Browsing by Author "LeRoith, Tanya"

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    Adenovirus transduction to express human ACE2 causes obesity-specific morbidity in mice, impeding studies on the effect of host nutritional status on SARS-CoV-2 pathogenesis
    Rai, Pallavi; Chuong, Christina; LeRoith, Tanya; Smyth, James W.; Panov, Julia; Levi, Moshe; Kehn-Hall, Kylene; Duggal, Nisha K.; Weger-Lucarelli, James (Elsevier, 2021-11-01)
    The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6 N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology or viral replication was observed between AdV transduced lean and obese groups, infected with SARS-CoV-2, but certain cytokines were expressed more significantly in infected obese mice compared to the lean ones. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection, as described here and elsewhere, may be inappropriate for nutrition studies.
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    Adrenocortical Challenge Response and Genomic Analyses in Scottish Terriers With Increased Alkaline Phosphate Activity
    Zimmerman, Kurt L.; Panciera, David L.; Hoeschele, Ina; Monroe, William E.; Todd, S. Michelle; Werre, Stephen R.; LeRoith, Tanya; Fecteau, Kellie; Lake, Bathilda B. (Frontiers, 2018-10-09)
    Scottish terriers (ST) frequently have increased serum alkaline phosphatase (ALP) of the steroid isoform. Many of these also have high serum concentrations of adrenal sex steroids. The study’s objective was to determine the cause of increased sex steroids in ST with increased ALP. Adrenal gland suppression and stimulation were compared by low dose dexamethasone (LDDS), human chorionic gonadotropin (HCG) and adrenocorticotropic hormone (ACTH) response tests. Resting plasma pituitary hormones were measured. Steroidogenesis-related mRNA expression was evaluated in six ST with increased ALP, eight dogs of other breeds with pituitary-dependent hyperadrenocorticism (HAC), and seven normal dogs. The genome-wide association of single nucleotide polymorphisms (SNP) with ALP activity was evaluated in 168 ST. ALP (reference interval 8–70 U/L) was high in all ST (1,054 U/L) and HAC (985 U/L) dogs. All HAC dogs and 2/8 ST had increased cortisol post-ACTH administration. All ST and 2/7 Normal dogs had increased sex steroids post-ACTH. ST and Normal dogs had similar post-challenge adrenal steroid profiles following LDDS and HCG. Surprisingly, mRNA of hydroxysteroid 17-beta dehydrogenase 2 (HSD17B2) was lower in ST and Normal dogs than HAC. HSD17B2 facilities metabolism of sex steroids. A SNP region was identified on chromosome 5 in proximity to HSD17B2 that correlated with increased serum ALP. ST in this study with increased ALP had a normal pituitary-adrenal axis in relationship to glucocorticoids and luteinizing hormone.We speculate the identified SNP and HSD17B2 gene may have a role in the pathogenesis of elevated sex steroids and ALP in ST.
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    Baculovirus stability in serum-free lyophilized and wet storage conditions
    Colandro, Michelle Elizabeth (Virginia Tech, 2013-09-10)
    The baculovirus expression vector system (BEVS) is an effective way to produce recombinant proteins for biopharmaceuticals. However baculovirus stocks are stored in subzero temperatures to maintain virus stability, and fetal bovine serum is commonly used in the storage solution. In an effort to lower transportation and storage costs, a storage formulation that can effectively store the baculovirus in above frozen temperatures without the use of FBS would be beneficial. In this study, DMSO, ethylene glycol, glycerol, sucrose, sorbitol, sucrose-phosphate, and sucrose-phosphate-glutamate were added to baculovirus stock at various concentrations to determine the most effective stabilizer for virus storage at 4°C. Of the seven additives studied, 1 M sorbitol most effectively preserved baculovirus stock over a period of 47 weeks stored in 4°C. Formulations that include sucrose, L-arginine, and Pluronic F68 were created to determine their effectiveness on virus stability in a freeze-dried state stored at room temperature. In a lyophilized state, 0.5 M sucrose maintained baculovirus stock stability after 5 weeks of storage. Lyophilized stocks not containing sucrose were no longer infective after 5 weeks.
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    Characterization of Atypical Hemolytic Ornithobacterium rhinotracheale Isolates and Comparison with the Normal Non-Hemolytic Phenotype
    Walters, Jessica Nicole (Virginia Tech, 2014-10-24)
    Ornithobacterium rhinotracheale (ORT) is a Gram-negative bacterium that causes respiratory disease in poultry characterized by rhinitis, tracheitis, and pneumonia with mortality averaging 2-3%. In the Shenandoah Valley of Virginia, the seroprevalence for ORT among turkey flocks as determined by enzyme-linked immunosorbent assay (ELISA) was found to be 70.9% (n=175). Additionally, the seroprevalence for hemorrhagic enteritis virus (vaccine induced), Bordetella avium, and paramyxovirus-1 was 100%, 74.8%, and 6.3% respectively. No significant interactions were detected. The type strain of ORT is characteristically non-hemolytic at least for 96 hours at 37°C on Columbia Blood Agar. In recent years, atypical isolates that rapidly produce hemolysis have been isolated with increasing frequency. A variety of in vitro tests were used to determine differences between representative isolates of the hemolytic (H) and non-hemolytic (NH) phenotypes. Findings suggest that the H isolate contains a 4 kb plasmid similar to that found in Reimerella anatipestifer. No plasmid was found in the NH isolate. Differences in growth characteristics and resistance to tetracyclines were also noted. No differences in proteins, biochemical characteristics or 16S rRNA sequences were found, the latter serving as confirmation that the isolates were both ORT. Embryo inoculation was used to assess virulence. No significant differences were observed and most embryos survived through to the day of hatch (pip) despite the fact that ORT could be re-isolated. In turkey poults however, the H phenotype did appear less virulent. A significant depression in weight gain was noted for birds inoculated intratracheally with the NH isolate at 7 days post-inoculation (dpi). NH inoculates also had significantly higher antibody levels on ELISA at 14 and 21 dpi and histopathological lesion scores for lung at 7, 14, and 21 dpi. The NH isolate could be re-isolated from NH-inoculated poults through 21 dpi; whereas the H isolate could only be re-isolated through 14 dpi. In conclusion, there are numerous differences between the NH and H isolates found in the field with the H isolate appearing less virulent and as such, making it a potential vaccine candidate. The phenotypic difference appears to correlate with this, but may not suffice to explain it.
