Medicine and Health Sciences Commons^™

Cellular Poly(C) Binding Proteins 1 And 2 Interact With Porcine Reproductive And Respiratory Syndrome Virus Nonstructural Protein 1Β And Support Viral Replication, Lalit Beura, Phat X. Dinh, Fernando A. Osorio, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine results in substantial economic losses to the swine industry worldwide. Identification of cellular factors involved in PRRSV life cycle not only will enable a better understanding of virus biology but also has the potential for the development of antiviral therapeutics. The PRRSV nonstructural protein 1 (nsp1) has been shown to be involved in at least two important functions in the infected hosts: (i) mediation of viral subgenomic (sg) mRNA transcription and (ii) suppression of the host’s innate immune response mechanisms. To further our understanding of the role of the viral …

Antagonistic Effects Of Cellular Poly(C) Binding Proteins On Vesicular Stomatitis Virus Gene Expression, Phat X. Dinh, Lalit Beura, Debasis Panda, Anshuman Das, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of PCBP2 in transfected cells suppressed VSV growth. Further studies revealed that PCBP2 negatively regulates overall viral mRNA accumulation and subsequent genome replication. Coimmunoprecipitation and immunofluorescence microscopic studies showed that …

Acute Fatty Liver Of Pregnancy: An Update On Mechanisms, Sathish Kumar Natarajan, Kavitha R. Thangaraj, Ashish Goel, C. E. Eapen, K. A. Balasubramanian, Anup Ramachandran

School of Veterinary and Biomedical Sciences: Faculty Publications

Acute fatty liver of pregnancy (AFLP), characterized by hepatic microvesicular steatosis, is a sudden catastrophic illness occurring almost exclusively in the third trimester of pregnancy. Defective fatty acid oxidation in the fetus has been shown to be associated with this disease. Since the placenta has the same genetic makeup as the fetus and as AFLP patients generally recover following delivery, we hypothesized that the placenta might be involved in pathogenesis of this disease. In an animal model of hepatic microvesicular steatosis (using sodium valproate), we found that microvesicular steatosis results in mitochondrial structural alterations and oxidative stress in subcellular organelles …

Role Of Il-17 And Th17 Cells In Hsv Induced Ocular Immunopathology, Amol Sahebrao Suryawanshi

Doctoral Dissertations

Herpes simplex virus (HSV) infection of the cornea leads to a blinding immuno-inflammatory condition of the eye also called stromal keratitis (SK). SK immunopathology is characterized by the infiltration of CD4⁺ T cells of Th1 phenotype as well as the development of new blood vessels into the normally avascular cornea. Studies in mouse models of SK have firmly established the role of CD4⁺ T cells, and particularly of Th1 phenotype, as the principal mediators of SK immunopathology. However, with the recent discovery of IL-17A and Th17 cells, the role of this cytokine as well as Th17 cells remains …

Glutaredoxin 2 Prevents H2o2-Induced Cell Apoptosis By Protecting Complex I Activity In The Mitochondria, Hongli Wu, Kuiyi Xing, Marjorie F. Lou

School of Veterinary and Biomedical Sciences: Faculty Publications

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200 μM hydrogen peroxide (H2O2) for 24 h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) …

Regulation Of Cytosolic Phospholipase A2 (Cpla2) And Its Association With Cell Proliferation In Human Lens Epithelial Cells, Yin Wang, Kui-Yi Xing, Marjorie F. Lou

School of Veterinary and Biomedical Sciences: Faculty Publications

PURPOSE. To investigate the molecular mechanism for cytosolic phospholipase A2 (cPLA2) regulation and its association to platelet-derived growth factor (PDGF)-induced cell proliferation.

METHODS. cPLA2 was examined using human lens epithelial (HLE) B3 cells. Reactive oxygen species (ROS) generation induced by PDGF was analyzed by luminescence assay. Cell proliferation was measured by cell counting and by BrdU assay. Human cPLA2 gene was cloned via RT-PCR followed by sitedirected mutagenesis to construct HLE B3 cells expressing either inactive cPLA2 enzyme with S228A mutation (S228A), or cPLA2 truncated at the calcium-binding C2 domain (C2D). Activity of cPLA2 was measured by arachidonic acid (AA) …

Immune Evasion Of Porcine Reproductive And Respiratory Syndrome Virus Through Glycan Shielding Involves Both Glycoprotein 5 As Well As Glycoprotein 3, Hiep Vu, Byungjoon Kwon, Kyoung-Jin Yoon, William W. Laegreid, Asit K. Pattnaik, Fernando A. Osorio

