Health Data Science, Department of Statistics, College of Science, Virginia Tech
3 The Charles E. Via, Jr. Department of Civil and Environmental Engineering, College of Engineering, Virginia 

Approximately 9 million people are injured annually from sports and recreation in the U.S., more than a third seek treatment in the emergency department, and several thousands are hospitalized for more severe injuries. In this study, we examined the association between historical redlining, a government-sanctioned racial discriminatory practice of the 1930s, and present-day sports and recreational injury (SRI) hospitalization in the U.S. We obtained 2011 SRI hospitalization data in the U.S. from the National Inpatient Sample (NIS) database, linked it to 1930s Home Owners' Loan Corporation (HOLC) redlining maps, and assigned U.S. hospitals to one of three HOLC grades (A+B - best/still desirable, C - definitely declining, and D - hazardous/redlined). Generalized linear mixed models, accounting for sample weight, stratified sampling, and patients clustered within hospitals, were used to examine these relationships. We found no association between HOLC grade and the risk of hospitalization for SRI among all racial/ethnic groups after adjusting for confounders; however, HOLC grade was associated with length of hospital stay (LOS) in Black, Hispanic and White patients, and total hospital charges per discharge for Black and Hispanic patients. Black patients who were hospitalized for SRI in historically redlined neighborhoods (grade D) had 38% longer LOS compared to those hospitalized in neighborhoods historically graded as "A+B: best/still desirable". In contrast, White and Hispanic patients who were hospitalized for SRI in historically redlined neighborhoods had 8% and 9% shorter LOS, respectively, compared to those hospitalized in "A+B: best/still desirable" neighborhoods. Total hospital charges per discharge were 29% and 12% lower for Black and Hispanic patients hospitalized in historically redlined neighborhoods compared to those hospitalized in neighborhoods historically graded as "A+B: best/still desirable", but no difference was observed among White patients. We also found that as the social vulnerability (i.e., the susceptibility of communities to the adverse impact of injury, disease outbreaks, and natural or human-caused disaster) of present-day neighborhood environment decreased, the impact of redlining on LOS weakened for Black patients but not for Hispanic and White patients. This study indicates that redlining, an indicator of structural racism, has a lasting impact on the length of stay and cost of hospitalization for SRI in the U.S. 

Support: GPSS Graduate Research Development Program, Office of Research and Graduate Studies 

P5 - Causal Inference to Scope Environmental Impact Assessment in MultiSector Systems: The Case of Trans-Border Hydropower Exports  

Amir Mortazavigazar, Ryan Calder 

Department of Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech

Decarbonization of the United States' electricity sector will require trillions of dollars of investment in generation and transmission infrastructure. The National Environmental Policy Act (NEPA) requires proponents of many major projects to complete environmental impact statements (EIS) that address reasonably foreseeable impacts, regardless of where these impacts occur. There has been controversy over the cause-effect relationships among electrical supply, electrical demand, apparent cost, and other variables given the complex interactions between them. Therefore, the range of environmental impacts attributable to new infrastructure projects is subject to frequent disagreements. In this work, we address increasing U.S. imports of Canadian hydropower in the context of falling prices and surplus generation. There has been controversy as to whether new transmission capacity stimulates new generation capacity and, thus, whether generation-side environmental and health impacts must be assessed in the scope of incremental transmission projects. We have developed a rich longitudinal database of variables related to generation capacity, export volume, retail prices, and climate over the period 1979-2021. We have applied a novel multivariable, wide neural network machine learning methodology to evaluate alternative causal models for the evolution of the electricity system and the role of new transmission infrastructure. We find no evidence that transmission capacity stimulates generation capacity. Rather, generation capacity growth in Canada is triggered primarily by domestic price signals and climate parameters, with trans-border transmission capacity developed primarily to absorb excess generation potential. This work supports a relatively narrow scope for EIS related to trans-border transmission projects. More generally, this analysis demonstrates how causal inference methods may help build consensus around the appropriate scope of EIS for highly interconnected energy and infrastructure projects. 

Support: Virginia-Maryland College of Veterinary Medicine Office of Research and Graduate Studies 

P7 - Deletion of NIK Increases Susceptibility to Colorectal Cancer through Diminished Intestinal Epithelial Cell Regeneration  

Holly A. Morrison 1, Audrey Rowe 1, Kristin Eden 1, Daniel E. Rothschild 1, Katherine Baumgarner 2, Stephan Brown 2, Eda Holf 3, Irving C. Allen 1  

1 Virginia Tech, Virginia Maryland College of Veterinary Medicine, Department of Biomedical Science and Pathobiology, Blacksburg, VA 24061
2 Via College of Osteopathic Medicine, Department of Cell Biology and Physiology, Spartanburg, SC 29303
3 Duke University, Department of Surgery, Durham, NC 27108 

