Diversity of Baculoviruses Isolated from Cutworms (Agrotis Spp.)
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Diversity of Baculoviruses Isolated from Cutworms (Agrotis spp.) Dissertation zur Erlangung des Grades Doktor der Naturwissenschaften am Fachbereich Biologie der Johannes Gutenberg-Universität Mainz Jörg Thomas Wennmann Mainz, 2014 Dekan: 1. Berichterstatter: 2. Berichterstatter: Tag der mündlichen Prüfung: 26.2.2015 Für meine Familie Content List of Abbreviations ................................................................................................................ 6 Chapter 1: General Introduction ............................................................................................ 8 An Introduction to Baculoviruses ........................................................................................... 8 Baculovirus Biology ............................................................................................................... 9 Classification and Nomenclature .......................................................................................... 11 The Baculovirus Infection Cycle .......................................................................................... 13 Baculovirus Genomics .......................................................................................................... 18 Application of Baculoviruses as Biological Bontrol Agents ................................................ 21 The Agrotis Baculovirus Complex ....................................................................................... 23 Thesis Outline .......................................................................................................................... 29 Chapter 2: The Genome Sequence of Agrotis segetum Nucleopolyhedrovirus B (AgseNPV-B) Reveals a New Baculovirus Species Within the Agrotis Baculovirus Complex ............................................................................................................. 32 Abstract ................................................................................................................................. 32 Introduction .......................................................................................................................... 33 Materials and Methods ......................................................................................................... 35 Results and Discussion ......................................................................................................... 38 Conclusions .......................................................................................................................... 62 Chapter 3: Detection and Quantitation of Agrotis Baculoviruses in Mixed Infections ................................................................................................................................. 64 Abstract ................................................................................................................................. 64 Introduction .......................................................................................................................... 65 Material and Methods ........................................................................................................... 67 Results .................................................................................................................................. 74 Discussion ............................................................................................................................. 82 Chapter 4: Competitive Interaction of the Agrotis segetum Granulovirus (AgseGV) and Agrotis segetum Nucleopolyhedrovirus B (AgseNPV-B) in Simultaneously Infected Cutworm Larvae .......................................................................... 86 Abstract ................................................................................................................................. 86 Introduction .......................................................................................................................... 87 Materials and Methods ......................................................................................................... 88 Results .................................................................................................................................. 92 Discussion ........................................................................................................................... 100 Chapter 5: General Discussion ............................................................................................ 104 References ............................................................................................................................. 110 Summary ............................................................................................................................... 124 Danksagung........................................................................................................................... 126 Curriculum Vitae ................................................................................................................. 128 LIST OF ABBREVIATIONS LIST OF ABBREVIATIONS (Baculovirus names and their abbreviations on pages 19 – 20; Table 1.1) % percent °C degree Celsius µg microgram µl microlitre µM micromolar aa amino acid AGERI Agricultural Genetic Engineering Research Institute B.t.a. Bacillus thuringiensis subspecies azawai bp base pair BV budded virus C cytosine cm centimeter ddH20 bidistilled water dH20 distilled water DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate ds double stranded e.g. for example et al. and others exp exponential function g gravitational; multiple of g G guanine Glu glutamic acid gran granulin GV granulovirus h hour ha hectare His histidine HRI Horticulture Research International ICTV International Committee on Nomenclature of Viruses JKI Julius Kühn Institute K-2-P Kimura-2-parameter kbp kilo base pairs LC10 10% lethal concentration LC50 median lethal concentration LD50 median lethal dose log logarithm LSC low speed centrifugation M molar 6 MBV Monodon baculovirus mM millimolar mm millimetre MNPV multiple nucleopolyhedrovirus MP maximum parsimony NCBI National Centre for Biotechnology Information ng nanogram NGS next generation sequencing nm nanometre no. number NPV nucleopolyhedrovirus nt nucletide NV nudivirus OB occlusion body ODV occlusion derived virus ORF open reading frame PCR polymerase chain reaction PFGE pulsed-field gel electrophoresis pH measure of acidity of basicityof aqueous solutions PM peritrophic membrane polh polyhedrin qPCR quantitative polymerase chain reaction REN restriction endonuclease RFLP restriction fragment length polymorphism RNA ribonucleic acid SCC sucrose cushion centrifugation SDS sodium dodecyl sulfate sec second SGHV salivary gland hypertrophy virus SGU sucrose gradient ultracentrifugation SNPV single nucleopolyhedrovirus spp. species pluralis; multiple species TA annealing temperature TAE tris base, acetic acid and EDTA buffer solution Taq DNA polymerase from Thermus aquaticus TBE tris base, boric acid and EDTA buffer solution TBR tree-bisection-reconnection TE tris base and EDTA buffer solution U enzyme unit UV ultraviolet light V volt v/v volume per volume w/w weight per weight WSSV white spot syndrome virus Xaa unspecific amino acid 7 CHAPTER 1: GENERAL INTRODUCTION CHAPTER 1: GENERAL INTRODUCTION An Introduction to Baculoviruses The description of diseased insect larvae goes back to the silk production in ancient China that based on the caterpillars of the silk moth Bombyx mori. In 1527, long time before any knowledge of microorganisms and viruses, an Italian poet described symptoms of diseased silk moth larvae, which are known today to be caused by baculoviruses (Benz, 1986). The craving of men for costly and fine silk let not only the silk moth and humans share a long part of history, but also baculoviruses that are our constant companion in a wide variety of fields, until today. One outstanding characteristic of this virus group encouraged research and their investigation: the formation of occlusion bodies (OBs). These virus protective protein coats are observable under the light microscope and aroused the interest of scientists around the world. Since the middle of the 20th century, the potential of baculoviruses as prospective biological control agents of insect pests was understood. First baculovirus based biocontrol agents were developed, increasing the interest in the biology and the molecular setup of these fascinating viruses (Miller, 1997). Since then, various baculovirus based biocontrol agents have been registered and applied for a sustainable control of insect pests in agriculture, horticulture and forestry. Stimulated by groundbreaking progress in cell culture techniques baculoviruses are further tested and applied in the fields of eukaryotic gene expression, gene therapy and vaccine production (for review van Oers, 2011). 8 Baculovirus Biology In several insect virus groups, including the baculoviruses (Baculoviridae), cypoviruses (Reoviridae) and entomopoxviruses (Poxviridae) embedding of virions within huge crystalline protein matrixes is found (Eberle et al., 2012; King et al., 2011). These so-called occlusion bodies (OBs) (Fig. 1.1A and B) protect the virions from hazardous environmental conditions, such as UV light, drought, excess humidity as well as enzymatic degradation. OBs are able to persist outside the host for a certain period of time (Rohrmann, 2013). Baculoviruses have been isolated from