School of Veterinary and Biomedical Sciences: Faculty Publications

Passive administration of porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing antibodies (NAbs) can effectively protect pigs against PRRSV infection. However, after PRRSV infection, pigs typically develop a weak and deferred NAb response. One major reason for such a meager NAb response is the phenomenon of glycan shielding involving GP5, a major glycoprotein carrying one major neutralizing epitope. We describe here a type II PRRSV field isolate (PRRSV-01) that is highly susceptible to neutralization and induces an atypically rapid, robust NAb response in vivo. Sequence analysis shows that PRRSV-01 lacks two N-glycosylation sites, normally present in wild-type (wt) PRRSV strains, …

A Mark–Recapture Technique For Monitoring Feral Swine Populations, Matthew M. Reidy, Tyler A. Campbell, David G. Hewitt

USDA Wildlife Services: Staff Publications

Techniques to monitor populations of feral swine (Sus scrofa) relative to damage control activities are needed on rangelands. Our objectives were to describe and assess a mark–recapture technique using tetracycline hydrochloride (TH) for monitoring feral swine populations. We established bait stations at study sites in southern and central Texas. During 1 d, we replaced normal soured corn bait with bait containing TH and counted the number of feral swine that consumed bait with observers. We conducted feral swine removal using box-style traps and helicopters, at which time we collected teeth for TH analysis. In southern Texas, we estimated …

Molecular Structures Of Prrsv That Contribute To Prrsv Protective Immunity, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

One of the objectives of the proposal is to delineate the regions of the two glycoproteins (GP2 and GP4) of PRRSV that interact with CD163. Other objectives of the proposal are to generate antibodies to these small regions of the glycoproteins as well as to the full-length proteins for future studies to determine if any of these antibodies possess PRRSV neutralizing activity.

To carry out the studies in the proposed objectives, we generated a series of mutants of GP2 as well as GP4 proteins in which various regions were specifically removed by manipulating the plasmids encoding these proteins. We then …

Low Levels Of Hydrogen Peroxide Stimulate Corneal Epithelial Cell Adhesion, Migration, And Wound Healing, Qing Pan, Wen-Ya Qiu, Ya-Nan Huo, Yu-Feng Yao, Marjorie F. Lou

School of Veterinary and Biomedical Sciences: Faculty Publications

PURPOSE. Intracellular reactive oxygen species have been reported to associate with growth factor and integrin signalings in promoting cell adhesion in many cell types. This study is to explore if exogenous H2O2 at low levels can be beneficial to cell adhesion, migration, and wound healing.

METHODS. Primary rabbit corneal epithelial cells treated with 0–70 M H2O2 were tested for viability by MTT assay, adhesion by centrifugation assay, focal contacts of vinculin and F-actin by immunofluorescence, activated Src(pY416), EGF receptor (pY845), vinculin(pY1065), FAK(pY397), and FAK(pY576) by immunoblotting. Cell migration was examined with 0–50 M H2O2 using the scratch wound technique. Corneal …

Factors Affecting Space Use Overlap By White-Tailed Deer In An Urban Landscape, W. David Walter, Jeff Beringer, Lonnie P. Hansen, Justin W. Fischer, Joshua J. Millspaugh, Kurt C. Vercauteren

USDA Wildlife Services: Staff Publications

Variation in the size and overlap of space use by white-tailed deer (Odocoileus virginianus) has broad implications for managing deer–human conflicts and disease spread and transmission in urban landscapes. Understanding which factors affect overlap of home range by various segments (i.e., age, sex) of an urban deer population has implications to direct contact between deer on disease epidemiology. We assessed size of home range and overlap of space use using the volume of intersection index (VI) for deer in an urban landscape by sex, age, season, and time of day. We found mean space use was larger for …

Rnai Screening Reveals Requirement For Host Cell Secretory Pathway In Infection By Diverse Families Of Negative-Strand Rna Viruses, Debasis Panda, Anshuman Das, Phat X. Dinh, Sakthivel Subramaniam, Debasis Nayak, Nicholas J. Barrows, James L. Pearson, Jesse Thompson, David L. Kelly, Istvan Ladunga, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genomewide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of …

Carbenoxolone Blocks The Light-Evoked Rise In Intracellular Calcium In Isolated Melanopsin Ganglion Cell Photoreceptors, Jayne R. Bramley, Erin M. Wiles, Patricia J. Sollars, Gary E. Pickard

School of Veterinary and Biomedical Sciences: Faculty Publications

Background: Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to widefield GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca²⁺ signals suggesting extensive …

Y-Like Retinal Ganglion Cells Innervate The Dorsal Raphe Nucleus In The Mongolian Gerbil (Meriones Unguiculatus), Liju Luan, Chaoran Ren, Benson Wui-Man Lau, Jian Yang, Gary E. Pickard, Kwok-Fai So, Mingliang Pu

School of Veterinary and Biomedical Sciences: Faculty Publications

Background: The dorsal raphe nucleus (DRN) of the mesencephalon is a complex multi-functional and multi-transmitter nucleus involved in a wide range of behavioral and physiological processes. The DRN receives a direct input from the retina. However little is known regarding the type of retinal ganglion cell (RGC) that innervates the DRN. We examined morphological characteristics and physiological properties of these DRN projecting ganglion cells.

