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Azu Td 8217412 Sip1 M.Pdf BUNYAVIRUS PERSISTENCE IN AEDES ALBOPICTUS CELL CULTURES. Item Type text; Dissertation-Reproduction (electronic) Authors FLORKIEWICZ, ROBERT ZIGMOND. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 03/10/2021 21:44:17 Link to Item http://hdl.handle.net/10150/185459 8217412 Florkiewicz, Robert Zigmond BUNY AVIRUS PERSIS1ENCE IN AEDES ALBOPlcruS CELL CULTURES The University ofArizona PH.D. 1982 University Microfilms Intern atio nal 300 N. Zeeb Road, Ann Arbor, MI 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark_...j_. 1. Glossy photographs or pages ~ 2. Colored illustrations, paper or print __ 3. Photographs with dark background ~ 4. Illustrations are poor copy __ 5. Pages with black marks, not original copy __ 6. Print shows through as there is text on both sides of page __ 7. Indistinct, broken or small print on several pages __ 8. Print exceeds margin requirements __ 9. Tightly bound copy with print lost in spine __ 10. Computer printout pages with indistinct print __ 11. Page(s) lacking when material received, and not available from school or author. 12. Page(s) seem to be missing in numbering only as text follows. 13. Two pages numbered . Text follows. 14. Curling and wrinkled pages __ 15. ~her____________________________________ _ University Microfilms International BUNYAVIRUS PERSISTENCE IN AEDES ALBOPICTUS CELL CULTURES by Robert Zigmond Florkiewicz A Dissertation Submitted to the Faculty of the DEPARTMENT OF CELLULAR AND DEVELOPMENTAL BIOLOGY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY WITH A MAJOR IN CELLULAR AND DEVELOPMENTAL BIOLOGY In the Graduate College THE UNIVERSITY OF ARIZONA 1 982 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Robert Zigmond Florkiewicz entitled Bunyavirus Persistence in Aedes Albopictus Cell Cultures and recommend that it be accepted as fulfilling the dissertation requirement for , 'I )/ <' t.:?/ Date Date Date Date Date I I Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation req)irelJlent. ,'/ j' . /j /// ;:" // / // • /ft:"7 ''--_L~';/,' ;;~ , (/)' / ("/l#· /" -;;~ , " \7 C.. 't.t.. .... ',.' ... / -A"; .Dissertatio Director Date I ! STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to bor­ rowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or re­ production of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the in­ terests of scholarship. In all other instances, however, permission must be obtained from the author. TO DAVID iii iv ACKNOWLEDGEMENTS There are an incredib~e number of people whose encouragement, assistance, criticism and patience I can never forget. Throughout my undergraduate education at Fairleigh Dickinson University, Rutherford, New Jersey; my graduate education at the University of Arizona; and during the time I was employed as an assistant scientist at both The Roche Institute of Molecular Biology, Nutley, New Jersey and the University of Arizona I am deeply indebted to the following people: Dr. Sarah Catovic, Dr. Emil Zebynei~ Dr. Brian McAuslian, Donna Diviney, Dr. Ann Skalka, Dr. Arthur Weissbach, Dr. Aaron Shatkin, Dr. Paul Bartl, Dr. Richard Adler, Dr. Barry Garfinkle, Dr. William McClements, Robert Welborn, Ralph Lardieri, Patricia Perkowski, Alfretta Danielewicz, Lynda Ogle, Louise Duhamel, Ysabel Contrearas, Hope Peralta, Lorie Ballesteros, Bill Lindenfeld, Esther Sherberg, Rydia Almy, Dr. Vasken Aposhian, Dr. Jack Rose, Dr. Kaoru Matsuda, Dr. Michael Parker, Gail Hewlett and Dr. James O'Leary. I truly thank my fellow graduate students for sharing the past four years. Particularly Keith Mott, Ali Behzadian and Hugh Mason. I would like to thank Pat Mason for proof-reading this dissertation. I would like to thank Evie Kurtz for typing and final preperation of the dissertation. I would like to thank Dr. Jennifer Hall, 'Dr. Neil Mendelson, Dr. Don Bourque and Dr. Larry McReynolds for agreeing to be members of my dissertation committee. I would like to thank Sandra Bevacqua, Laura Wakeford and Kim Tau for tolerating me in the lab and my occasional chauvinism. There are those who are most difficult to thank. Marty, as dissertation advisor you have certainly aged listening to me. You have helped me through many difficult times. You have been my friend, I thank you. From my heart and soul I give my thanks and my love to David Florkiewicz, Denise Florkiewicz, Melanie Spriggs, Ewa Przybytkowska, Kathleen Storm, Mom and Dad. Without you I could not continue. THANK YOU ALL. v TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS. vii LIST OF TABLES ix ABSTRACT .. x 1. INTRODUCTION 1 Virus .. 