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    Characterization of the Capsular Polysaccharide of Haemophilus parasuis and its Application in the Diagnosis and Prevention of Glasser's Disease
    Hyman, Anne Catherine Michalenka (Virginia Tech, 2015-04-20)
    Haemophilus parasuis is a Gram-negative bacterium responsible for Glasser's Disease in pigs, though little is known regarding its antigenic or virulence factors. Our goals were to characterize the H. parasuis capsular polysaccharide (CP), determine its role in serotype-specificity and virulence, determine if CP is immunogenic, and develop diagnostic and protective products to prevent rampant H. parasuis infection within swine herds. Material from H. parasuis was purified using carbohydrate isolation techniques and compared to CPs from other Pasteurellaceae. Rabbits were immunized with CPs to generate antisera for microscopy, immunoassays, and bactericidal assays. CP antisera were conjugated to latex particles to create an agglutination assay for detection and typing of H. parasuis. CP was conjugated to Cholera Toxin B, and used to immunize mice and piglets before challenge with H. parasuis to determine its protective efficacy against Glasser's Disease. Broth-grown cells expressed CP, which reacted with antisera in microscopy and immunoassays. Broth-grown H. parasuis cells were serum-resistant unless homologous anti-CP serum was present. In contrast, agar-grown cells did not react with antisera in immunoassays, and cells were susceptible to killing by normal swine serum. CP was not expressed on the surface of agar-grown cells unless supplemented with bicarbonate. The addition of bicarbonate also contributed to the variability in CP quantity and upregulation of genes in the CP locus. Sensitized latex particles agglutinated strongest with homologous H. parasuis CPs, cells, and agar-grown cell lysates, but also reacted weakly with higher concentrations of heterologous CPs. The latex beads did not agglutinate with non-H. parasuis swine bacterial pathogens. Mice immunized with the CP-CTB conjugate produced a significantly higher IgG2/Th2 response than unimmunized mice or mice immunized with only CP, and immunized mice had fewer bacteria in their tissues that unimmunized mice. The CP conjugate produced a robust IgG antibody response to CP when used to immunized piglets, but because the control animals also survived H. parasuis challenge, the protective efficacy remains inconclusive. Therefore, the H. parasuis CP is the antigen that confers serotype identity, and can be implemented in methods and help direct future research in disease prevention and serotype tracking in H. parasuis infections.
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    Conjugated Linoleic Acid in the treatment of murine autoimmune glomerulonephritis
    Hammond, Sarah Elizabeth (Virginia Tech, 2015-10-15)
    Conjugated linoleic acid (CLA) has been shown to reduce inflammation via Peroxisome Proliferator-Activated Receptor (PPAR)-γ in inflammatory disorders such as Crohn's Disease and Inflammatory Bowel Disease. We sought to determine whether CLA isomers would reduce inflammation via PPAR-γ in cultured mesangial cells, and in murine models of anti-glomerular basement membrane (anti-GBM) glomerulonephritis and Systemic Lupus Erythematosus (SLE). SV40-transformed mouse mesangial cells (MES13) were cultured with pure CLA isomers (c9,t11 or t10,c12-CLA or a 50:50 mixture prior to immune stimulation with lipopolysaccharide and interferon-γ. Next, cultured mesangial cells were transfected with small interfering RNA (siRNA) targeting PPAR-γ and treated with CLA isomers prior to immune stimulation. ELISA, qPCR, Western blot, and Griess reaction were performed to measure cytokine production, mRNA expression, induced nitric oxide synthase (iNOS) and nitrite production, respectively. Next, myeloid-specific (LysM creR2+) PPAR-γ knockout mice were treated with CLA prior to the induction of anti-GBM glomerulonephritis and evaluated for disease. Finally, NZM2410/J mice (a natural model of SLE) were treated with c9,t11-CLA and evaluated for disease progression. Treatment with CLA reduced IL-6 production in cultured mesangial cells, but not in siRNA-treated mesangial cells, supporting a PPAR-γ-mediated mechanism. CLA treatment increased both Transforming Growth Factor (TGF-β) and Interleukin-1 Receptor Antagonist (IL-1RA) mRNA expression independent of PPAR--γ. While CLA treatment reduced nitrite production and iNOS production to some degree, this was an inconsistent finding. Conversely, in the induced anti-GBM mouse model, CLA treatment increased mesangial cell IL-6 mRNA expression, reduced TGF-β expression, and had no effect on IL-1RA. Moreover, NZM2410/J mice that were fed a c9,t11-CLA-supplemented diet had reduced survival times, increased renal inflammation and increased serum IgG2a relative to controls. Taken together, these studies indicate that the in vitro MES13 cell line does not translate to the in vivo mouse model of anti-GBM induced glomerulonephritis. Furthermore, while CLA may have beneficial effects in other mouse models, it worsens disease in NZM2410/J mice. Findings from these models should be interpreted with caution.