Methodology/Principal Findings: The Mongolian gerbils are highly visual rodents with a diurnal/crepuscular activity rhythm. It has been widely used as experimental animals of various studies including seasonal affective disorders and depression. Young adult gerbils were used …

Challenges And Opportunities For Toxicology In Mexico, Rodrigo Franco, Balam Muñoz

School of Veterinary and Biomedical Sciences: Faculty Publications

We have developed this Special Issue titled “Challenges and Opportunities for Toxicology in Mexico” with the aim to highlight not only the challenges toxicology research faces in Mexico, but also the efforts and drive towards cutting edge research to address them. Altogether, this special issue should be a valuable source of information for all readers with an interest in toxicology and human health.

Mexico is an important economy worldwide and is a major exporter of several supplies and commodities. Because of this, its industry is a source of occupational exposure to several chemical derivatives (Nieusma 2011). The situation of occupational …

Roles Of The 15-Kda Selenoprotein (Sep15) In Redox Homeostasis And Cataract Development Revealed By The Analysis Of Sep 15 Knockout Mice, Marina V. Kasaikina, Dmitri E. Fomenko, Vyacheslav M. Labunskyy, Salil A. Lachke, Wenya Qiu, Juliet A. Moncaster, Jie Zhang, Mark W. Wojnarowicz, Sathish Kumar Natarajan, Mikalai I. Malinouski, Ulrich Schweizer, Petra A. Tsuji, Bradley A. Carlson, Richard L. Maas, Marjorie F. Lou, Lee E. Goldstein, Dolph L. Hatfield, Vadim N. Gladyshev

School of Veterinary and Biomedical Sciences: Faculty Publications

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteinerich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable …

Intrinsically Photosensitive Retinal Ganglion Cells, Gary E. Pickard, Patricia J. Sollars

School of Veterinary and Biomedical Sciences: Faculty Publications

Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond to light in the absence of all rod and cone photoreceptor input. The existence of these ganglion cell photoreceptors, although predicted from observations scattered over many decades, was not established until it was shown that a novel photopigment, melanopsin, was expressed in retinal ganglion cells of rodents and primates. Phototransduction in mammalian ipRGCs more closely resembles that of invertebrate than vertebrate photoreceptors and appears to be mediated by transient receptor potential channels. In the retina, ipRGCs provide excitatory drive to dopaminergic amacrine cells and ipRGCs are coupled to GABAergic amacrine cells via gap …

The Impact Of Tricaine Methanesulfonate, 2-Phenoxyethanol, And Carvone-Methyl Salicylate On The Innate Immune Response Of Zebrafish (Danio Rerio), Charles R. Wulff

Honors Theses

Anesthesia plays a vital role in the maintenance of aquaculture species, where it is used to minimize stress during complex handling tasks such as transport, assessment, and harvesting. However, anesthetics have been shown to suppress the innate immune response, which could impact immunity and increase risk of infection. Tricaine methanesulfonate (MS-222) and 2-Phenoxyethanol (2-PE) represent two of the most commonly used anesthetics in aquaculture, with R-(+)-carvone, in the form of carvone-methyl salicylate (CMS) has recently been proposed as an alternative anesthetic for food fish. These three anesthetics were used to assess the influence of anesthetics on the immune system of …

Long-Term Clinicopathological Characteristics Of Alpacas Naturally Infected With Bovine Viral Diarrhea Virus Type Ib, D. Bedenice, Edward J. Dubovi, Clayton Kelling, Jamie N Henningson, Christina L. Topliff, N. Parry

School of Veterinary and Biomedical Sciences: Faculty Publications

Background: Substantial bovine viral diarrhea virus (BVDV)-related production losses in North American alpaca herds have been associated with BVDV type Ib infection.

Objectives: To classify and differentiate the long-term clinicopathological characteristics of BVDV type Ib infection of al- paca crias, after natural virus exposure. We hypothesized that persistently infected (PI) alpacas specifically demonstrate growth retardation, clinicopathological evidence of opportunistic infections, and early mortality.

