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

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

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

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

Deciphering Single Nucleotide Polymorphisms and Evolutionary Trends in Isolates of the Cydia pomonella granulovirus

Six complete genome sequences of Cydia pomonella granulovirus (CpGV) isolates from Mexico (CpGV-M and CpGV-M1), England (CpGV-E2), Iran (CpGV-I07 and CpGV-I12), and Canada (CpGV-S) were aligned and analyzed for genetic diversity and evolutionary processes. The selected CpGV isolates represented recently identified phylogenetic lineages of CpGV, namely, the genome groups A to E. The genomes ranged from 120,816 bp to 124,269 bp. Several common differences between CpGV-M, -E2, -I07, -I12 and -S to CpGV-M1, the first sequenced and published CpGV isolate, were highlighted. Phylogenetic analysis based on the aligned genome sequences grouped CpGV-M and CpGV-I12 as the most derived lineages, followed by CpGV-E2, CpGV-S and CpGV-I07, which represent the most basal lineages. All of the genomes shared a high degree of co-linearity, with a common setup of 137 (CpGV-I07) to 142 (CpGV-M and -I12) open reading frames with no translocations. An overall trend of increasing genome size and a decrease in GC content was observed, from the most basal lineage (CpGV-I07) to the most derived (CpGV-I12). A total number of 788 positions of single nucleotide polymorphisms (SNPs) were determined and used to create a genome-wide SNP map of CpGV. Of the total amount of SNPs, 534 positions were specific for exactly one of either isolate CpGV-M, -E2, -I07, -I12 or -S, which allowed the SNP-based detection and identification of all known CpGV isolates

Using Next Generation Sequencing to Identify and Quantify the Genetic Composition of Resistance-Breaking Commercial Isolates of Cydia pomonella Granulovirus

The use of Cydia pomonella granulovirus (CpGV) isolates as biological control agents of codling moth (CM) larvae is important in organic and integrated pome fruit production worldwide. The commercially available isolates CpGV-0006, CpGV-R5, and CpGV-V15 have been selected for the control of CpGV resistant CM populations in Europe. 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. The genetic background of the three isolates was investigated with next generation sequencing (NGS) tools by comparing their nucleotide compositions to whole genome alignments of five CpGV isolates representing the known genetic diversity of the CpGV genome groups A to E. Based on the distribution of single nucleotide polymorphisms (SNPs) in Illumina sequencing reads, we found that the two isolates 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 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

Monitoring Insect Transposable Elements in Large Double-Stranded DNA Viruses Reveals Host-to-Virus and Virus-to-Virus Transposition

International audienceAbstract The mechanisms by which transposable elements (TEs) can be horizontally transferred between animals are unknown, but viruses are possible candidate vectors. Here, we surveyed the presence of host-derived TEs in viral genomes in 35 deep sequencing data sets produced from 11 host–virus systems, encompassing nine arthropod host species (five lepidopterans, two dipterans, and two crustaceans) and six different double-stranded (ds) DNA viruses (four baculoviruses and two iridoviruses). We found evidence of viral-borne TEs in 14 data sets, with frequencies of viral genomes carrying a TE ranging from 0.01% to 26.33% for baculoviruses and from 0.45% to 7.36% for iridoviruses. The analysis of viral populations separated by a single replication cycle revealed that viral-borne TEs originating from an initial host species can be retrieved after viral replication in another host species, sometimes at higher frequencies. Furthermore, we detected a strong increase in the number of integrations in a viral population for a TE absent from the hosts’ genomes, indicating that this TE has undergone intense transposition within the viral population. Finally, we provide evidence that many TEs found integrated in viral genomes (15/41) have been horizontally transferred in insects. Altogether, our results indicate that multiple large dsDNA viruses have the capacity to shuttle TEs in insects and they underline the potential of viruses to act as vectors of horizontal transfer of TEs. Furthermore, the finding that TEs can transpose between viral genomes of a viral species sets viruses as possible new niches in which TEs can persist and evolve