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    Cross-protection and Potential Animal Reservoir of the Hepatitis E Virus
    Sanford, Brenton Joel (Virginia Tech, 2012-05-29)
    HEV is an important public health concern due largely to water-borne outbreak. Recent research confirms individual cases of zoonotic transmission due to human exposure to contaminated animal meats. At least four recognized and two putative genotypes of mammalian HEV have been reported: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 are zoonotic. In addition to humans, strains of HEV have been genetically identified from pigs, chickens, rats, mongoose, deer, rabbits and fish. The current experimental vaccines are all based on a single strain of HEV, even though multiple genotypes of HEV are co-circulating in some countries and thus an individual may be exposed to more than one genotype. Therefore, it is important to know if prior infection with a genotype 3 swine HEV will confer protective immunity against subsequent exposure to genotypes 3 and 4 human and swine HEV. In the first study, specific-pathogen-free pigs were divided into 4 groups of 6 each. Pigs in the three treatment groups were each inoculated with a genotype 3 swine HEV, and 12 weeks later, challenged with the same genotype 3 swine HEV, a genotype 3 human HEV, and a genotype 4 human HEV, respectively. Sera from all pigs were tested for HEV RNA and IgG anti-HEV, and fecal samples were also tested for HEV RNA each week. The pigs inoculated with swine HEV became infected as evidenced by fecal virus shedding and viremia, and the majority of pigs also developed IgG anti-HEV prior to challenge at 12 weeks post-inoculation. After challenge, viremia and fecal virus shedding of challenge viruses were not detected, suggesting that prior infection with a genotype 3 swine HEV prevented pigs from developing viremia and fecal virus shedding after challenge with homologous and heterologous genotypes 3 and 4 HEV, respectively. Immunogenic epitopes are located within the open reading frame 2 (ORF 2) capsid protein and recombinant ORF 2 antigens are capable of preventing HEV infection in non-human primates and chickens. In the second study we expressed and purified N-truncated ORF 2 antigens based on swine, rat, and avian HEV strains. Thirty pigs were randomly divided into groups of 6 pigs each and initially vaccinated with 200µg swine ORF 2 antigen, rat ORF 2 antigen, avian ORF 2 antigen, or PBS buffer (positive and negative control groups) and booster with the same vaccine 2 weeks later. At 4 wks, after confirming seroconversion to IgG anti-HEV antibody with ELISA, all groups except the negative control were challenged with swine genotype 3 HEV (administered intravenously). The protective and cross-protective abilities of these antigens were determined following swine genotype 3 challenge by evaluating both serum and fecal samples for HEV RNA using nested RT-PCR and IgG anti-HEV using ELISA. The results from these two studies have important implications for future development of an effective HEV vaccine. As a part of our ongoing efforts to search for potential animal reservoirs for HEV, we tested goats from Virginia for evidence of HEV infection and showed that 16% (13/80) of goat sera from Virginia herds were positive for IgG anti-HEV. Importantly, we demonstrated that selected goat sera were capable of neutralizing HEV in cell culture. Subsequently, in an attempt to genetically identify the HEV-related agent from goats, we conducted a prospective study in a closed goat herd with known anti-HEV seropositivity and monitored a total of 11 kids from the time of birth until 14 weeks of age for evidence of HEV infection. Seroconversion to IgG anti-HEV was detected in 7 out of the 11 kids, although repeated attempts to detect HEV RNA by a broad-spectrum nested RT-PCR from the fecal and serum samples of the goats that had seroconverted were unsuccessful. In addition, we also attempted to experimentally infect laboratory goats with three well-characterized mammalian strains of HEV but with no success. The results indicate that a HEV-related agent is circulating and maintained in the goat population in Virginia and that the goat HEV is likely genetically very divergent from the known HEV strains.
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    Determining ideal staple size for small intestinal surgery in cats
    Hiebert, Elizabeth C. (Virginia Tech, 2022-03-08)
    Background: The use of stapling equipment for intestinal surgery in cats is rarely reported, and appropriate staple sizes for cat intestine are unknown. Objective: To determine staple cartridge sizes for thoracoabdominal (TA) and endoscopic gastrointestinal anastomosis (EndoGIA) that will simultaneously prevent leakage of small intestinal contents while also allowing for sufficient vascular permeability past the staple lines for intestinal healing. Methods: Two sizes of EndoGIA cartridges (2.0/2.5/3.0mm and 3.0/3.5/4.0mm) and two sizes of TA cartridges (2.5mm and 3.5mm), applied in a transverse manner across fresh cadaveric cat jejunum, were evaluated via intestinal burst pressure testing for maximum intraluminal pressure prior to leaking, and via infusion of an intravascular dye at normal arterial pressures to determine percentage of vascular patency past the staple lines. Vascular patency was compared not only from pre-and post-staple segments of the same intestinal sample, but also EndoGIA vascular patency was evaluated against TA vascular patency. Results: Two cats met study criteria. All samples had intraluminal burst pressures over twice the chosen minimum (of 30mmHg). Vascular patency post- staple line ranged from 0-90.8%, with the most consistently high numbers noted with the TA 3.5mm cartridges. No EndoGIA cartridge had a post- staple line vascular patency higher than 31.1%, and no intravascular dye was noted in any post- staple line sample in the EndoGIA 2.0/2.5/3.0mm group. Conclusions: While statistical analysis of the dataset was unable to be performed due to low numbers of samples for comparison, both intestinal intraluminal burst pressure trends and intravascular dye patterns suggested both the TA 3.5mm cartridge and (to a lesser extent) the 3.0/3.5/4.0mm EndoGIA cartridge could provide the ideal combination of intraluminal seal without restriction of vascular access for healing. The intravascular dye infusion technique, developed during this research, shows promise as a future instrument to determine vascular patterns around intestinal implants in cadaveric cat specimens.