Patients with a medical history of Inflammatory Bowel Disease are predisposed to colorectal cancer with greater than 20% developing colitis-associated colorectal cancer (CAC). Diminished regulation of intestinal epithelial cell (IEC) proliferation is a critical feature in the development of colorectal cancer. However, loss of stem cell regulation in crypts is widely predicted to be the cell-of-origin for malignant transformation. We hypothesize that NIK and proper noncanonical NF-xB signaling is essential for protection against colorectal cancer and functions through the epithelial cell compartment rather than the stem cell compartment. Here, we show that NF-xB inducing kinase (NIK) attenuates colorectal cancer through the regulation of IEC regeneration and differentiation mediated by noncanonical NF-xB signaling. Murine models with IEC-specific NIK deletion have increased susceptibility to inflammation-induced tumorigenesis following the chemical induction of AOM/DSS. Mechanistic studies using crypts and organoids further revealed an imbalance of proper apoptosis and proliferation signaling, with colonic crypts having increased accumulation of mature, non-dividing IECs. This suggests a model for Top-down tumorigenesis as terminally differentiated IECs lack proper turnover and compensate for decreased regenerative capacity from the stem cell niche by acquiring stem-cell like features. Our work has clinical relevancy as human CAC biopsy samples have attenuated noncanonical NF-xB signaling, including significantly decreased gene expression of NIK (MAP3K14) and effector chemokines produced (CXCL12, CXCL13, CCL19, CCL21). Here, we present a novel role for noncanonical NF-xB signaling in regulating IEC regeneration and differentiation in protecting against CAC development. 

Support: Supported by grants from the iTHRIVE Pilot Translational and Clinical Studies Program, VCOM Center for One Health Research, Virginia Tech Carilion Research Acceleration Program, and the Virginia-Maryland College of Veterinary Medicine. 

P17 - Sex Linked Growth Disorder and Aberrant Pituitary Gene Expression in Nestin-Cre Mediated Egr1 Conditional Knockout Mice  

Cody Swilley 1,2, Yuze Zheng 1, Lin Yu 1,3, Xiguang Xu 1,2, Min Liu 1,2, Kurt Zimmerman 2, Hehuang Xie 1,2,3,4,5,6*

1 Epigenomics and Computational Biology Lob, Fralin Life Sciences Institute of Virginia Tech, VA
2 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, VA
3 Genetics, Bioinformotics and Computational Biology program, Virginia Tech, Blacksburg, VA
4 Department of Biological Sciences, College of Science, Virginia Tech, Blacksburg, VA
5 Translational Biology, Medicine and Health Program, Virginia Tech, Blacksburg, VA
6 School of Neuroscience, Virginia Tech, Blacksburg, VA

Genes regulating hormone release are essential for maintaining metabolism and energy balance. Egr1 encodes a transcription factor regulating hormone production and release, and the decreased growth hormone has been reported in Egr1 complete knockout mice. Reduction in growth hormone level has also been observed in Nestin-Cre mouse, a frequently used model to study nervous system. Currently, it remains elusive how Egr1 loss or Nestin-Cre driver disrupts pituitary gene expression. Here we compared the growth curves and pituitary gene expression profiles for Nestin-Cre mediated Egr1 conditional knockout (Egr1cKO) mice together with their controls. Reduced body weight was observed for both Nestin-Cre and EgrlcKO mice and the loss of Egr1 has slightly severer impact on females than on male mice. RNA-seq data analyses revealed that the sex-related differences were amplified in Nestin-Cre mediated Egr1 conditional knockout mice. In addition, in males, the influence of Egr1cKO on pituitary gene expression may be overridden by the Nestin-Cre driver. Differential genes associated with Nestin-Cre driver were significantly enriched for genes related to growth factor activity and binding. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in neuronal cell lineage have distinct impacts on pituitary gene expression in a sex-specific manner.

Support: This work was supported by NIH grant T32OD028239, NS094574, MH120498, the Center for One Health Research at the Virginia-Maryland College of Veterinary Medicine and the Edward Via College of Osteopathic Medicine, and the Fralin Life Sciences Institute faculty development Jund for H X.  