Animals: Thirty-five crias naturally exposed to BVDV (18 acute, 3 chronic, 14 PIs), and 19 healthy cohort controls of 5 northeastern alpaca farms were prospectively evaluated over 2 years (September 2005–September 2008).

Methods: Observational cohort-control study.

Results: …

Consensus-Based Reporting Standards For Diagnostic Test Accuracy Studies For Paratuberculosis In Ruminants, Ian A. Gardner, Søren S. Nielsen, Richard J. Wittington, Michael T. Collins, Douwe Bakker, Beth Harris, Srinand Sreevatsan, Jason E. Lombard, Raymond Sweeney, David R. Smith, Jerrie Gavalchin, Shigetoshi Eda

School of Veterinary and Biomedical Sciences: Faculty Publications

The Standards for Reporting of Diagnostic Accuracy (STARD) statement was developed to encourage complete and transparent reporting of key elements of test accuracy studies in human medicine. The statement was motivated by widespread evidence of bias in test accuracy studies and the finding that incomplete or absent reporting of items in the STARD checklist was associated with overly optimistic estimates of test performance characteristics. Although STARD principles apply broadly, specific guidelines do not exist to account for unique considerations in livestock studies such as herd tests, potential use of experimental challenge studies, a more diverse group of testing purposes and …

Regulated Commercial Harvest To Manage Overabundant White-Tailed Deer: An Idea To Consider?, Kurt C. Vercauteren, Charles W. Anderson, Timothy R. Van Deelen, David Drake, W. David Walter, Stephen Vantassel, Scott E. Hygnstrom

USDA Wildlife Services: Staff Publications

Declines in hunter recruitment coupled with dramatic growth in numbers of white-tailed deer (Odocoileus virginianus) have challenged our ability to manage deer populations through regulated hunting. We review the efficacy of current regulated hunting methods and explain how they are unable to reduce deer numbers sufficiently in some environments. Regulated commercial harvest would provide an additional tool to help state wildlife agencies manage overabundant populations of white-tailed deer. We outline potential means to govern regulated commercial deer harvest and explain how it is compatible with the North American Model of Wildlife Conservation. We identified several benefits, including reduced …

Gender Differences In Cns Autoimmunity Induced By Mimicry Epitope For Plp 139–151 In Sjl Mice, Chandirasegara Massilamany, Sivasubramani Thulasingam, David Steffen, Jay Reddy

Jay Reddy Publications

Development of multiple sclerosis (MS) is more prevalent in females than in males, but the underlying mechanisms are not clear. Microbial infections have been suspected as triggers of MS and it is not known whether gender differences in reactivity to environmental antigens contribute to the disease pathogenesis. We demonstrated that ACA 83–95, a mimicry epitope from Acanthamoeba castellanii for proteolipid protein (PLP) 139–151, induces clinical signs of encephalomyelitis in both male and female SJL mice. Conversely ACA 83–95-induced effector cells from males fail to induce disease in female mice. Although we found no gender differences in the frequencies of antigen-specific …

Molecular And Genomic Approaches To Understanding Host-Virus Interactions In Shaping The Outcome Of Equine Arteritis Virus Infection, Yun Young Go

University of Kentucky Doctoral Dissertations

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis, a disease of equids. During natural outbreaks of the disease, EAV can cause abortion in pregnant mares and persistent infection in stallions. Understanding how host cellular proteins interact with viral RNA and viral proteins, as well as their role in viral infection, will enable better characterization of the pathogenesis of EAV and establishment of persistent infection in stallions. Accordingly, we hypothesized that both viral factors and host genetically related factors could influence the outcome of EAV infection in horses. To test this hypothesis, we first combined contemporary molecular …

Genetic Immunization In The Horse: The Potential For Enhanced Immune Responses With Deacylated Polyethyleneimine (Pei) And Immunostimulatory Cytokines As Vaccine Adjuvants, Deborah Lee Even

Theses and Dissertations--Veterinary Science

DNA vaccines in larger animals, such as horses, are generally less effective and elicit significantly weaker immune responses, than in small animal model systems. To provide optimal protection against pathogenic microorganisms, the induction of both humoral and cellular immune responses from DNA vaccination may be necessary. One limitation to DNA immunization in the horse is the difficulty in generating high levels of antigen-specific antibody and CTL responses. Previous work in the laboratory has demonstrated that expression constructs containing native sequences encoding the surface unit (SU) envelope glycoprotein (pCiSU) of the Equine Infectious Anemia Virus (EIAV) are ineffective at stimulating immune …