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    Determining the Pathogenesis and Enzootic Transmission of Usutu Virus
    Kuchinsky, Sarah (Virginia Tech, 2022-09-02)
    Usutu virus (USUV) is an emerging zoonotic virus within the Flaviviridae family that can cause neurological disease in humans and wild birds. USUV is maintained in an enzootic cycle between wild birds, primarily passerine species, and ornithophilic mosquitoes, predominantly Culex spp. mosquitoes. Since its first isolation in 1959 in South Africa, USUV has spread throughout sub-Saharan Africa and Europe. Its emergence into Europe was marked by large die-offs, or epizootics, of the Eurasian blackbird (Turdus merula), as well as an increase in human cases. This dissertation sought to understand whether USUV has evolved to become more pathogenic in humans or transmissible in birds. We compared the pathogenesis of five different USUV isolates, four recent isolates: Spain 2009, Netherlands 2016, Senegal 2003, Uganda 2012, and South Africa 1959, in an interferon α/β receptor knockout (Ifnar-/-) mouse model. We observed significant mortality, high viral levels in serum and tissues in all USUV strains except for the Netherlands 2016 strain. Eighteen non-synonymous mutations were identified throughout the genome of Netherlands 2016 strain compared to the other USUV isolates. To further understand USUV infection in wild birds, we developed a physiologically relevant model of infection using juvenile chickens. In juvenile chickens, we found that the European strains were characterized by more pathogenesis and higher viral titers in tissues compared to the African strains. This work established the first viremic bird model of USUV infection. Passerine birds have been suggested to be important for USUV maintenance, however a species competent for transmission has not been identified. We first determined that wild-caught house sparrows (Passer domesticus) and Culex quinquefasciatus mosquitoes were susceptible to Netherlands 2016 and Uganda 2012 USUV strains. Following an infectious feed to assess enzootic transmission, house sparrows were able to transmit both USUV strains to Cx. quinquefasciatus mosquitoes, with the Netherlands 2016 strain being more infectious compared to the Uganda 2012 strain. The collection of these chapters provides great insights on the pathogenesis of distinct USUV strains, disease presentation in birds, and enzootic transmssion of USUV. Additionally, they indicate that USUV emergence in the United States is entirely feasible.
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    Development and validation of a negative-strand-specific reverse transcription-PCR assay for detection of a chicken strain of hepatitis E virus: Identification of nonliver replication sites
    Billam, P.; Pierson, F. W.; Li, W.; LeRoith, Tanya; Duncan, R. B.; Meng, Xiang-Jin (American Society for Microbiology, 2008-06-18)
    As a positive-strand RNA virus, hepatitis E virus ( HEV) produces an intermediate negative-strand RNA when it replicates. Thus, the detection of negative-strand viral RNA is indicative of HEV replication. The objective of this study was to develop a negative-strand-specific reverse transcription-PCR ( RT-PCR) assay for the identification of extrahepatic sites of HEV replication. Briefly, a 494-bp fragment within the orf1 gene of a chicken strain of HEV ( designated avian HEV) was amplified and cloned into a pSK plasmid. A synthetic negative-strand viral RNA was generated from the plasmid by in vitro transcription and was used to standardize the assay. A nested set of primers was designed to amplify a 232-bp fragment of the negative-strand viral RNA. The assay was found to detect up to 10 pg and 10(-5) pg of negative-strand HEV RNA in first- and second-round PCRs, respectively. The standardized negative-strand-specific RT-PCR assay was subsequently used to test 13 conveniently obtained tissue specimens collected sequentially on different days postinoculation from chickens experimentally infected with avian HEV. In addition to the liver, the negative-strand-specific RT-PCR assay identified replicative viral RNA in gastrointestinal tissues, including the colorectal, cecal, jejunal, ileal, duodenal, and cecal tonsil tissues. The detection of replicative viral RNA in these tissues indicates that after oral ingestion of the virus, HEV replicates in the gastrointestinal tract before it reaches the liver. This is the first report on the identification of extrahepatic sites of HEV replication in animals after experimental infection via the natural route. The assay should be of value for studying HEV replication and pathogenesis.