P19 - Regulation of peripheral-derived immune cells in the remodeling of pre-existing pial collateral vessels following ischemic stroke 

J. Jing,  X. Wang, and M. Theus

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, VA

Stroke is the fifth leading cause of neurological morbidity and mortality in the United States. Approximately, 85% of which are diagnosed as ischemic resulting from arterial obstruction of the middle cerebral artery (MCA) in the pial surface of the brain. Pre­existing pial collateral vessels connect the MCA branches to the anterior or posterior arteries on both dorsal hemispheres, they represent vascular redundancies that help retrogradely re-supply cerebral blood flow to the ischemic tissue following vascular occlusion. Remodeling of pial collateral vessels (arteriogenesis) is crucial to restore blood perfusion into the ischemic penumbra and reduce tissue damage. Increased fluid shear stress induced by altered blood flow results in the activation of the endothelium and subsequent recruitment of peripheral-derived immune cells (PDis), which triggers the outward growth of collateral vessels. While little is known about the cell-type specific contribution of PDis in the remodeling process, previous results showed that peri-vascular macrophages are critical regulators of arteriogenesis. Our recent studies indicate that EphA4 receptor tyrosine kinase serves as a suppressor of arteriogenesis in ischemic stroke and limits the pro-resolving phenotype of monocyte/macrophages (MMs). The objective of our current study is to explore the novel role of EphA4 in regulating PDis-mediated pial collateral remodeling in a mouse model of permanent middle cerebral artery occlusion (pMCAO). Our initial findings, using bone marrow chimeric EphA4 knockout mice and the selective arteriole labeling technique, vessel painting, demonstrate that PDI-specific EphA4 negatively regulates arteriogenesis after pMCAO. We hypothesize that selective expression of EphA4 on PDI cells, specifically MMs, influences their recruitment and functions to restrict pial collateral remodeling, cerebral blood flow restoration and tissue protection following pMCAO. Overall, these studies will give an insightful understanding of EphA4 rn mediating PDI-mediated arteriogenesis after pMCAO and investigate if MMs-specific EphA4 in the peripheral circulation contributes to the acute neurovascular inflammation during arteriogenesis. 

Support: RO1 NS112541 

P21 - A Trp with Brucella abortus

Tristan Stoyanof, Clay Caswell 

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, VA

As one of the primary building blocks in biology, amino acids can quickly become a key asset for bacteria to infect a host. Brucella abortus, an intracellular pathogen, is able to synthesize tryptophan (Trp), but it is very energetically demanding to produce. In order to be efficient, Trp biosynthesis is repressed by a variety of methods when environmental Trp levels are above a certain threshold. In these conditions, transcriptional attenuation of trpE leads to a truncated mRNA product (rnTrpL) that has new regulatory functions, including the repression of the trpDC operon. In the closely-related bacterium Sinorhizobium meliloti, rnTrpL also regulates genes unrelated to Trp biosynthesis, including those involved with quorum sensing and efflux pumps. It is unknown what regulatory targets rnTrpL has in B. abortus, and few phenotypes have been observed when trpL is deleted. Despite a lack of virulence defects in both macrophage and mouse models of infection, a growth defect in the trpL mutant was observed when grown in a defined medium with very low Trp concentrations. Although this is counterintuitive to the proposed model, current work is focused on ensuring the trpL mutation did not affect trpE expression and defining the TrpL regulon via RNA sequencing. 

Support: NIH

P23 - MinION™ Next-Generation Sequencing for Rapid Identification and Distinction of 

PRRSV Wildtype and Vaccine Strains

Maria Buman Ruiz Diaz and Kevin K. Lahmers 

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, VA

Background: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a 15,000-base pair, single-stranded RNA virus with remarkable mutational ability. It has evaded routine control methods since it's discovery in the 1980s and is considered endemic in all US commercial swine herds, annually costing $641 million. PRRSV is classified into PRRSV-1 (European, EU) and PRRSV-2 (North American, NA) subtypes, with an indeterminate number of strains and lineages within each subtype. 

The disease is commonly diagnosed by clinical signs and either antibody ELISA tests or PCR tests using Open Reading Frame 5 (ORF5), notorious for its variability amongst strains. Antibody tests cannot distinguish between naturally infected pigs and vaccinated pigs, and PCR can only differentiate EU vs NA strains, but not between their corresponding vaccines. The goal of this study is to develop a novel diagnostic test using Oxford Nanopore Technology (ONT) MinION™ next-generation sequencing (NGS) to quickly and accurately detect individual strains of PRRSV in pigs regardless of vaccine status, and potentially identify multiple genotypes in a sample. 

MinION™ uses nanopore technology capable of sequencing 20 million base pairs or more within 24 hours. DNA samples are end-prepped with special targets that anchor to a gel and are ratcheted though a nanopore three nucleotides at a time, creating changes in the electrical current which are then interpreted by ONTs open-source software, Guppy™ , thus creating highly accurate reads. These nucleotide sequences are then aligned and searched with a genome sequence bank such as BLAST, to identify wild type strains, vaccine strains, or previously undescribed strains. 

Hypothesis: PCR amplification paired with ONT sequencing will be able to differentiate PRRSV-2 genotypes in mixed samples. 

Preliminary Conclusions: Primer pairs bound to cDNA, but not for the expected regions. We suspect this may be due to non-specific binding of the designed primers. However, we plan to continue testing with animal serum and decide if these primers may be useful for diagnostic testing. Many publications have cited the use of MinION™ sequencing for diagnostic testing, including field testing during the 2014 Ebola virus outbreak. Therefore, we presume Mini ON™ will be capable of detecting PCR amplicon samples and differentiating between strains within the same sample. 

