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.

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