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    Development of subunit vaccines against porcine reproductive and respiratory syndrome virus (PRRSV)
    Hu, Jianzhong (Virginia Tech, 2012-08-06)
    Since emerging in Europe and the US, PRRS has spread globally and become the most significant infectious disease currently devastating the swine industry. In the US alone, the economic losses caused by this disease amount to more than 560 million US dollars every year. Modified-live PRRSV vaccines (MLV) are the most effective option currently available for the control of the disease. MLVs can confer solid protection against homologous re-infection and have significant effects in reducing viral shedding. But the vaccine efficacy varies upon heterologous challenge. None of the current vaccines are able to completely prevent respiratory infection, transplacental transmission, as well as pig-to-pig transmission of the virus. More importantly, the intrinsic risk of MLV vaccine to revert to virulent virus under farm conditions poses a great safety concern. The unsatisfactory efficacy and safety of current PRRSV vaccines drives the continuous efforts of developing a new generation of vaccines. The strategy we focus on for novel PRRSV vaccine development is subunit vaccine. The reasons for choosing this strategy are: 1) subunit vaccines only contain the immunogenic fragments of a pathogen. Administration of such pathogen fragments eliminates the risk of pathogens reverting back to their virulent form as in the case of modified live vaccines. 2) Subunit vaccines have advantages in terms of vaccine production since a well-defined pathogen fragment can more easily be produced consistently. To achieve of our goal of developing safe and efficacious subunit vaccines against PRRSV, three projects were completed. First, a scalable process for purification of PRRSV particles from cell culture was developed. This process produced purified viral particles for ELISA and cell-based assays used in vaccine development. Second, a plant-made oral subunit vaccine against PRRSV was developed. Administration of the plant-made vaccine, the vaccinated animals produced virus-specific serum and intestine mucosal antibodies with neutralization activity, as well as cellular immune responses with a preference of virus-specific IFN-γ production. Since neutralization antibodies and virus-specific IFN-γ response are the crucial factors contributing to protection against PRRSV infection, the plant-made oral subunit vaccine strategy is an attractive strategy for developing a new generation of the vaccine to control PRRS disease. Third, a chimeric protein consisting of the ectodomains of viral M and GP5 proteins was expressed and purified. The protein product showed a single band on a silver-stained gel and contained an endotoxin level of less than 10 EU/mg protein. In addition, the purified protein showed expected bioactivities. It was antigenic, could bind to a cellular receptor for the virus (heparan sulfate), and could block virus infection of susceptible cells. Therefore, the chimeric protein is a promising subunit vaccine candidate against PRRSV.
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    Diagnostic accuracy of stereotactic brain biopsy for intracranial neoplasia in dogs: Comparison of biopsy, surgical resection, and necropsy specimens
    Kani, Yukitaka; Cecere, Thomas E.; Lahmers, Kevin K.; LeRoith, Tanya; Zimmerman, Kurt L.; Isom, Scott; Hsu, Fang-Chi; Debinski, Waldemar; Robertson, John L.; Rossmeisl, John H. Jr. (American College of Veterinary Internal Medicine, 2019-05)
    Background Stereotactic brain biopsy (SBB) is a technique that allows for definitive diagnosis of brain lesions. Little information is available regarding the diagnostic utility of SBB in dogs with intracranial diseases. Objective To investigate the diagnostic accuracy (DA) of SBB in dogs with brain tumors. Animals Thirty-one client-owned dogs that underwent SBB followed by surgical resection or necropsy examinations. Methods Retrospective observational study. Two pathologists blinded to SBB and reference standard diagnoses reviewed histologic specimens and typed and graded tumors according to World Health Organization and revised canine glioma classification criteria. Agreement between tumor type and grade from SBB were compared to reference standards and assessed using kappa statistics. Patient and technical factors associated with agreement also were examined. Results Stereotactic brain biopsy specimens were obtained from 24 dogs with gliomas and 7 with meningiomas. Tumor type agreement between SBB and the reference standard was observed in 30/31 cases (kappa = 0.95). Diagnostic concordance was perfect for meningiomas. Grade agreement among gliomas was observed in 18/23 cases (kappa = 0.47). Stereotactic brain biopsy underrepresented the reference standard glioma grade in cases with disagreement. The DA of SBB was 81%, with agreement noted in 56/69 biopsy samples. Smaller tumors and fewer SBB specimens obtained were significantly associated with diagnostic discordance. Conclusions and Clinical Importance The DA of SBB readily allows for the diagnosis of common brain tumors in dogs. Although glioma grade discordance was frequent, diagnoses obtained from SBB are sufficient to currently inform therapeutic decisions. Multiple SBB specimens should be collected to maximize DA.
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    Dietary epicatechin improves survival and delays skeletal muscle degeneration in aged mice
    Si, Hongwei; Wang, Xiaoyong; Zhang, Longyun; Parnell, Laurence D.; Admed, Bulbul; LeRoith, Tanya; Ansah, Twum-Ampofo; Zhang, Lijuan; Li, Jianwei; Ordovas, Jose M.; Si, Hongzong; Liu, Dongmin; Lai, Chao-Qiang (2019-01)
    We recently reported that epicatechin, a bioactive compound that occurs naturally in various common foods, promoted general health and survival of obese diabetic mice. It remains to be determined whether epicatechin extends health span and delays the process of aging. In the present study, epicatechin or its analogue epigallocatechin gallate (EGCG) (0.25% w/v in drinking water) was administered to 20-mo-old male C57BL mice fed a standard chow. The goal was to determine the antiaging effect. The results showed that supplementation with epicatechin for 37 wk strikingly increased the survival rate from 39 to 69%, whereas EGCG had no significant effect. Consistently, epicatechin improved physical activity, delayed degeneration of skeletal muscle (quadriceps), and shifted the profiles of the serum metabolites and skeletal muscle general mRNA expressions in aging mice toward the profiles observed in young mice. In particular, we found that dietary epicatechin significantly reversed age-altered mRNA and protein expressions of extracellular matrix and peroxisome proliferator-activated receptor pathways in skeletal muscle, and reversed the age-induced declines of the nicotinate and nicotinamide pathway both in serum and skeletal muscle. The present study provides evidence that epicatechin supplementation can exert an antiaging effect, including an increase in survival, an attenuation of the aging-related deterioration of skeletal muscles, and a protection against the aging-related decline in nicotinate and nicotinamide metabolism.Si, H., Wang, X., Zhang, L., Parnell, L. D., Admed, B., LeRoith, T., Ansah, T.-A., Zhang, L., Li, J., Ordovas, J. M., Si, H., Liu, D., Lai, C.-Q. Dietary epicatechin improves survival and delays skeletal muscle degeneration in aged mice.
