DOCSLIB.ORG

A Functional Analysis of the Non-Coding Rnas of Murine Gammaherpesvirus 68

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Functional Analysis of the Non-Coding Rnas of Murine Gammaherpesvirus 68 A functional analysis of the non-coding RNAs of murine gammaherpesvirus 68 Nila Roy Choudhury Submitted for the degree of Doctor of Philosophy The University of Edinburgh 2010 Declaration I declare that this thesis has been composed by me and that all work included in this thesis is my own except where otherwise stated. No part of this work has been submitted, or will be submitted for any other degree of professional qualification. Nila Roy Choudhury 2010 Centre for Infectious Diseases The Roslin Institute and Royal (Dick) School of Veterinary Studies University of Edinburgh Summerhall Edinburgh EH9 1QH ii Acknowledgments I would like to thank my supervisors Dr Bernadette Dutia, Dr Simon Talbot and Professor Alastair Aitken for their help and advice during my PhD. As my main advisor Dr Bernadette Dutia provided guidance and advice at every stage of my work and I am very grateful to have been given the opportunity to do my PhD in her research group. A big thank you also to Dr Andrew Cronshaw for all his help with the cICAT technique and Amy Buck for her help with the qRT-PCR for miRNAs and general advice. I would also like to thank the rest of the people in the group and all the people in the tower block for creating a fantastic working environment. Thank you to my family, especially my parents who always encouraged me to work for what I wanted and not give up. Finally, Ewan, thank you for being there supporting me throughout my PhD, encouraging me and believing in me. iii Abstract Murine gammaherpesvirus 68 (MHV-68) is used as a model for the study of gammaherpesvirus infection and pathogenesis. In the left region of the genome MHV-68 encodes four unique genes, eight viral tRNA-like molecules (vtRNAs) and nine miRNAs. The vtRNAs have a predicted cloverleaf-like secondary structure like cellular tRNAs and are processed into mature tRNAs with the addition of 3’ CCA termini, but are not aminoacylated. Their function is unknown; however they have been found to be expressed at high levels during both lytic and latent infection and are packaged in the virion. The miRNAs are expressed from the vtRNA primary transcripts during latent infection. All herpesviruses examined to date have been found to express miRNAs. These are thought to aid the viruses in avoiding the host immune response and to establish and maintain latency. The aim of this project was to investigate the functions of the vtRNAs and miRNAs of MHV-68. MHV-76 is a natural deletant mutant lacking the unique genes, vtRNAs and miRNAs. This virus was previously used in our lab to construct two insertion viruses encoding vtRNAs1-5 and miRNAs1-6. The only difference between MHV-76 and the insertion viruses is therefore the vtRNAs and miRNAs. The B-cell line NS0 was latently infected with the various viruses and the infected cells characterised. In situ hybridisation and antibody staining showed that all viruses infect the same proportion of cells. The insertion viruses were confirmed to express the vtRNAs during latency by RT-PCR. In addition, using Northern blot analysis the insertion viruses were shown to express miRNA1 during lytic infection of fibroblast cells; however, not during latent infection of NS0 cells. The lack of miRNA1 expression during latency was confirmed using qRT-PCR and miRNAs3-6 were found to be expressed at a lower level than in MHV-68 infected cells. Replication and reactivation kinetics of latently infected NS0 cells showed that introduction of vtRNAs and miRNAs into MHV-76 causes a reduction in reactivation and production of lytic virus. To determine if the reduction in reactivation was caused by the miRNAs, they were introduced into infected cells by transfection. Transfection of miRNAs1-6 into MHV-76 infected cells or miRNA1 into insertion virus infected cells did not lead to an increase or decrease in iv reactivation. It was confirmed by qRT-PCR that the transfection did result in miRNA levels higher than in insertion virus infected cells. Further, down-regulation of miRNAs using a