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Host-Virus Interactions in the Pacific White Shrimp, Litopenaeus Vannamei Duan Sriyotee Loy Iowa State University

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Host-Virus Interactions in the Pacific White Shrimp, Litopenaeus Vannamei Duan Sriyotee Loy Iowa State University Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2014 Host-virus interactions in the Pacific white shrimp, Litopenaeus vannamei Duan Sriyotee Loy Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Virology Commons Recommended Citation Loy, Duan Sriyotee, "Host-virus interactions in the Pacific white shrimp, Litopenaeus vannamei" (2014). Graduate Theses and Dissertations. 13777. https://lib.dr.iastate.edu/etd/13777 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Host-virus interactions in the Pacific white shrimp, Litopenaeus vannamei by Duan Sriyotee Loy A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Veterinary Microbiology Program of Study Committee: Lyric Bartholomay, Co-Major Professor Bradley Blitvich, Co-Major Professor D.L. Hank Harris Cathy Miller Michael Kimber Iowa State University Ames, Iowa 2014 Copyright © Duan Sriyotee Loy, 2014. All rights reserved. ii TABLE OF CONTENTS CHAPTER 1: GENERAL INTRODUCTION...…………………………………………...1 Introduction…………………………………………………………………………………………1 Dissertation Organization…………………………………………………………………………3 Literature Review ................................................................................................................ 3 References ........................................................................................................................ 23 CHAPTER 2: CHARACTERIZATION OF NEWLY REVEALED SEQUENCES IN THE INFECTIOUS MYONECROSIS VIRUS GENOME IN LITOPENAEUS VANNAMEI ............................................................................................................... 33 Abstract ............................................................................................................................. 33 Introduction ....................................................................................................................... 35 Materials and Methods ...................................................................................................... 37 Results and Discussion ..................................................................................................... 47 Acknowledgments ............................................................................................................. 53 References ........................................................................................................................ 54 CHAPTER 3: VARIATION IN VIRULENCE IN GEOGRAPHICALLY DISTINCT ISOLATES OF INFECTIOUS MYONECROSIS VIRUS IN THE PACIFIC WHITE SHRIMP, LITOPENAEUS VANNAMEI .................................................................... 71 Abstract ............................................................................................................................. 71 Introduction ....................................................................................................................... 72 Materials and Methods ...................................................................................................... 74 Results .............................................................................................................................. 78 Discussion ......................................................................................................................... 81 References ........................................................................................................................ 84 CHAPTER 4: COMPARATIVE ANALYSIS OF SUPPRESSION OF IMMEDIATE EARLY, EARLY AND LATE GENES OF WHITE SPOT SYNDROME VIRUS ........ 96 Abstract ............................................................................................................................. 96 Introduction ....................................................................................................................... 97 Materials and Methods .................................................................................................... 100 Results ............................................................................................................................ 104 Discussion ....................................................................................................................... 107 Acknowledgements ......................................................................................................... 111 References ...................................................................................................................... 112 CHAPTER 5: DIRECT DELIVERY OF DOUBLE STRANDED RNA INTO THE FOREGUT LUMEN OF LITOPENAEUS VANNAMEI DEMONSTRATES PROTECTION AGAINST WHITE SPOT SYNDROME VIRUS AND INFECTIOUS MYONECROSIS VIRUS ......................................................................................... 120 Abstract ........................................................................................................................... 120 Introduction ..................................................................................................................... 121 Materials and Methods .................................................................................................... 124 Results ............................................................................................................................ 128 Discussion ....................................................................................................................... 130 Acknowledgements ......................................................................................................... 131 References ...................................................................................................................... 132 iii CHAPTER 6: GENERAL CONCLUSIONS ............................................................ 141 ACKNOWLEDGEMENTS ....................................................................................... 145 1 CHAPTER1: GENERAL INTRODUCTION Introduction The global population is predicted to expand from the current size to an estimated 7 billion to 9.1 billion by 2050. Aquatic food products, the fastest growing of the food-producing sectors, are predicted to supply more than 50% of this sector by 2015 (Stentiford et al. 2012). The aquaculture industry continues to grow rapidly, particularly in the case of cultured marine shrimp. Indeed, cultured shrimp harvest was an estimated 3.5 million metric tons representing a sale value of $14.6 billion USD in 2009 (Moss et al. 2012). In the early era of shrimp farming expansion (1970s-1980s), Penaeus monodon was the major in culture species, and the industry relied on wild caught stocks to produce postlarvae (PL) and adult broodstock to supply seed for the industry (Moss et al. 2012). However, this is a high-risk practice because wild-caught animals can be carriers of diseases that then can be introduced into culture facilities. The industry largely has shifted from using wild seed stocks of P. monodon to using specific pathogen-free (SPF) domesticated lines of Litopenaeus vannamei, a North American species. L. vannamei was introduced to Asian crustacean fisheries in the late 1990s and soon became the dominant species in Asia and other shrimp farming regions (Flegel 2012, Lightner et al. 2012). Aquaculture practices have also changed from a traditional extensive farming to semi-intensive or intensive systems that are more efficient production systems. The adoption of L. vannamei in culture systems worldwide, along with effective biosecurity measures and intensive farming techniques, have resulted in 2 rapid growth of the global shrimp farming industry over the last four decades (Flegel 2012, Moss et al. 2012). This growth has been hampered only by pandemic viral diseases that have devastated harvests and yields; therefore, they have also greatly impacted the economics of the shrimp industry worldwide (Flegel 2012, Lightner et al. 2012). Thus, development of strategies to prevent and control viral diseases is of tremendous interest and importance for the sustainability of this industry. The research described in this dissertation emphasizes two virus pathogens: 1) Infectious myonecrosis virus (IMNV) and 2) White spot syndrome virus (WSSV) that have caused more than $16 billion in losses combined since the first outbreaks (Lightner et al. 2012). The first manuscript of this dissertation describes the characterization of newly revealed sequences in the IMNV genome. This paper provides evidence that additional sequences exist in the IMNV genome, resulting in an overall genome length that is 8226 base pairs (bp) as compared to the original description of 7561 bp. The second manuscript describes variation in virulence in geographically distinct isolates of IMNV from Brazil and Indonesia. The third manuscript describes the utilization of RNAi to silence WSSV by targeting early gene expression and that is more effective at suppressing virus replication than suppressing late genes. This result provides a new target antiviral for WSSV. The fourth manuscript presents the development of direct delivery methodology of double-stranded
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