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    Discovery of Novel Strains of Animal Hepatitis E Viruses in the United States: Antigenic and Genetic Characterization, Cross-Species Infection, and Public Health Implications
    Cossaboom, Caitlin Marie (Virginia Tech, 2015-03-17)
    Hepatitis E virus (HEV) is an important human pathogen, with pigs and likely other animal species serving as natural reservoirs. There are currently four recognized HEV genotypes that infect humans within the genus Hepevirus of the family Hepeviridae. Genotypes 1 and 2 are human viruses that are associated with waterborne and fecal-oral transmission in developing countries, while genotypes 3 and 4 have been identified in humans and other animal species and are zoonotic and endemic in both industrialized and developing countries. In my dissertation research, we identified the first strain of HEV from rabbits in the United States. We subsequently determined the complete genome sequence of the virus. Phylogenetic analyses of the full-length sequence indicated that U.S. rabbit HEV is a distant member of the zoonotic genotype 3, thus raising a potential concern for zoonotic infection. In order to investigate the cross-species potential of rabbit HEV, we then determined its antigenic cross-reactivity with other animal strains of HEV. Additionally, we demonstrated that the novel rabbit HEV could cross species barriers and infect pigs under experimental conditions. Finally, we attempted to determine the risk factors and sources of foodborne HEV infection in the United States. We detected HEV for the first time from non-liver pork commercial products in the United States and demonstrated consumption of undercooked meat a risk factor for HEV infection. HEV sequences of genotype 3 origin were detected from pork products purchased from grocery stores in Southwest Virginia. Approximately 6.3% (21/335) of university students tested seropositive for HEV antibodies and, importantly, those with a history of consuming undercooked meats were 13 times more likely to be seropositive. These results further underscore the importance of cooking pork thoroughly and using proper hygiene when preparing meals.
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    Dynamic Programming of Innate Immunity in Health and Disease
    Yuan, Ruoxi (Virginia Tech, 2016-11-02)
    Whether innate immune cells may be adapted into potential memory states has becoming an important question in the field of immunity. Although previous conceptual paradigm failed to acknowledge this important question, emerging clinical and basic observations have started to shed intriguing clues to shake the previous dogma regarding innate immunity of being "simple", "raw", "first-line defense with no memory". We have aimed to further address this fundamental issue in this dissertation work, under the close guidance of Dr. Liwu Li. We have chosen to use the model system of Toll-Like-Receptor (TLR) signaling networks within primary monocytes. TLRs play fundamental roles in sensing pathogen-associated molecular patterns (PAMPs) and modulation of innate immunity. Lipopolysaccharide (LPS), an endotoxin found on the cell membrane of gram-negative bacteria, is the ligand of TLR4 and induces a range of inflammatory as well as anti-inflammatory responses. Higher dosages of LPS were known to cause robust yet transient expression of pro-inflammatory mediators. On the other hand, the effects of super-low dose LPS, commonly manifested in humans with adverse health conditions, have been largely ignored in the basic research field. Super-low dose LPS may skew host immune environment into a mild non-resolving pro-inflammatory state, which is a risk factor for inflammatory diseases such as atherosclerosis, compromised wound healing, and elevated risks for sepsis. Our central hypothesize is that monocytes may be adapted by super-low dose LPS into a non-resolving low-grade inflammatory state conducive for the pathogenesis of inflammatory diseases. We have employed both in vitro cell culture system as well as in vivo disease models to test this hypothesis. For the in vitro system, we have cultured primary murine monocytes with increasing signal strength of LPS. Monocyte phenotypes such as the expression of key inflammatory mediators including cytokines, chemokines, and cellular surface markers were studied. Potential molecular and cellular mechanisms were examined. We revealed a novel low-grade inflammatory monocyte phenotype termed ML adapted by super-low dose LPS, mediated through IRF5. For the in vivo system, we have employed both acute and chronic models of inflammation. For the chronic model, we have tested the effects of super-low dose LPS on monocyte polarization in vivo, as well as its contribution to the pathogenesis of atherosclerosis. Furthermore, we have tested the effects of programmed monocytes on wound healing. For the acute model, we have tested the effects of pre-conditioning with super-low dose LPS on the subsequence risks of sepsis elicited by cecal ligation and puncture. We have demonstrated aggravated atherosclerosis, compromised wound healing, and increased sepsis mortality in mice pre-conditioned with super-low dose LPS. Taken together, our findings reveal that monocytes can be differentially programmed into distinct states, depending on the signal strength of LPS. The differential programming and adaptation of monocytes can occur both in vitro and in vivo, and may bear profound pathological consequences.
