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Use of Next Generation Sequencing to Improve Identification and Quantification of Baculovirus Diversity

Gueli Alletti, Gianpiero (2018)
Use of Next Generation Sequencing to Improve Identification and Quantification of Baculovirus Diversity.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

The family of Baculoviridae comprises occluded double-stranded DNA viruses with rod-shaped, enveloped virions infecting larval stages of the insect orders Lepidoptera, Diptera and Hymoptera. Due to their narrow host range, their high virulence to target insects and the absence of adverse effects to human health and the environment, several baculovirus species are already in use as biological control agents in pest control. One possibility for their use, is for control of cutworms. Cutworms are severe soil pests of many agricultural and horticultural crops that live near or underneath the soil surface and feed polyphagous on seedlings, stems, roots and other parts of the plant and among other species they also comprise the genus Agrotis. In the past, four baculovirus species were isolated from Agrotis segetum and A. ipsilon, namely: Agrotis segetum nucleopolyhedrovirus A (AgseNPV-A), Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B), Agrotis ipsilon nucleopolyhedrovirus (AgipNPV) and Agrotis segetum granulovirus (AgseGV). All these species are promising candidates for a baculovirus based plant protection product for the control of cutworms. The focus here was laid on AgseGV and AgseNPV-B, as these species have already been extensively investigated at the Julius Kühn Institute in Darmstadt. The European isolate AgseGV-DA was identified as a slow-killing type I granulovirus due to the low dose mortality response and the tissue tropism of infection restricted solely to the fat body of infected larvae. The fully NGS sequenced genome of AgseGV-DA showed no sequence variation and was more than 99% identical to those of the Chinese isolates AgseGV-XJ and AgseGV-L1. The AgseGV-DA genome is 131,557 bp in length and contains 149 putative open reading frames, including 37 baculovirus core genes and the per os infectivity factor ac110 as well as one putative non-hr like origin of replication. AgseGV has a distinct enhancin gene, with a distant relation to the enhancins from the genus Betabaculovirus. Although all three isolates belong to the species Agrotis segetum granulovirus, AgseGV-DA was proposed as the type isolate due to the complete sequence and pathology description to the International Committee for Virus Taxonomy (ICTV). The permissivity of the insect cell culture AiE1611T was evaluated for AgseGV-DA and AgseNPV-B which is the prerequisite for in vitro experiments. While AgseGV and AgseNPV-B can co-infect susceptible larvae, it has been demonstrated that AiE1611T is only permissive for AgseNPV-B but not for AgseGV, as evidenced by two experiments with baculovirus derived DNA transfected cells and in infections with hemolymph containing budded virus from infected larvae. AgseNPV-B produced large numbers of occlusion bodies and the virus was selected in one round of plaque purification for additional experiments. Among the twelve genetical and morphological identical isolates, one isolate termed PP2 was used in particular to investigate the virus stability in ten consecutive passages in AiE1611T cells. The cell culture was further used to generate a recombinant AgseNPV-B clone, the bacmid bAgseNPV-B. AgseNPV-B PP2 showed endured a high genomic stability during the passages, while the dose-mortality responses in larvae were scattering over ten passages. A loss of virulence was observed right after the first round of passaging, then the activity of PP2 remained stable over ten passages. This was observed by the absence of few polyhedra phenotypes in phase contrast microscopy and by next-generation sequencing (NGS) of five selected passages. NGS sequencing revealed that defective particles were absent over ten passages. The few single nucleotide polymorphisms (SNPs) detected by this approach were mostly within homologous repeat sequences, which make a correct mapping of the short sequencing reads difficult due to their repetitive nature. Thus, no conclusions were drawn from these SNPs. The proof of method for detecting large deletions was given by sequencing the bacmid bAgseNPV-B (chapter V). This bacmid was deleted by roughly 43 kb in the AgseNPV-B genome but still possesses the full bacterial recombinant DNA inserted by homologous recombination into hr6 of AgseNPV-B. The deletion additionally affected 42 orfs and two hrs. In consequence, AiE1611T cells could be transfected with DNA of bAgseNPV-B and showed cytopathological effects, however the infection was blocked at an early stage with missing DNA replication and no spreading of virus infection was observed. This is possibly correlated to the deletion of lef-1, lef-2 and me53 in bAgseNPV-B The second baculovirus example presented here, is the use of Cydia pomonella granulovirus (CpGV). CpGV has a narrow host range and is highly virulent against the Lepidopteran pest species Cydia pomonella (codling moth; CM) and to a lower extent to a very few closely related Tortricids and has been developed and intensively used as a commercial biocontrol agent of CM in virtually all pome fruit production areas. CpGV comprises several isolates and extensive phylogenetic has brought evidence that all isolates can be divided into five genome groups or lineages. The basics for this classification are distinct trends on the genome level such as, insertions and deletions, and as focused here genome group specific SNPs. Data-sets generated of NGS of commercially available CpGV isolates were combined with their responses to codling moth types expressing different types (I - III) of CpGV resistance. In infection experiments, CpGV-0006 and CpGV-R5 were able to break type I resistance and to a lower extent also type III resistance, whereas CpGV-V15 overcame type I and the rarely occurring type II and type III resistance. Based on the distribution of SNPs in Illumina sequencing reads it was found that both CpGV-0006 and CpGV-R5 have highly similar genome group compositions, consisting of about two thirds of the CpGV genome group E and one third of genome group A. In contrast, CpGV-V15 is composed of about equal parts of CpGV genome group B and E. According to the identified genetic composition of these isolates, their efficacy towards different resistance types can be explained and predictions on the success of resistance management strategies in resistant CM populations can be made for future CpGV isolates based in this.