P33 - PERSISTENCE OF SARCOCYSTIS NEU RONA, AND HISTOPATHOLOGIC CHANGES IN HORSES WITH EPM 

Lauren Helber 1, Alayna N. Hay 2, Bettina Wagner 3, Caroline M. Leeth 2, Tanya LeRoith 4, Thomas E. Cecere 4, Kevin K. Lahmers 4, Frank M. Andrews 5, Stephen R. Werre 6, Amy L. Johnson 1, Carol K. Clark 8, Nicola Pusterla 9, Stephen M. Reed 10, David S. Lindsay 4, Sandra D. Taylor 11, Krista E. Estell 12, Martin Furr 13, Robert J. Mackay 14, Fabio Del Piero 15, Mariano Carossino 15, Kacey Pandaleon 1, Savannah Weatherford 1, Sharon G. Witonsky 1

1 Department of Large Animal Clinical Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech Blacksburg, VA, USA.
2 Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, USA.
3 Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY, USA.
4 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA.
5 Equine Health Studies Program, Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.
6 Department of Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA.
7 Department of Clinical Studies, University of Pennsylvania, School of Veterinary Medicine, New Bolton Center, Kennett Square, PA, USA.
8 Peterson and Smith Equine Hospital, Ocala, FL, USA.
9 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA.
10 Rood and Riddle Equine Hospital, Lexington, KY, USA.
11 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA.
12 Department of Large Animal Clinical Sciences, Marion duPont Scott Equine Medical Center, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Leesburg, VA, USA.
13 Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, USA.
14 Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL, USA
15 Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA

EPM is a devastating neurologic disease in horses and neurologic deficits can persist long past acute disease. Until the underlying mechanisms of immunopathology and neuropathology are better elucidated, the ability for horses to recover and remain healthy is limited. Therefore, to unravel these mechanisms, the objective of this study was to define the immunopathologic changes associated with Sarcocystis neurona infection. EPM­ affected and control horses were enrolled based on our case definitions. Immunofluorescence, cytokine, and chemokine analysis, as well as immunohistochemistry (IHC) and polymerase chain reaction (PCR) for S. neurona were performed. S. neurona was identified in all EPM-affected horses (n=9), including those previously treated with anti­protozoal medications (n=4). S. neurona was identified by IHC at a significantly greater frequency than PCR. Horses chronically affected with EPM had increased degenerative histopathologic changes in their CNS compared to acutely affected horses. Degenerative changes including scarring, spheroid formation, and neuron degeneration. Based on our findings, we propose that S. neurona has the ability to persist even after antiprotozoal treatment, which may result in degenerative changes. Due to the limited case numbers with a diverse treatment history and chronicity of disease, larger studies are warranted to further define the immuno- and neuropathology associated with S. neurona infection, including assessing whether IHC for S. neurona is a more accurate diagnostic technique.

Support: IRC, VHIB 

P35 - Lansoprazole potentiates amphotericin B activity against multi-drug resistant Candida auris targeting cytochrome bc1 complex  

Ehab A. Salama 1, Yehia Elgammal 1, Aruna Wijeratne 2, Mohamed N. Seleem 1 

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA.
2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, United States 

Candida auris has emerged as a problematic fungal pathogen associated with high morbidities and mortalities. Amphotericin B is the most effective and broad-spectrum antifungal agent used for treatment of invasive fungal candidiasis with extremely rare resistance among clinical isolates. However, the clinical efficacy of this drug has been impacted recently with the emergence of C. auris which possessed extraordinary resistant profile against all available antifungal drugs, including amphotericin B. There is an urgent need for novel antifungal agents or co-drugs capable of restoring/enhancing the antifungal activity of amphotericin B and reducing its toxicity. In this study, by screening a panel of -3,400 FDA-approved drugs we identified the proton pump inhibitor, lansoprazole, as a potent enhancer for the activity of amphotericin B against C. auris. Lansoprazole exhibited potent synergistic interactions with amphotericin B against 18/20 (90%) C. auris isolates with ΣFICI ranged from 0.25 to 0.5. Proteome Integral Solubility Alteration (PISA) assay revealed that lansoprazole inhibits an essential target in the yeast cytochrome system (Rieske protein of the mitochondrial cytochrome bc1 complex) leading to increase in the oxidative stress in the fungal cells which consequently augment the oxidative damaging effect of amphotericin B on C. auris cells. The target was confirmed with the rotenone rescue assay and transcriptome sequencing (RNA-Seq) analysis. Most importantly, lansoprazole restored the in vivo efficacy of amphotericin Bin an immunocompromised mouse model, resulting in a 1.7-log (-98%) CFU reduction in the kidney burden of C. auris. In conclusion, our results identified lansoprazole as a potent enhancer to the antifungal activity of amphotericin B in addition to identification of mitochondrial cytochrome bcl as a novel drug target to overcome the antifungal resistance in C. auris.