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    Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.
    Rothschild, Daniel E.; Zhang, Yao; Diao, Na; Lee, Christina K.; Chen, Keqiang; Caswell, Clayton C.; Slade, Daniel J.; Helm, Richard F.; LeRoith, Tanya; Li, Liwu; Allen, Irving C. (2016-12-03)
    Aberrant inflammation is a hallmark of inflammatory bowel disease (IBD) and colorectal cancer. IRAK-M is a critical negative regulator of TLR signaling and overzealous inflammation. Here we utilize data from human studies and Irak-m(-/-) mice to elucidate the role of IRAK-M in the modulation of gastrointestinal immune system homeostasis. In human patients, IRAK-M expression is up-regulated during IBD and colorectal cancer. Further functional studies in mice revealed that Irak-m(-/-) animals are protected against colitis and colitis associated tumorigenesis. Mechanistically, our data revealed that the gastrointestinal immune system of Irak-m(-/-) mice is highly efficient at eliminating microbial translocation following epithelial barrier damage. This attenuation of pathogenesis is associated with expanded areas of gastrointestinal associated lymphoid tissue (GALT), increased neutrophil migration, and enhanced T-cell recruitment. Further evaluation of Irak-m(-/-) mice revealed a splice variant that robustly activates NF-κB signaling. Together, these data identify IRAK-M as a potential target for future therapeutic intervention.
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    Eph-mediated restriction of cerebrovascular arteriogenesis
    Okyere, Benjamin (Virginia Tech, 2019-04-26)
    Stroke is a leading cause of morbidity and long-term neurological disability in the U.S. Ischemic stroke, which accounts for approximately 90% of all strokes, is the result of an occlusion in the arteriole cerebrovascular network. No effective treatment options exist to provide neuroprotection from occlusion, and limited success has been seen clinically when attempting to restore blood flow to vulnerable neural tissue regions. Enhancement of pial collateral remodeling (Arteriogenesis) has recently been shown to improve blood flow and mitigate neural tissue damage following stroke (1-3). Arteriogenesis is the remodeling of pre-existing arteriole vessel which are able to re-route blood to blood-deprived regions of tissue. Arteriogenesis requires endothelial cell (EC) and smooth muscle cell proliferation, extracellular matrix degradation and recruitment of circulating bone marrow-derived cells (4-6). Unlike spouting angiogenesis, which requires weeks following occlusion to develop, arteriogenesis begins as early as 24-48hrs post-stroke (7, 8) and can expeditiously enhance blood flow to ischemic regions, making it an attractive target for therapeutic intervention. Our preliminary studies, in an EphA4 global knockout mouse model, indicated that EphA4 receptor tyrosine kinase severely limits pial arteriole collateral formation. The preliminary work also showed that activation of EC EphA4 receptor in vitro inhibited vascular formation. Additionally, ECs lining the collateral vessel have been shown to play a role in collateral remodeling (9). Taken together, the objective of this dissertation was to elucidate the cell autonomous role of the EphA4 receptor and given the central role of the EC in collateral remodeling, we postulated that EphA4 receptor on ECs the limits pial collateral formations. Using a cell-specific loss-of-function approach, we tested the hypothesis that EC-specific EphA4 plays an important role in pial collateral development and remodeling after induced stroke. The results from this dissertation show that (1) EphA4 expression on ECs suppress the formation of pial collaterals during development and limits EC growth via suppression of p-Akt in vitro (2) EC-specific EphA4 ablation leads to increased collateral remodeling, enhanced blood flow recovery, tissue protection and improved neurological behavioral outcomes after stroke and (3) Mechanistically, EphA4 limits pial collateral remodeling via attenuation of the Tie2/Angiopoietin-2 signaling pathway. The work presented in this dissertation demonstrate that EphA4 can be targeted therapeutically to increase pial collateral remodeling to alleviate neurological deficits after ischemic stroke.
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    Epidemiology and Pathophysiology of Clostridial Dermatitis (Cellulitis) in Turkeys
    Lighty, Megan Elizabeth Folk (Virginia Tech, 2015-10-01)
    Clostridial dermatitis (CD) is a multifactorial disease of rapidly-growing turkeys. Clostridium septicum (Cs) has been identified as the primary cause, although C. perfringens (Cp) has also been implicated. Pathogenesis is not fully understood; however, it is hypothesized that Clostridia translocate from the gastrointestinal tract and spread hematogenously to capillary beds of skeletal muscles. Intense genetic selection has produced a rapidly growing bird that is heavier and less active. This may predispose birds to development of CD due to positional restriction of blood flow to the caudal breast and medial thigh. Subsequent reduction in oxygen tension within these tissues produces conditions conducive to germination, proliferation, and toxin production by previously trapped, non-replicative Clostridia. Studies were undertaken to investigate the epidemiology and pathophysiology of CD. Retrospective epidemiologic investigations evaluated incidence, risk factors, and economic impact of CD. Cs and Cp qPCR were performed on blood and tissue samples to demonstrate hematogenous spread in asymptomatic birds. Studies assessed the effect of prolonged recumbency by measuring oxygen saturation and surface temperature in dependent tissues. Tissues from CD cases were evaluated for Cs and Cp alpha toxin mRNA (CsA and CpA). Analyses were conducted to determine associations between these toxins and severity of histopathologic lesions. Whole genome sequencing was performed on the Cs type strain to identify other toxin genes. Flock type, breed, weight at time of processing, and stocking density affected disease incidence. Detection of Clostridium spp. in intestine, liver, and muscle from asymptomatic turkeys without cutaneous trauma implies hematogenous spread from an endogenous source. Focal polyphasic myonecrosis in dependent muscles of asymptomatic turkeys suggests an underlying predisposition to development of CD. Recumbency appeared to be associated with decreased perfusion to these tissues. Cs DNA was present in asymptomatic birds without corresponding CsA mRNA expression suggesting that organisms were present in a quiescent form. CsA was associated with CD while CpA did not appear to be involved in pathogenesis. Genome sequencing identified several coding regions which may correspond to other potentially active Cs toxins. These results support the proposed mechanism of pathogenesis and provide targets for further investigation of disease pathophysiology and vaccine development.