siRNA against DICER did not lead to a reduction in reactivation. This supports the hypothesis that the vtRNAs rather than the miRNAs are responsible for the reduction of reactivation seen in insertion virus latently infected cells. To determine the effect of the non-coding RNAs on protein expression, NS0 cells latently infected with MHV-76 and insertion virus were analysed using cleavable ICAT and 1-D PAGE cleavable ICAT. In an ICAT analysis the proteins are labelled and the levels of individual proteins in two samples can be compared using mass spectrometry. These techniques were optimised and several proteins with differences in expression between the viruses were identified. It was, however, difficult to determine any specific functions of the non-coding RNAs from the data. v Contents Title i Declaration ii Acknowledgments iii Abstract iv Contents vi List of Figures xi List of Tables xiv Abbreviations xv 1. Chapter One: Introduction 1 1.1. Herpesviruses 2 1.1.1. Classification 2 1.1.1.1. Alphaherpesvirinae 4 1.1.1.2. Betaherpesvirinae 4 1.1.1.3. Gammaherpesvirinae 4 1.1.2. Herpesvirus structure 5 1.1.3. Herpesvirus genome 7 1.1.4. Herpesvirus life cycle 9 1.1.4.1. Entry 9 1.1.4.2. Gene expression 12 1.1.4.3. Replication 12 1.1.4.4. Assembly and egress 13 1.1.4.5. Latency 13 1.2. Gammaherpesviruses 14 1.2.1. Epstein-Barr virus 14 1.2.1.1. EBV latency 15 1.2.1.1.1. Latency transcripts 17 1.2.1.2. EBV infection and associated diseases 19 1.2.2. Kaposi’s sarcoma-associated herpesvirus 22 1.2.2.1. KSHV genome 23 1.2.2.2. KSHV epidemiology 23 1.2.2.3. KSHV latency 24 1.2.2.3.1. Latency transcripts 25 1.2.2.4. KSHV infection and associated diseases 27 1.2.2.4.1. Kaposi’s sarcoma 27 1.2.2.4.2. Primary effusion lymphoma 28 1.2.2.4.3. Multicentric Castleman’s disease 28 1.3. Murine gammaherpesvirus-68 29 1.3.1. Discovery and classification 29 1.3.2. MHV-68 virion 30 vi 1.3.3. MHV-68 genome 30 1.3.4. MHV-68 life cycle 31 1.3.4.1. Entry 31 1.3.4.2. Gene expression 31 1.3.4.3. Viral DNA replication 33 1.3.4.4. Assembly and egress 34 1.3.5. MHV-68 primary infection 34 1.3.6. MHV-68 latency 35 1.3.6.1. Latency in vitro 36 1.3.6.2. Latency transcripts 37 1.3.7. MHV-68 evasion of the host’s immune system 38 1.3.8. MHV-68 pathogenesis 38 1.3.9. MHV-76 and other related viruses 39 1.3.10. Left-hand end of MHV-68 40 1.3.10.1. M1 40 1.3.10.2. M2 42 1.3.10.3. M3 43 1.3.10.4. M4 43 1.3.10.5. vtRNAs and miRNAs 44 1.4. Non-coding RNAs 45 1.4.1. Viral non-coding RNA molecules 45 1.4.1.1. Alphaherpesvirus non-coding RNA molecules 45 1.4.1.1.1. HSV LATs 45 1.4.1.2. Gammaherpesvirus non-coding RNA molecules 47 1.4.1.2.1. EBV EBERs 47 1.4.1.2.2. HVS U RNAs 50 1.4.1.2.3. KSHV PAN RNAs 51 1.4.1.3. Adenovirus non-coding RNAs 52 1.4.1.3.1. VAI and VAII 52 1.4.2. tRNAs 53 1.4.2.1. tRNA structure 53 1.4.2.2. tRNA expression 55 1.4.2.3. tRNA functions 56 1.4.2.3.1. Translation 56 1.4.2.3.2. Amino acid starvation 56 1.4.2.4. tRNA genes and chromatin 57 1.4.2.5. tRNA functions during viral infections 58 1.4.2.6. tRNA-like molecules 58 1.4.2.6.1. Plant virus tRNA-like molecules 59 1.4.2.6.2. Transfer messenger RNA 59 1.4.2.6.3. The threonyl-tRNA synthetase gene of E.coli 60 1.4.3. miRNAs 60 1.4.3.1. miRNA biogenesis 60 1.4.3.2. miRNA functions 63 1.4.3.3. Viruses and miRNAs 64 1.5. Project outline 69 vii 2. Chapter Two: Materials and methods 70 2.1. Cell Culture techniques 71 2.1.1. Maintenance of cell lines 71 2.1.2. Harvesting and counting cells 71 2.1.3. Cytospins 71 2.1.4. Transfection 72 2.2. Virological methods 72 2.2.1. Preparation of virus stocks 72 2.2.2. Virus titration 73 2.2.3. Infection of NS0 cells for virus characterisation studies 73 2.2.4. Infective centre assay 74 2.2.5. GFP labelled viruses 74 2.2.6. Cloning of infected cells 74 2.2.7. Staining for lytic proteins 75 2.3. In situ hybridisation 75 2.3.1. Generation of labelled RNA probe 75 2.3.2. In situ hybridisation of cytospins 77 2.4. DNA isolation and manipulation 78 2.4.1. Plasmid preps 78 2.5. RNA isolation and manipulation 79 2.5.1. microRNA isolation 79 2.5.2. DNase treatment of RNA 79 2.5.3. Reverse transcription of RNA 79 2.5.4. In vitro transcription of vtRNAs 80 2.6. Polymerase chain reaction (PCR) 80 2.6.1. Standard PCR 80 2.6.2.