Support: NIH 

P39 - Single Dose Pharmacokinetics of Pimobendan in Healthy Adult Horses  

Catherine Jula, Jennifer Davis, Harold McKenzie Ill  

Department of Large Animal Clinicial Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA

Background: Pimobendan is an inodilator approved for treatment of canine cardiac disease. The pharmacokinetics have not been investigated in horses. 

Hypothesis/Objectives: This study evaluated the pharmacokinetics of oral Vetmedin (V) in horses. Additional objectives were to determine the bioequivalence of compounded pimobendan capsules (C) and suspension (S) and the effects of sample site on plasma drug concentrations. Animals: Six privately-owned healthy horses (5 mares, 1 gelding). Methods: All horses received a single 0.5mg/kg dose of pimobendan via oral syringe. The initial two horses received C, S, or V using a crossover design with a minimum 1-week washout period. Samples were collected simultaneously from lateral thoracic and jugular catheters before and after drug administration at predetermined times. Differences between formulation and sample site were analyzed by one-way ANOVA. Four horses received (V) only with jugular samples collected. Analysis was by LC-MS/MS and noncompartmental pharmacokinetics for (V). Results: No significant differences were noted between formulations or sample site (P > 0.05). Concentrations in compounded formulations were 88% (S) and 90% (C) of label. For V, mean (±SD) maximum plasma concentration (Cmax) was 4.96 ± 2.13ng/mL at 2.17 ± 0.98hr, and area under the curve (AUC0-∞) was 22.1 ± 8.8hr*ng/mL. Concentration of the active metabolite (O-desmethylpimobendan) was below the limit of detection (0.07ng/mL) for all samples. Conclusions and clinical importance: At 0.5mg/kg PO, pimobendan plasma concentrations were considerably lower than reported in dogs. There was no evidence of oral transmucosal absorption. Pimobendan is poorly absorbed in horses, regardless of formulation, and appears unlikely to have clinical effects. 

Support: Virginia Tech Foundation  

P9 - The role of glial inflammatory mediators on neural dysregulation induced by SARS-­CoV-2 infection  

Brittany Heath 1,2, Kylene Kehn-Hall 1,2  

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
2 Center for Emerging, Zoonotic, and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA 

Neurological effects due to post-acute sequalae of SARS-CoV-2 infection (neuro-PASC) will impact public health spanning decades. Alterations in the central nervous system (CNS) have been observed in a subset of COVID-19 patients presenting with neurologic manifestations akin to Alzheimer's Disease and Parkinson's Disease. Millions of people worldwide are currently affected by a neurodegenerative disease with the number of cases expected to rise. The causal relationship of COVID-19 and neurodegenerative diseases remain to be determined, yet it is critical to promptly identify the mechanisms of neural dysfunction due to neuro-PASC. To address this gap, we aim to identify pathways in which SARS-CoV-2 infection leads to glial activation and subsequent neuronal injury and/or death. Here, we tested the direct infection of human astrocytes, microglia, and neuroblastoma cells by SARS-CoV-2. The release of infectious virus, absolute quantification of viral RNA, and fold changes in gene expression were measured. Astrocytes infected with SARS-CoV-2, maintained constant viral titers (PFU / mL) and intracellular viral RNA up to 48 hours post-infection (hpi). In contrast, microglia rapidly cleared SARS-CoV-2 infectious virus at 48hpi and intracellular viral RNA decreased at 24hpi. Next, we pre-screened cellular RNA for three markers of inflammation using RT­qPCR. CXCL10, IL1 , and IL6 were chosen due to the elevated circulating cytokines in patient serum, and due to their strong correlation with disease severity and clinical prognosis in COVID-19 patients. In astrocytes, significant increased expression of CXCL10 and IL6 was observed at 9hpi and 24hpi, respectively. Microglia exhibited significant upregulation of CXCL10 at 72hpi in absence of infectious virus. Direct infection of neuroblastoma cells with SARS-Co V-2 did not impact cell viability as determined by CellTiter-Glo. Our findings validate SARS-CoV-2 ability to modulate glial activity in the presence or absence of infectious virus. Ongoing studies aim to identify secreted inflammatory markers from SARS-CoV-2 infected glial cells and their relation to neuronal viability as determined by multiplex-ELISA and CellTiter-Glo, respectively.  