Typ des Eintrags: Dissertation
Erschienen: 2018
Autor(en): Gueli Alletti, Gianpiero
Art des Eintrags: Erstveröffentlichung
Titel: Use of Next Generation Sequencing to Improve Identification and Quantification of Baculovirus Diversity
Sprache: Englisch
Referenten: Jehle, Prof. Dr. Johannes ; Thiel, Prof. Dr. Gerhard
Publikationsjahr: 2018
Ort: Darmstadt
Datum der mündlichen Prüfung: 7 Dezember 2018
URL / URN: https://tuprints.ulb.tu-darmstadt.de/8287
Kurzbeschreibung (Abstract):

The family of Baculoviridae comprises occluded double-stranded DNA viruses with rod-shaped, enveloped virions infecting larval stages of the insect orders Lepidoptera, Diptera and Hymoptera. Due to their narrow host range, their high virulence to target insects and the absence of adverse effects to human health and the environment, several baculovirus species are already in use as biological control agents in pest control. One possibility for their use, is for control of cutworms. Cutworms are severe soil pests of many agricultural and horticultural crops that live near or underneath the soil surface and feed polyphagous on seedlings, stems, roots and other parts of the plant and among other species they also comprise the genus Agrotis. In the past, four baculovirus species were isolated from Agrotis segetum and A. ipsilon, namely: Agrotis segetum nucleopolyhedrovirus A (AgseNPV-A), Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B), Agrotis ipsilon nucleopolyhedrovirus (AgipNPV) and Agrotis segetum granulovirus (AgseGV). All these species are promising candidates for a baculovirus based plant protection product for the control of cutworms. The focus here was laid on AgseGV and AgseNPV-B, as these species have already been extensively investigated at the Julius Kühn Institute in Darmstadt. The European isolate AgseGV-DA was identified as a slow-killing type I granulovirus due to the low dose mortality response and the tissue tropism of infection restricted solely to the fat body of infected larvae. The fully NGS sequenced genome of AgseGV-DA showed no sequence variation and was more than 99% identical to those of the Chinese isolates AgseGV-XJ and AgseGV-L1. The AgseGV-DA genome is 131,557 bp in length and contains 149 putative open reading frames, including 37 baculovirus core genes and the per os infectivity factor ac110 as well as one putative non-hr like origin of replication. AgseGV has a distinct enhancin gene, with a distant relation to the enhancins from the genus Betabaculovirus. Although all three isolates belong to the species Agrotis segetum granulovirus, AgseGV-DA was proposed as the type isolate due to the complete sequence and pathology description to the International Committee for Virus Taxonomy (ICTV). The permissivity of the insect cell culture AiE1611T was evaluated for AgseGV-DA and AgseNPV-B which is the prerequisite for in vitro experiments. While AgseGV and AgseNPV-B can co-infect susceptible larvae, it has been demonstrated that AiE1611T is only permissive for AgseNPV-B but not for AgseGV, as evidenced by two experiments with baculovirus derived DNA transfected cells and in infections with hemolymph containing budded virus from infected larvae. AgseNPV-B produced large numbers of occlusion bodies and the virus was selected in one round of plaque purification for additional experiments. Among the twelve genetical and morphological identical isolates, one isolate termed PP2 was used in particular to investigate the virus stability in ten consecutive passages in AiE1611T cells. The cell culture was further used to generate a recombinant AgseNPV-B clone, the bacmid bAgseNPV-B. AgseNPV-B PP2 showed endured