1 Arthropod Transmission of Virus. 7 Bunyavirus Molecular Biology . 9 Bunyavirus Reassortants ••...•. 12 Analysis of Tissue Culture Systems 15 Persistence. • . • 17 ts Mutants . • . • . 18 Defective Interfering. 19 Present Study .•. 24 2. MATERIALS AND METHODS. 26 Cells. • . 26 Viruses. • . 27 Virus Purification 27 Interacellular Extraction. 28 Radiolabeling. • • . 28 Gel Electrophoresis. • 29 Virus Growth Curves. • . 29 Infectious Center Assay. 30 Bacillus Thuringiensis var. Israelensis Toxin Assay. 30 Synthesis of DNA Representing Bunyavirus Genome RNA. 31 Preparation of pBR322 DNA and Annealing with Bunyavirus Specific DNA • • • • • . • . • • . • . • 33 Preparation of Bacteria and Transformation 33 Preparation of Cloned DNA and Analysis 34 3. RESULTS. ••• 36 Primary Infections and Growth Curves 36 Characteristics of Virus Released During Aedes Albopictus Cell Infections . 40 Virus Specific RNA Profiles •..• 42 Are Cells Persistently Infected? 47 vi TABLE OF CONTENTS--Continued Page Interference • • . • . 59 DI Particles ••.•.•••....••.... 63 Persistent Virus Return to Vertebrate Cells •• 64 Comparative Effects of Bacillus Thuringiensis vac. Isralensis Cytotoxin ••..... 65 Molecular Cloning of Bunyavirus Genome. 71 4. DISCUSSION • • 83 Summa ry . • . ..••.. 83 Virus Host Relationship ••.•.•••....•.. 84 Characteristics of Infections with Virus from PI Cells 86 Interference • • • • . • 90 ts Mutants . • . • . • • . 94 Superinfections •..•..•.. 97 Virus Growth Analysis . • . .. ••• 101 Characteristics of Toxin Response .....•. 103 Comparisons between Cell Culture and the Insect Systems .• 105 Conclusions 110 REFERENCES . 114 vii LIST OF ILLUSTRATIONS Figure Page 1. Aedes albopictus Cells Infected With Inkoo or Uukuneimi Virus . 37 2. BHK Cell Culture Infections with Inkoo or Uukuneimi Vi rus . • . 39 3. Pl aque Morphology 41 4. RNA Profiles from Inkoo Infected Aedes albopictus Cell Cultures .... 44 5. Standard Virus RNA Profile 46 6. Uukuneimi Persistently Infected Aedes albopictus Cell Superinfections ........ -.-.---. 48 7. Inkoo Persistently Infected Aedes albopictus Cell Superinfections . 49 8. California Serogroup Virus Superinfection of Inkoo Persistently Infected Aedes albopictus Cells 51 9. California Serogroup Virus Superinfection of Uukuneimi Persistently Infected Aedes albopictus Cells ... .. 52 10. Superinfections of Inkoo Persistently Infected Aedes albopictus cells at Passage 150 . 54 11. Superinfections of Uukuneimi Persistently Infected Aedes albopictus Cells at Passage 150. 55 12. Aedes albopictus Cell Infections With Additional California Serogroup Viruses . 56 13. Virus Stability. 58 14. Co-infection of Aedes albopictus Cell Cultures 60 15. Co-infection of BHK Cell Cultures .. 62 viii LIST OF ILLUSTRATIONS--Continued 16. Intracellular RNA Profile From BHK Cells Infected With Inkoo PI Virus •...•. 66 17. Outline of Cloning Strategy. 72 18. Virus Purification .•... 73 19. Polynucleotide Phosphorylase Directed Po1yadenylation of RNA • • . • . • • • . • • • • • 75 20. Alkaline Gel Analysis of DNA Synthesis 78 2l. Pst Digestion of Uukuneimi Clones .•. 81 22. A Representative Arbovira1 Life Cycle • 106 ix LIST OF TABLES Table Page 1. Virus Identification ....••......•. 5 2. Adsorption of Toxin by Preincubation with Cells 68 3. Effect of BTl Toxin on Mosquito Cells in Culture. 70 x ABSTRACT Some viruses which infect plants, animals and man are transmitted by an intermediary arthropod vector. The viruses for which this is true are termed arboviruses (Arthropod-borne-viruses). In many instances the virus delivered to the new host results in the establishment of a disease state and/or the death of the infected host. In all instances, however, the arthropod (invertebrate) vector is apparently
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