Load more

Recommended publications
  • Cyprinus Carpio
    Académie Universitaire Wallonie - Europe Université de Liège Faculté de Médecine Vétérinaire Département des Maladies Infectieuses et Parasitaires Service d’Immunologie et de Vaccinologie Etude des portes d’entrée de l’Herpèsvirus cyprin 3 chez Cyprinus carpio Study of the portals of entry of Cyprinid herpesvirus 3 in Cyprinus carpio Guillaume FOURNIER Thèse présentée en vue de l’obtention du grade de Docteur en Sciences Vétérinaires Année académique 2011-2012 Académie Universitaire Wallonie - Europe Université de Liège Faculté de Médecine Vétérinaire Département des Maladies Infectieuses et Parasitaires Service d’Immunologie et de Vaccinologie Etude des portes d’entrée de l’Herpèsvirus cyprin 3 chez Cyprinus carpio Study of the portals of entry of Cyprinid herpesvirus 3 in Cyprinus carpio Promoteur : Prof. Alain Vanderplasschen Guillaume FOURNIER Thèse présentée en vue de l’obtention du grade de Docteur en Sciences Vétérinaires Année académique 2011-2012 « La science progresse en indiquant l'immensité de l'ignoré. » Louis Pauwels Remerciements Liège, le 15 février 2012 L’accomplissement d’une thèse est un long et palpitant voyage en océan où se mélangent la curiosité, le doute, la persévérance, et la confiance… en soi bien sûr, mais surtout envers toutes les personnes qui, par leurs conseils, leur aide, leur soutien m’ont permis de mener cette thèse à bien. Je tiens ici à remercier mes collègues, amis et famille qui ont été tantôt les phares, tantôt les boussoles, toujours les fidèles compagnons de cette aventure. Je commencerais par adresser mes plus sincères remerciements à mon promoteur, le Professeur Alain Vanderplasschen, qui m’avait déjà remarqué en amphithéâtre pour ma curiosité, à moins que ce ne soit pour mon irrésistible coiffure..
    [Show full text]
  • Molecular Identification and Genetic Characterization of Cetacean Herpesviruses and Porpoise Morbillivirus
    MOLECULAR IDENTIFICATION AND GENETIC CHARACTERIZATION OF CETACEAN HERPESVIRUSES AND PORPOISE MORBILLIVIRUS By KARA ANN SMOLAREK BENSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Kara Ann Smolarek Benson I dedicate this to my best friend and husband, Brock, who has always believed in me. ACKNOWLEDGMENTS First and foremost I thank my mentor, Dr. Carlos Romero, who once told me that love is fleeting but herpes is forever. He welcomed me into his lab with very little experience and I have learned so much from him over the past few years. Without his excellent guidance, this project would not have been possible. I thank my parents, Dave and Judy Smolarek, for their continual love and support. They taught me the importance of hard work and a great education, and always believed that I would be successful in life. I would like to thank Dr. Tom Barrett for the wonderful opportunity to study porpoise morbillivirus in his laboratory at the Institute for Animal Health in England, and Dr. Romero for making the trip possible. I especially thank Dr. Ashley Banyard for helping me accomplish all the objectives of the project, and all the wonderful people at the IAH for making a Yankee feel right at home in the UK. I thank Alexa Bracht and Rebecca Woodruff who have been with me in Dr. Romero’s lab since the beginning. Their continuous friendship and encouragement have kept me sane even in the most hectic of times.