a high genomic stability during the passages, while the dose-mortality responses in larvae were scattering over ten passages. A loss of virulence was observed right after the first round of passaging, then the activity of PP2 remained stable over ten passages. This was observed by the absence of few polyhedra phenotypes in phase contrast microscopy and by next-generation sequencing (NGS) of five selected passages. NGS sequencing revealed that defective particles were absent over ten passages. The few single nucleotide polymorphisms (SNPs) detected by this approach were mostly within homologous repeat sequences, which make a correct mapping of the short sequencing reads difficult due to their repetitive nature. Thus, no conclusions were drawn from these SNPs. The proof of method for detecting large deletions was given by sequencing the bacmid bAgseNPV-B (chapter V). This bacmid was deleted by roughly 43 kb in the AgseNPV-B genome but still possesses the full bacterial recombinant DNA inserted by homologous recombination into hr6 of AgseNPV-B. The deletion additionally affected 42 orfs and two hrs. In consequence, AiE1611T cells could be transfected with DNA of bAgseNPV-B and showed cytopathological effects, however the infection was blocked at an early stage with missing DNA replication and no spreading of virus infection was observed. This is possibly correlated to the deletion of lef-1, lef-2 and me53 in bAgseNPV-B The second baculovirus example presented here, is the use of Cydia pomonella granulovirus (CpGV). CpGV has a narrow host range and is highly virulent against the Lepidopteran pest species Cydia pomonella (codling moth; CM) and to a lower extent to a very few closely related Tortricids and has been developed and intensively used as a commercial biocontrol agent of CM in virtually all pome fruit production areas. CpGV comprises several isolates and extensive phylogenetic has brought evidence that all isolates can be divided into five genome groups or lineages. The basics for this classification are distinct trends on the genome level such as, insertions and deletions, and as focused here genome group specific SNPs. Data-sets generated of NGS of commercially available CpGV isolates were combined with their responses to codling moth types expressing different types (I - III) of CpGV resistance. In infection experiments, CpGV-0006 and CpGV-R5 were able to break type I resistance and to a lower extent also type III resistance, whereas CpGV-V15 overcame type I and the rarely occurring type II and type III resistance. Based on the distribution of SNPs in Illumina sequencing reads it was found that both CpGV-0006 and CpGV-R5 have highly similar genome group compositions, consisting of about two thirds of the CpGV genome group E and one third of genome group A. In contrast, CpGV-V15 is composed of about equal parts of CpGV genome group B and E. According to the identified genetic composition of these isolates, their efficacy towards different resistance types can be explained and predictions on the success of resistance management strategies in resistant CM populations can be made for future CpGV isolates based in this.

Alternatives oder übersetztes Abstract:
Alternatives AbstractSprache

Die Familie Baculoviridae umfasst doppelsträngige DNA Viren mit stabförmigen, umhüllten Virionen, welche Larvenstadien der Insektenordnungen Lepidoptera, Diptera und Hymenoptera infizieren. Aufgrund ihres oftmals sehr engen Wirtsspektrums und dem Fehlen von negativen Einflüssen auf die menschliche Gesundheit und die Umwelt, werden bereits einige Baculovirusarten in biologischen Pflanzenschutzmitteln verwendet.