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    Equine Herpesvirus Type 1: Filling Gaps Toward Improved Outbreak Management
    Saklou, Nadia Talal (Virginia Tech, 2023-09-06)
    Equine herpesvirus type 1 (EHV-1) is a common pathogen of horses that typically causes upper respiratory disease, however is also associated with late-term abortion, neonatal foal death and neurologic disease. Once a horse is infected, the virus concentrates to local lymphoid tissue, where it becomes latent. The virus can recrudesce during times of stress, which can lead to the initiation of devastating outbreaks. Some variants of EHV-1 have been associated with more severe disease outcomes. Appropriate outbreak management focuses on minimizing the movement of potentially exposed horses. This approach lacks a strategy for prevention at the level of latency largely due to a knowledge paucity in regards to carriage rate of latent EHV-1. Biosecurity decisions are also dependent on awaiting currently-available diagnostic testing that often take several days for results. Thus, our work has been focused on understanding the carriage rate of the latent virus in different geographic regions as well as improving diagnostic efficiency, both of which are essential for improving the management of EHV-1 disease. Loop mediated isothermal amplification (LAMP) is a method that amplifies nucleic acid rapidly at a constant temperature and is minimally affected by inhibitors that are often found in clinical samples. This procedure can be followed by multiple detection methods. A new, efficient sequencing method, called nanopore sequencing, has been developed in a handheld device, called MinION, that provides thorough output in a timely manner. When combined with LAMP, it has been referred to as LAMPore. The first objective of our work was to estimate the prevalence of latent EHV-1 and compare the frequency of each variant in the submandibular lymph nodes from horses in Virginia. Our second objective was to perform direct DNA sequencing of EHV-1 using the mobile MinION sequencer in combination with LAMP viral enrichment. Our findings demonstrated a low apparent prevalence of latent EHV-1 DNA in submandibular lymph nodes in this population of horses in Virginia as well as successful detection and identification of EHV-1 in equine nasal swab samples using LAMPore sequencing.
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    Evaluating the Expression of Angiogenic Mediators in a Mouse Model of Tumor Metastasis
    Crawford, Natalie M. (Virginia Tech, 2008-07-03)
    Solid tumors typically require angiogenesis, the development of new blood vessels, for growth and metastasis. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating receptors on host endothelial cells. One such receptor, Flt-1, occurs as either a membrane bound or a secreted form (sFlt-1) that can inhibit angiogenic signaling. Previous studies have shown that variation in mRNA expression of VEGF and its receptors KDR, sFlt-1 and Flt-1 occurs in pathological angiogenesis, i.e. metastatic tumorigenesis. We hypothesize that the ratio of sFlt-1:Flt-1 mRNA will be altered in the presence of solid tumors. The objective of this study was to evaluate the expression of sFlt-1 and Flt-1 mRNAs in a mouse metastatic tumor model using CT26.CL25 cells. CT26.CL25 cells are VEGF-producing murine colon carcinoma cells transfected with the lacZ gene, which expresses B-galactosidase activity. These cells, injected intravenously, form tumor nodules in the lung. A pilot study revealed development of lung nodules in mice nine days after intravenous injection with 105 cells. In a second study, twenty-five 10- week-old female Balb/c mice were injected intravenously, via tail vein, with 2 x 105cells, and fifteen with vehicle control. Lung nodules developed in all mice injected with cells. Tissues were harvested by routine necropsy and either formalin-fixed for routine histology/histochemistry or stored for quantitative RT-PCR (QPCR) analysis of gene expression. Under microscopic evaluation, sections of lungs stained with Hematoxylin & Eosin (H&E) revealed nodules composed of polygonal neoplastic cells. cDNA from lungs (14 tumor-bearing, 10 controls) and cultured CT26.CL25 cells was analyzed by QPCR using primers and TaqMan probes directed against sFlt-1, Flt-1, KDR, VEGFA, PlGF (Placental Growth Factor), Angiotensin Converting Enzyme (ACE), 18S ribosomal RNA and neoR (neomycin phosphotransferase). We observed an increased sFlt-1:Flt-1 ratio in tumor-bearing versus control lungs, suggesting that tumor-derived signals may influence sFlt-1 and Flt-1 expression differentially. Additionally, there was increased expression of Flt-1, sFlt-1 and KDR in tumors versus controls, but not in VEGF expression in tumors versus controls. Interestingly, expression of PlGF was increased in tumors versus controls, suggesting its role as an enhancer of tumor progression in the presence of other angiogenic factors. Together, these findings indicate that solid tumor angiogenesis results from an intricate balance of various angiogenic factors.