    [Show full text]
  • Diversity of Large DNA Viruses of Invertebrates ⇑ Trevor Williams A, Max Bergoin B, Monique M
    Journal of Invertebrate Pathology 147 (2017) 4–22 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip Diversity of large DNA viruses of invertebrates ⇑ Trevor Williams a, Max Bergoin b, Monique M. van Oers c, a Instituto de Ecología AC, Xalapa, Veracruz 91070, Mexico b Laboratoire de Pathologie Comparée, Faculté des Sciences, Université Montpellier, Place Eugène Bataillon, 34095 Montpellier, France c Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands article info abstract Article history: In this review we provide an overview of the diversity of large DNA viruses known to be pathogenic for Received 22 June 2016 invertebrates. We present their taxonomical classification and describe the evolutionary relationships Revised 3 August 2016 among various groups of invertebrate-infecting viruses. We also indicate the relationships of the Accepted 4 August 2016 invertebrate viruses to viruses infecting mammals or other vertebrates. The shared characteristics of Available online 31 August 2016 the viruses within the various families are described, including the structure of the virus particle, genome properties, and gene expression strategies. Finally, we explain the transmission and mode of infection of Keywords: the most important viruses in these families and indicate, which orders of invertebrates are susceptible to Entomopoxvirus these pathogens. Iridovirus Ó Ascovirus 2016 Elsevier Inc. All rights reserved. Nudivirus Hytrosavirus Filamentous viruses of hymenopterans Mollusk-infecting herpesviruses 1. Introduction in the cytoplasm. This group comprises viruses in the families Poxviridae (subfamily Entomopoxvirinae) and Iridoviridae. The Invertebrate DNA viruses span several virus families, some of viruses in the family Ascoviridae are also discussed as part of which also include members that infect vertebrates, whereas other this group as their replication starts in the nucleus, which families are restricted to invertebrates.
    [Show full text]
  • Evidence to Support Safe Return to Clinical Practice by Oral Health Professionals in Canada During the COVID-19 Pandemic: a Repo
    Evidence to support safe return to clinical practice by oral health professionals in Canada during the COVID-19 pandemic: A report prepared for the Office of the Chief Dental Officer of Canada. November 2020 update This evidence synthesis was prepared for the Office of the Chief Dental Officer, based on a comprehensive review under contract by the following: Paul Allison, Faculty of Dentistry, McGill University Raphael Freitas de Souza, Faculty of Dentistry, McGill University Lilian Aboud, Faculty of Dentistry, McGill University Martin Morris, Library, McGill University November 30th, 2020 1 Contents Page Introduction 3 Project goal and specific objectives 3 Methods used to identify and include relevant literature 4 Report structure 5 Summary of update report 5 Report results a) Which patients are at greater risk of the consequences of COVID-19 and so 7 consideration should be given to delaying elective in-person oral health care? b) What are the signs and symptoms of COVID-19 that oral health professionals 9 should screen for prior to providing in-person health care? c) What evidence exists to support patient scheduling, waiting and other non- treatment management measures for in-person oral health care? 10 d) What evidence exists to support the use of various forms of personal protective equipment (PPE) while providing in-person oral health care? 13 e) What evidence exists to support the decontamination and re-use of PPE? 15 f) What evidence exists concerning the provision of aerosol-generating 16 procedures (AGP) as part of in-person
    [Show full text]
  • This Thesis Has Been Submitted in Fulfilment of the Requirements for a Postgraduate Degree (E.G
    This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: • This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. • A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. • This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. • The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. • When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Marek’s Disease Virus Pathogenesis and Latency Gillian Hunter M Phil The University of Edinburgh 2012 i DECLARATION I declare that all work included in this thesis has been composed by myself. Contributions to the work of this thesis by colleagues are fully acknowledged. No part of this work has been or will be submitted for any other degree or qualification. Gillian Hunter January 28, 2011 Centre for Infectious Diseases The Roslin Institute Royal (Dick) School of Veterinary Studies Summerhall Tower Block University of Edinburgh Edinburgh EH9 1QH ii ABSTRACT Marek’s Disease virus (MDV) is a highly contagious, widespread and persistent neoplastic herpesvirus causing extensive lymphoblastic tumours in chickens. The virus is shed in feather dust and spread through inhalation. Vaccines are available to protect against the effects of MDV but not replication of the virus and subsequent contamination of the environment leading to flock exposure.