Ein möglicher Einsatz ist die Bekämpfung von Erdraupen. Erdraupen stellen ernstzunehmende Bodenschadorganismen im Garten- und Ackerbau dar und umfassen neben anderen Arten auch die Gattung Agrotis. Sie leben nahe oder unterhalb der Oberfläche wo sie sich polyphag von den Setzlingen, Stämmen, Wurzeln und anderen Pflanzenteilen ernähren. In jüngster Vergangenheit wurden aus den Arten Agrotis segetum und A. ipsilon vier Baculovirusarten isoliert: Agrotis segetum nucleopolyhedrovirus A (AgseNPV-A), Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B), Agrotis ipsilon nucleopolyhedrovirus (AgipNPV) und Agrotis segetum granulovirus (AgseGV). All diese Arten sind vielversprechende Kandidaten für biologische Insektizide gegen Erdraupen auf Basis von Baculoviren. Der Fokus dieser Arbeit lag jedoch auf AgseGV und AgseNPV-B aufgrund der langjährigen Forschungserfahrung mit diesen beiden Arten am Julius-Kühn-Institut in Darmstadt.

Aufgrund seiner geringen Mortalität in A. segetum Larven und des beobachteten Zelltropismus, der aufe eine Infektion des Fettkörpers und des Mitteldarmepitheliums beschränkt ist, wurde das europäische Isolat AgseGV-DA als langsam wirkendes Typ I Granulovirus identifiziert. Die vollständige mit NGS sequenzierte Genomsequenz beinhaltete lediglich einen homogenen Genotyp und war mehr als 99% identisch zu den beiden chinesischen Isolaten AgseGV-XJ und AgseGV-L1. Das AgseGV-DA Genom hat eine Größe von 131,557 bp und enthält 149 mögliche offene Leserahmen (orfs), unter anderem 37 Baculoviruskerngene und den Infektionsfaktor ac110, sowie einen möglichen non-hr Replikationsursprung. AgseGV besitzt für ein spezielles enhancin Gen, welches eine entfernte Verwandtschaft zu jenen enhancin Genen aufweist, die bisher in der Gattung Betabaculovirus entdeckt wurden. Wenngleich alle drei AgseGV Isolate derselben Spezies angehören, so wurde AgseGV-DA als das speziesbeschreibende Isolat vom Internationalen Komitee für Virustaxonomie (ICTV) wegen seiner vollständigen Beschreibung der Genomsequenz und Pathologie ausgewählt.

Die Permissivität der Insektenzellkultur AiE1611T gegenüber AgseGV-DA und AgseNPV-B, wurrde untersucht, da sie die Grundvoraussetzung jedwelcher in vitro Experimente darstellt. Während AgseGV und AgseNPV-B anfällige Larven koinfizieren könne, zeigte sich in Experimenten mit transfizierter DNA und infektiöser Hämolymphe von infizierten Larven, dass AiE1611T Zellen lediglich für AgseNPV-B permissiv sind, aber jedoch nicht für AgseGV. In AiE1611T Zellen wurden vielfache Okklusionskörper von AgseNPV-B produziert und das Virus wurde in einer Runde eines „Plaque Purification“ Assay für die weiteren Experimente selektiert. Von zwölf genotypisch und morphologisch identischen Isolaten, wurde eines, PP2 genannt, benutzt um die Virusstabilität in zehn aufeinanderfolgenden Passagen in AiE1611T Zellen untersucht. Weiterhin wurde die Zellkultur für die Untersuchung eines rekombinanten Virusklons, dem Bacmid bAgseNPV-B, benutzt. AgseNPV-B PP2 erwies sich als genomisch stabil, wohingegen die Infektionsrate in Larven über die Passagen schwankte. Während in der ersten Passage ein Verlust der Virulenz zu verzeichnen war, blieb diese im weiteren Verlauf stabil. Gleichzeitig wurde über den Verlauf von zehn Passagen das Auftreten sogenannter „few polyhdera“ Mutanten weder mikroskopisch noch in der Genomanalyse beobachtet. Die wenigen Einzelnukleotidpolymorphismen (SNPs), welche mit diesem Verfahren ermittelt wurden, lagen im Bereich von „homologous repeat“ Sequenzen (hrs). Eine exakte Zuordnung der kurzen Sequenzierungs-Reads war deshalb aufgrund der repetitiven Natur dieser hrs nicht möglich, weswegen keine weiteren Schlüsse aus diesen SNPs gezogen wurden. Die Methodenvalidierung für die Detektion von Deletionen gelang mithilfe des Bacmids bAgseNPV-B. Dieses Bacmid war um schätzungsweise 43 kb im AgseNPV-B Genom deletiert, besaß aber weiterhin die bakterielle rekombinante DNA, welche durch homologe Rekombination im hr6 eingesetzt wurde. Die Deletion umfasste 42 orfs und zwei hrs. Als Konsequenz konnten AiE1611T zwar transfiziert werden, was an dem cytopathologischen Effekt ersichtlich war, die Infektion wurde jedoch zu einem frühen Zeitpunkt blockiert und verteilte sich nicht auf benachbarte Zellen. Dies korreliert wahrscheinlich mit dem Fehlen der AgseNPV-B Gene lef-1, lef-2 und me53.