Support: Office of Research and Graduate Studies   

P11 - Single-Cell RNA Sequencing Reveals Disease Stage-Dependent Transcriptomic Profiles of Regulatory B Cells in Systemic Lupus Erythematosus

Razan Alajoleen 1, Andrea Daamen 2, Prakash R. Timilesena 3, Song Li 3, Peter E. Lipsky 2, Xin Luo 1   

1 Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States
2 RILITE Foundation, Charlottesville, VA, United States
3 School of Plant and Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States 

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with abnormal activation of immune cells. Regulatory B (Breg) cells are a B cell subset that negatively regulate immune responses via secretion of immunoregulatory cytokines interleukin (IL)-10, IL-35, and transforming growth factor (TGF)-B. We had previously shown that in lupus-prone MRL/lpr mice, pre-disease Breg cells were more potent in suppressing autoimmunity than active-disease Breg cells. In this study, using single-cell RNA (scRNA) sequencing, we profiled -10,000 Breg cells from female MRL/lpr mice at the pre-disease (6-8 weeks of age) vs. active-disease (10-12 weeks of age) stages. Based on known markers of Breg subsets, scRNA analysis identified respective clusters as transitional-2 marginal zone precursor B cells (T2-MZP), marginal zone B cells (MZB), transitional 1 B cells (T1), germinal center B cells (GC), and Bl B cells. Our data showed that the pre-disease Breg cells were predominantly T2-MZP and MZB cells. Follicular B cells outside the marginal zone, on the other hand, are significantly increased in the active-disease stage. Two long noncoding RNAs (lncRNAs), Malatl and Xist, were significantly increased in active-disease Breg cells, where the expression of Lglas3, Slpi, and Spp1 was also increased. Notably, active-disease Breg cells exhibited an exhausted phenotype with increased expression of T-bet (Tbx21). Collectively, our findings suggest that the SLE disease stage-dependent functions of Breg cells are regulated at the transcriptional level. In future experiments, we will determine whether the immunosuppressive functions of active-disease Breg cells can be restored by knocking down the differentially overexpressed genes.   

Support: NIH funding: 1R01AR073240 and 1R21AI156197   

P13 - A High-Fat Diet Promotes Lupus Nephritis in Lupus-Prone Mice in a Sex- and CX3CR1-Dependent Manner

Caitlin Armstrong 1,2, Ran Lu 1,2, Xavier Cabana-Puig 1, Teresa Southard 1, Tanya LeRoith 1, Michael Appiah 1, Fernando Gutierrez Garcia 1, Razan Alajoleen 1, James C. Testerman 1, Liwu Li 2, Christopher M. Reilly 3, Xin Luo 1  

1 Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA, USA 
2 Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA 
3 Edward Via College of Osteopathic Medicine, Blacksburg, VA, USA 

The pathogenesis of SLE, which affects multiple organs systems, is poorly understood. Lupus nephritis (LN) is a common complication of SLE that occurs when there is kidney involvement. SLE, like many autoimmune conditions, has a strong female sex bias of disease incidence. Previous work in our lab indicated a protective role of a chemokine receptor, CX3CR1, in the pathogenesis of SLE using female mice from a well-characterized murine model of SLE (MRL/lpr). Here, a littermate breeding strategy was used to generate male and female MRL/lpr mice with either Cx3crl+/+ or Cx3crl+/- genotypes. Animals from each sex/genotype category were fed either an inflammation-promoting high-fat diet (HFD) or a control diet (CD). We hypothesized that a HFD promotes LN in MRL/lpr mice in a sex-and CX3CR1-dependent manner. Weekly assessment of proteinuria levels revealed higher proteinuria levels in female Cx3crl+/- mice fed a HFD compared with diet- and genotype-matched controls. Proteinuria levels in male mice were unaffected by both Cx3crl +/- genotype and diet. Similarly, gross organ weights of both kidneys and renal lymph nodes were increased for female mice in the HFD diet condition compared with the diet-matched controls. No effect of Cx3crl+/- genotype was observed for either sex on either gross organ weight. Scoring of formalin-fixed, paraffin-embedded sections from the kidneys of female mice established an interactive effect of HFD diet and Cx3crl+/-genotype on multiple histopathology indices of glomerular and tubulointerstitial damage. Collectively, these data support our hypothesis. Additional work is needed to determine the underlying mechanism(s) driving the observed effects.

Support: NIH grants AR067418, AR073240, and HL163948. ID IGEP. BMVS

P15 - HDAC6 knockout mitigates pristane-induced lupus

Dao Xu and Christopher M. Reilly  

Department of Biomedical Sciences and Pathobiology and Edward Via College of Osteopathic Medicine 