    [Show full text]
  • Cyprinid Herpesvirus 3
    1 © 2015. This manuscript version is made available under the CC-BY-NC-ND 4.0 license 2 http://creativecommons.org/licenses/by-nc-nd/4.0/ 3 doi:10.1016/bs.aivir.2015.03.001 4 Running title: Cyprinid herpesvirus 3 5 Title: Cyprinid herpesvirus 3, an archetype of fish alloherpesviruses 6 Authors and Affiliations 7 Maxime Boutier 1, Maygane Ronsmans 1, Krzysztof Rakus 1, Joanna Jazowiecka-Rakus 1, 8 Catherine Vancsok 1, Léa Morvan 1, Ma. Michelle D. Peñaranda 1, David M. Stone 2, Keith 9 Way 2, Steven J. van Beurden 3, Andrew J. Davison 4 and Alain Vanderplasschen 1* 10 11 1 Immunology-Vaccinology (B43b), Department of Infectious and Parasitic Diseases, 12 Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary 13 Medicine, University of Liège, B-4000 Liège, Belgium. 14 2 The Centre for Environment, Fisheries and Aquaculture Science, Weymouth Laboratory, 15 Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, United Kingdom. 16 3 Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 17 1, 3584CL Utrecht, The Netherlands. 18 4 MRC - University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 19 5JR, United Kingdom. 20 21 22 * Corresponding author. Mailing address: Immunology-Vaccinology (B43b), Department of 23 Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, 24 B-4000 Liège, Belgium. Phone: 32-4-366 42 64 - Fax: 32-4-366 42 61 25 E-mail: [email protected] 26 Author’s contacts (see affiliations above) 27 28 Maxime Boutier: [email protected] ; +32 4 366 42 66 29 Maygane Ronsmans: [email protected] ; +32 4 366 42 66 30 Krzysztof Rakus: [email protected] ; +32 4 366 42 66 31 Joanna Jazowiecka-Rakus: [email protected] ; +32 4 366 42 66 32 Catherine Vancsok: [email protected] ; +32 4 366 42 66 33 Léa Morvan: [email protected] ; +32 4 366 42 66 34 Ma.
    [Show full text]
  • Documento Completo Descargar Archivo
    UNIVERSIDAD NACIONAL DE LA PLATA FACULTAD DE CIENCIAS VETERINARIAS Trabajo de Tesis realizado como requisito para optar al título de DOCTOR EN CIENCIAS VETERINARIAS RESPUESTA INMUNE LOCAL EN LA PATOGENIA DEL ABORTO INDUCIDO POR HERPESVIRUS EQUINO 1 Autor: Méd. Vet. BRAVI María Emilia DIRECTORAS: Dra. Galosi Cecilia M. y Dra. Zanuzzi Carolina N. Cátedras de Virología e Histología y Embriología. FCV-UNLP Miembros del jurado: Dra. Dolcini Guillermina Dra. Monteavaro Cristina Dra. Rambeaud Magdalena 2018 II A mis hijos, Catalina y Ciro Bravi, María Emilia III De todo quedaron tres cosas: la certeza de que estaba siempre comenzando, la certeza de que había que seguir y la certeza de que sería interrumpido antes de terminar. Hacer de la interrupción un camino nuevo, hacer de la caída, un paso de danza, del miedo, una escalera, del sueño, un puente, de la búsqueda,…un encuentro. Fernando Pessoa Bravi, María Emilia IV Agradecimientos En primer lugar quiero agradecer a mis directores, quienes me guiaron y siempre tuvieron gran dedicación y compromiso para transmitir sus conocimientos, formando un excelente grupo de trabajo, pero sobretodo de personas y a quienes admiro muchísimo por su trayectoria; A Ceci, por darme la oportunidad de conocer el mundo de la investigación y por confiar en mí, por ponerle amor antes que todo, amor a lo que uno hace y con quien trabaja, enseñando con el ejemplo. Por ser tan buena persona y comprensiva, por sus sabias palabras y grandes consejos. Para mí fue un orgullo ser su becaria; la última de un largo camino. A Caro, por brindarme su apoyo y honestidad, por sus consejos y el tiempo que dedicó siempre con gran responsabilidad, compromiso y cariño.