Bei dem zweiten exemplarischen Baculovirus das hier behandelt wurde, handelt es sich um das Cydia pomonella granulovirus (CpGV). CpGV besitzt ein sehr enges Wirtsspektrum und wirkt sehr effizient gegen die lepidoptären Schädlinge Cydia pomonella (Apfelwickler, CM) und im geringeren Maße auch gegen nahe artverwandte Wickler. Aufgrund dessen wurde CpGV erfolgreich zu einem biologischen Insektizid entwickelt, welches nahezu im gesamten Obstbau Verwendung findet. CpGV umfasst einige Isolate und die intensive phylogenetische Forschung an CpGV konnte unter anderem beweisen, dass man die Isolate in fünf Genomgruppen/-typen unterteilen kann. Die Unterteilung gibt bestimmte Trends wieder, unter anderem Insertionen und Deletionen und abstammungslinienspezifische SNPs, welche hier im Fokus stehen. Datensätze aus NGS Sequenzierungen der kommerziellen Isolate CpGV-0006, CpGV-R5 und CpGV-V15 wurden bezüglich ihrer genomischen Zusammensetzung mit ihrer Wirkung in verchiedenen Larven Populationen mit den Resistenztypen I - III kombiniert. In Infektionen, waren die Isolate CpGV-0006 und CpGV-R5 resistenzbrechend für die Typ-I Resistenz und ebenso in geringerem Umfang für die Typ-III Resistenz. Aufgrund der SNP Verteilung der Illumina Sequenzen konnte gefolgert werden, dass beide Isolate genomisch beinahe identisch waren und zu zwei Dritteln aus Genomtyp E und zu einem Drittel aus Genomtyp A bestanden. Im Gegensatz dazu besteht CpGV-V15 zu etwa gleichen Teilen aus den Genomytpen B und E und bricht alle drei Resistenztypen. Die unterschiedliche Wirksamkeit lässt sich mit der Zusammensetzung aus verschiedenen Genomtypen erklären, was für zukünftige CpGV Isolate verwendet werden kann um ihren möglichen Erfolg in der Bekämpfung resistenter Apfelwicklerpopulationen vorherzusagen.

Deutsch
URN: urn:nbn:de:tuda-tuprints-82879
Sachgruppe der Dewey Dezimalklassifikatin (DDC): 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
Fachbereich(e)/-gebiet(e): 10 Fachbereich Biologie
Hinterlegungsdatum: 16 Dez 2018 20:55
Letzte Änderung: 16 Dez 2018 20:55
PPN:
Referenten: Jehle, Prof. Dr. Johannes ; Thiel, Prof. Dr. Gerhard
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 7 Dezember 2018
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