Systemic lupus erythematosus (SLE) is an autoimmune disease often occurred in women. SLE is characterized by extreme production of pathogenic antibodies and overmany inflammation. Histone deacetylase 6 (HDAC6) belongs to class IIb histone deacetylase. It has been demonstrated that selective HDAC6 inhibitor suppresses inflammation in lupus­prone mice. It is known that C57BL/6 mice develop lupus-like disease following pristane injection. Here in this study, sex and age-matched wild type and HDAC6 knockout mice on the C57BL/6 background were selected and they were injected with 0.5 ml pristane or PBS intraperitoneally (i.p.) at 8 weeks of age. 10 days later, the mice were euthanized. At the endpoint, we measured body and spleen weight, collected the serum, and harvested peritoneal cells and splenocytes for flow cytometry. We found pristane treatment increased the spleen weight and there was no difference between WT mice and HDAC6 knockout mice. Moreover, there was no difference in T cell or B cell populations in the splenocytes as shown by flow cytometry results. Pristane treatment motivated the population of inflammatory monocytes ( CD11 b+Ly6C++) and neutrophils ( CD11 b +Ly6G+). The recruitment of these inflammatory monocytes and neutrophils in the peritoneum was significantly decreased in HDAC6 knockout mice compared to the WT mice. In addition, pristane treatment promoted the interferon (IFN) signature genes, including Isg15, Cxcl10, Irf9, Irf7, Mx1, and Oas 1a. qPCR results showed that these IFN signature genes were decreased in HDAC6 knockout mice compared to the WT mice. In brief, our study shows that HDAC6 knockout inhibits the recruitment of inflammatory monocytes and neutrophils in the peritoneum in early inflammation response to pristane. HDAC6 deletion also inhibited the expression of IFN signature genes after pristine stimulation. 

Support: NIH

P25 - Counteracting Asp erg illus species antifungal resistance via combinational therapies

Nicolas Burns, Ehab Salama, Mohamed Seleem   

Department of Biomedical Sciences and Pathobiology and Center for One Health Research

Aspergillus fumigatus annually contributes to more than 300,000 infections globally, with an average mortality of 65%. Invasive pulmonary infections arising from Aspergillus fumigatus are life-threatening fungal infections for immunosuppressed and cancer patients. However, the disease is much broader in scope, with cases of COVID-19 infections also containing aspergillus infections, raising the specter of more infections that are undiagnosed. Triazoles, such as voriconazole, itraconazole, posaconazole, are primary antifungals for first-line therapies for infections arising due to Aspergillus sp. Our predominant issue is the ever increasing incidence of broadly antifungal resistant strains of A. fumigatus. These incidents are and will continue to increase and trigger alarm due to treatment failure and high mortality. Mortality for these infections can range from 40-90%. In recognition of this threat alongside other fungal pathogens, the CDC has generated a watch list. Aspergillus fumigatus has been placed within the critical priority group due to its rate of occurrence, predation of the ill and rapidly growing resistance. Therefore, our group is searching for a co-drug capable of enhancing frontline antifungal potency. Here our group sought to determine the mechanisms behind interactions between HIV protease inhibitors and frontline azole drugs. We challenged various pathogenic and clinically important A. fumigatus isolates. Through this extensive investigation lopinavir was identified to work with powerful synergy when in tandem with itraconazole and posaconazole against A. fumigatus isolates. Lopinavir demonstrated synergistic relationships with itraconazole against 16 A. fumigatus isolates and 4 Aspergillus species, niger, flavus and brasiliensis; ΣFICI values were between 0.188 and 0.375. An indifference effect was noted, with an ΣFICI ranging from 0.53 to 1.125. Surprisingly, once lopinavir was in growth media with posaconazole, effective synergistic outcomes were determined to be present in 22 isolates, their ΣFICI range was 0.091 to 0.188; an indifference effect was noted in 3 azole-resistant isolates, 0.53 to 0.62. Furthermore, lopinavir has shown itself capable of re-sensitizing azole-resistant A. fumigatus isolates, 731 and 733 from the CDC. These results generate novel routes for further investigation and tenable translatable treatments that can drastically reduced the azole payload necessary to remove Aspergillus fumigatus from an infected host.

Support: NIAID

P27 - Using mouse coronavirus to determine the impact of obesity on coronavirus disease severity and identify biomarkers of severe disease outcome

Pallavi Rai, Jeffrey M. Marano, Sheryl Coutermarsh-Ott, James Weger-Lucarelli   

Department of Biomedical Sciences and Pathobiology 

Corona virus disease 2019 ( COVID-19) has been shown to cause more severe or even fatal disease in the elderly and individuals with co-morbidities. Obesity is an important co­morbidity impacting -13% adults globally and 43% adults in the U.S. and has been associated with increased risk of hospitalizations in COVID-19 patients. Current knowledge on the impact of co-morbidities is based on epidemiological evidence and/ or infections of non-natural hosts with SARS-CoV-2, but COVID-19 disease outcome is determined by complex host-virus interactions including host's immune response to the virus. Therefore, a more relevant model is needed to establish a causal link between co­morbidities and coronavirus disease severity, which can then be used to better understand coronavirus pathogenesis and to identify biomarkers associated with severe disease outcomes. To that end, we infected lean and diet-induced obese (DIO) mice with mouse hepatitis virus (MHV)-1, a mouse coronavirus, and found that obese mice had more severe disease in terms of weight loss, mortality, and lung histopathology. To identify biomarkers of severe disease, we performed RNA sequencing (RNASeq) analysis on blood samples collected at 2 days-post-infection (dpi) and these can be used in timely diagnosis and risk stratification of patients for effective utilization of health care resources. 