    [Show full text]
  • The-Dictionary-Of-Virology-4Th-Mahy
    The Dictionary of VIROLOGY This page intentionally left blank The Dictionary of VIROLOGY Fourth Edition Brian W.J. Mahy Division of Emerging Infections and Surveillance Services Centers for Disease Control and Prevention Atlanta, GA 30333 USA AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier Academic Press is an imprint of Elsevier 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 32 Jamestown Road, London NW1 7BY, UK Copyright © 2009 Elsevier Ltd. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or trans- mitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Departmentin Oxford, UK: phone (ϩ44) (0) 1865 843830; fax (ϩ44) (0) 1865 853333; email: [email protected]. Alternatively visit the Science and Technology website at www.elsevierdirect.com/rights for further information Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloguing in Publication Data A catalogue record for this book is available from the Library of Congress ISBN 978-0-12-373732-8 For information on all Academic Press publications visit our website at www.elsevierdirect.com Typeset by Charon Tec Ltd., A Macmillan Company.
    [Show full text]
  • Ostreid Herpesvirus-1 Infection in Pacific Oysters (Crassostrea Gigas) - New Zealand
    Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Ostreid herpesvirus-1 infection in Pacific Oysters (Crassostrea gigas) - New Zealand A thesis presented in partial fulfilment of the requirements for the degree Of Master of Veterinary Studies At Massey University Palmerston North, New Zealand Rudolfo Bueno 2015 Institute of Veterinary, Animal and Biomedical Sciences Massey University Palmerston North, New Zealand 2015 i Abstract Ostreid herpesvirus-1 (OsHV-1) was associated with summer mortalities in New Zealand Pacific oysters in 2010-2011. During the mortality investigation, a cohort of Pacific oyster spat, negative with OsHV-1 from a South Island hatchery, were followed forward after transfer to a grow-out farm with high oyster mortalities in the North Island. One important finding in this short longitudinal study was the temporality of OsHV-1 nucleic acid detection by real time PCR assay and onset of Pacific oyster mortality. The research described in this thesis was undertaken to further support the causal link between OsHV-1 infection and oyster mortality. To achieve this aim, an in situ hybridisation (ISH) assay was developed to elucidate OsHV-1 infection in Pacific oysters collected from the same short prospective study. OsHV-1 presence and distribution in spat indicated by ISH signal were then correlated with the existence of any histopathological findings in oyster tissues. Hybridisation of the labelled probe with the target region in the OsHV-1 genome on infected cells produced dark blue to purplish black cell precipitates during colorimetric detection.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2017/0165230 A1 RUDD Et Al
    US 201701 65230A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0165230 A1 RUDD et al. (43) Pub. Date: Jun. 15, 2017 (54) USE OF GSK-3 INHIBITORS OR A63L/433 (2006.01) ACTIVATORS WHICH MODULATE PD-1 OR A6IR 9/00 (2006.01) T-BET EXPRESSION TO MODULATE T (52) U.S. Cl. CELL IMMUNITY CPC .......... A61K 31/404 (2013.01); A61K 31/433 (2013.01); A61K 9/0053 (2013.01); A61 K (71) Applicant: Christopher RUDD, Montreal (CA) 38/10 (2013.01); A61K 31/426 (2013.01); A6 IK3I/506 (2013.01) (72) Inventors: Christopher RUDD, Montreal (CA): Dae Choon LEE, Springfield, MO (57) ABSTRACT (US); David Mark ROTHSTEIN, Pittsburgh, PA (US); Young Mee LEE, The present application generally relates to the discovery Springfield, MO (US) that glycogen synthase kinase 3 (GSK-3) is an upstream signalling molecule that controls PD-1 transcription and (21) Appl. No.: 15/302,589 Tbet expression by