Support: NSF

P29 - Can deep learning (CNN) detect minimal residual disease (MRD) in dogs treated for lymphoma?

Christina Pacholec 1, Nikolaos Dervisis 2, Hehuang Xie 1, Kevin Lahmers 1, Priscila Beatriz da Silva Serpa 1, Kurt Zimmerman 1 

1 Department of Biomedical Sciences and Pathobiology 
2 Department of Small Animal Clinical Sciences 

The focus of this study is to demonstrate that a convoluted neural network (CNN), using cytology images from dogs undergoing treatment for lymphoma, can be used to establish minimal residual disease status similar to which is typically done using molecular testing.

Support: Veterinary Memorial Fund (VMF) and American Kennel Club (AKC) 

P31 - Ritonavir, a promising cost-effective amphotericin synergist against cryptococcal meningitis infection 

Naur Alkashef, Mohamed Seleem  

1 Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA

Cryptococcus neoformans causing life-threatening meningitis and represents an alarming global health threat associated with high mortality among immunocompromised individuals and HIV patients. The current arsenal of antifungal drugs to combat the growing problem of cryptococcus is very limited. Amphotericin Bis the front line treatment for cryptococcal meningitis. However, the treatment with amphotericin Bis commonly associated with severe adverse effects. In this study, we used the combinatorial approach to minimize the toxicity and to enhance the efficacy of amphotericin B against C. neoformans. We evaluate the HIV-protease inhibitor, ritonavir, as a potential co-drug to work synergistically and to enhance the effectiveness of amphotericin B treatment. Ritonavir exhibits a potent in-vitro synergistic interactions when combined with amphotericin B against 100% ( 15/ 15) of the tested C. neoformans isolates with a fractional inhibitory concentration index (ΣFICI) ranging from 0.07 to 0.31. Notably, the combination of ritonavir with amphotericin Bled to killing of all tested isolates within 3 hours as measured by time killing assays. As a part of the involved mechanistic study, ritonavir significantly interferes with glucose transport in C. neoformans reducing its uptake by 52%. These data highlight the potential of antifungal combination between amphotericin B and ritonavir to combat C. neoformans infections. Furthermore, these data will provide insight into the potential clinical usefulness of ritonavir because it is commonly administered in HIV-infected patients and cryptococcus is a leading cause of morbidity and mortality in those patients. 

Support: NIH

P41 - Functional Characterization of TgTKL 1 Kinase in Toxoplasma gondii Pathogenesis 

Dim a Hajj Ali and Rajshekhar Y. Gaji 

1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA

Toxoplasma gondii is an important human pathogen that causes miscarriage in pregnant women, blindness and cognitive impairment in newborn children. Available treatments are compromised by toxic side effects, inability to treat the chronic form of the disease, which urges the need to develop new therapeutic strategies. Therefore, identifying parasite's factors involved in pathogenesis could lead to finding new drug targets. Tyrosine Kinase­Like (TKL) family kinases are predicted to play critical roles in Toxoplasma's growth, and they are poorly studied. We focused on TgTKL1, and our studies showed that this kinase is important for parasite growth in vitro and virulence in vivo. TgTKL1 contains four different domains: RNI, Enhanced Disease Resistance 1 (EDR1) domain, kinase domain and Nuclear Localization Signal (NLS) motif and contributions of these domains to TgTKLl's functioning remains unknown. To define the role of these domains in TgTKL1's function, we generated different mutant strains using CRISPR-Cas9. To determine significance of kinase domain, we successfully generated a kinase mutant strain and interestingly, it displayed defects in growth and virulence like the null mutant. RNA seq analysis showed that invasion related genes are downregulated in the kinase mutant including TgSUB1 (subtilisin 1), a protease required for microneme proteins processing during host-cell invasion. Accordingly, TgTKL1 kinase mutant displayed impaired processing of micronemal proteins revealing that kinase activity is crucial for TgTKL1 function. Furthermore, we generated mutants in the NLS motif to determine if localization to the nucleus is critical for TgTKL1 function. Additionally, we are also in the process of generating RNI and EDR1 deletion mutants and once these strains are generated, they will be subjected to different phenotypic assays including growth, invasion, microneme secretion and virulence. Moreover, multiple approaches including quantitative phosphoproteomics, immunoprecipitation and proximity ligation assays will be used to understand the signaling pathways mediated by TgTKL1. 

Support: NIAID