immune cells and in particular T-cells. Based on this discovery, and in view of the known immu (22) PCT Fed: Apr. 9, 2015 nosuppressive effect of PD-1 on immunity and the promot ing effect of Tbet on T cell immunity, the present invention (86) PCT No.: PCT/B2O15/OS2606 relates to the use of GSK-3 inhibitors to promote immunity, S 371 (c)(1), including cytotoxic T cell immunity in Subjects in need (2) Date: Oct. 7, 2016 thereof, especially subjects with chronic conditions wherein inhibiting PD-1 expression and/or blockade or Tbet up Related U.S. Application Data regulation is therapeutically desirable Such as cancer and infectious conditions.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 9,687,536 B2 Nagata Et Al
    US009687536B2 (12) United States Patent (10) Patent No.: US 9,687,536 B2 Nagata et al. (45) Date of Patent: Jun. 27, 2017 (54) METHODS AND COMPOSITIONS FOR (56) References Cited INTRANASAL DELVERY U.S. PATENT DOCUMENTS (75) Inventors: Ryoichi Nagata, Kagoshima (JP); 8.337,817 B2 * 12/2012 Nagata ................. A61K9/0043 Shunji Haruta, Kagoshima (JP) 424/46 8,827,946 B2 * 9/2014 Tsutsui ................. A61K9/0043 (73) Assignees: SHIN NIPPON BIOMEDICAL 604/212 LABORATORIES, LTD., 9,101,539 B2 * 8/2015 Nagata ................. A61K9/0043 2003/0092145 A1 5/2003 Jira et al. Kagoshima-Shi, Kagoshima (JP): THE 2003/0186271 A1 10/2003 Hwang et al. CHEMO-SERO-THERAPEUTC 2004/0042972 A1 3/2004 Truong-leet al. RESEARCH INSTITUTE, Kita-Ku, 2006/0292.173 Al 12/2006 Macadam 2007/0275O14 A1 11/2007 YuSibov et al. Kumamoto-Shi, Kumamoto (JP) 2008/0206281 A1* 8, 2008 Look .................... A61K 39,155 424,211.1 (*) Notice: Subject to any disclaimer, the term of this 2008/0226729 A1 9, 2008 Sullivan et al. patent is extended or adjusted under 35 2009. O155351 A1 6/2009 Heil et al. U.S.C. 154(b) by 0 days. 2009/0232894 A1* 9, 2009 Chouvenc ............ A61K 9, 1623 424/489 2010.0068223 A1 3/2010 Scheffczik (21) Appl. No.: 13/640,623 2010/0178331 A1* 7/2010 Nagata ................. A61K9/0043 514/1.1 (22) PCT Fed: Apr. 15, 2011 2013/0273 120 A1* 10/2013 Nagata ................. A61K9/0043 424/400 (86) PCT No.: PCT/UP2011/002225 2015/0017212 A9* 1/2015 Nagata ................. A61K9/0043 424/400 S 371 (c)(1), (2), (4) Date: Dec.
    [Show full text]
  • The Role of Viral Glycoproteins and Tegument Proteins in Herpes
    Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2014 The Role of Viral Glycoproteins and Tegument Proteins in Herpes Simplex Virus Type 1 Cytoplasmic Virion Envelopment Dmitry Vladimirovich Chouljenko Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Veterinary Pathology and Pathobiology Commons Recommended Citation Chouljenko, Dmitry Vladimirovich, "The Role of Viral Glycoproteins and Tegument Proteins in Herpes Simplex Virus Type 1 Cytoplasmic Virion Envelopment" (2014). LSU Doctoral Dissertations. 4076. https://digitalcommons.lsu.edu/gradschool_dissertations/4076 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. THE ROLE OF VIRAL GLYCOPROTEINS AND TEGUMENT PROTEINS IN HERPES SIMPLEX VIRUS TYPE 1 CYTOPLASMIC VIRION ENVELOPMENT A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Interdepartmental Program in Veterinary Medical Sciences through the Department of Pathobiological Sciences by Dmitry V. Chouljenko B.Sc., Louisiana State University, 2006 August 2014 ACKNOWLEDGMENTS First and foremost, I would like to thank my parents for their unwavering support and for helping to cultivate in me from an early age a curiosity about the natural world that would directly lead to my interest in science. I would like to express my gratitude to all of the current and former members of the Kousoulas laboratory who provided valuable advice and insights during my tenure here, as well as the members of GeneLab for their assistance in DNA sequencing.
    [Show full text]