Biological and molecular characterization of Rachiplusia nu single nucleopolyhedrovirus, a promising biocontrol agent against the South American soybean pest Rachiplusia nu.

Rachiplusia nu (Lepidoptera: Noctuidae) is a key soybean pest in Argentina. Current management of this moth relies mainly on the use of synthetic insecticides and transgenic plants. In search of biological control-based alternatives, a baculovirus from R. nu (hereafter RanuNPV) was characterized and its insecticidal properties tested under laboratory conditions. RanuNPV occlusion bodies (OBs) were nearly tetrahedral, averaging 1.0 ±â€¯0.2 µm in their longest edge and containing singly enveloped nucleocapsids. Histopathology of infected late-instar larvae revealed broad tissue tropism, where fat bodies and epidermis were the most affected organs. Phylogenetic analysis of concatenated polh, lef-8 and lef-9 partial sequences classified RanuNPV as a new species that clusters with other group II alphabaculoviruses infecting larvae of Plusiinae. Bioassays performed with R. nu neonates determined the median lethal dosage to be approximately 2.5 OBs/larva; most insects died within 4-5 days post inoculation showing typical baculovirus-induced liquefaction. No effects were observed in other lepidopteran species assayed, including Spodoptera frugiperda, Cydia pomonella and Diatraea saccharalis. High pathogenicity and host specificity make RanuNPV a good candidate for controlling R. nu.

Biological and molecular features of Nosema rachiplusiae sp. n., a microsporidium isolated from the neotropical moth Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae).

Light, electron microscopy and DNA analyses were performed to characterize a microsporidium infecting Rachiplusia nu larvae from a laboratory rearing in Argentina. Diplokaryotic spores were oval and measured 3.61 ± 0.29 × 1.61 ± 0.14 µM (fresh). The spore wall was composed of an electron-dense exospore and an electron-lucent endospore, ca. 30 nm and 100-120 nm thick, respectively. The polar filament was arranged in a single rank of 10-12 coils (typically 11). Microsporidian cells were found in the cytoplasm, next to the endoplasmic reticulum (especially the prespore stages) and generally surrounded by electron-lucent spaces. The infection was polyorganotropic; the fat body appeared as the most heavily invaded tissue, followed by tracheal matrix and epidermis. A molecular phylogeny based on the small (SSU) and large subunit (LSU) ribosomal RNA genes clearly placed the new isolate within the "Nosema bombycis clade". Considering both SSU and LSU concatenated partial sequences, the microsporidium from R. nu showed 99.5% nucleotide similarity with N. bombycis and 99.8% with its closest relative, a microsporidium isolated from Philosamia cynthia. According to its genetic and biological features, the R. nu isolate is proposed as the new species Nosema rachiplusiae sp. n., expanding the limited knowledge on microsporidia associated to endemic South-American moths.

Molecular Identification of Helicoverpa armigera (Lepidoptera: Noctuidae: Heliothinae) in Argentina and Development of a Novel PCR-RFLP Method for its Rapid Differentiation From H. zea and H. gelotopoeon.

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae: Heliothinae) is among the most voracious global pests of agriculture. Adults of this species were identified recently in northern Argentina by dissection of male genitalia. In this work, a rapid and simple molecular tool was designed to distinguish H. armigera from the morphologically similar indigenous bollworms Helicoverpa zea (Boddie) and Helicoverpa gelotopoeon (Dyar), regardless of the life stage. Amplification of partial COI gene with a new primer pair, and subsequent digestion with endonuclease HinfI, yielded different RFLP profiles for the three main Helicoverpa pests currently present in South America. The method was validated in Helicoverpa specimens collected across Argentina, whose identity was further corroborated by COI sequencing and phylogenetic analysis. The data reported here constitute the first molecular confirmation of this pest in the country. The survey revealed the occurrence of H. armigera in northern and central Argentina, including the main soybean- and maize-producing area.

Analysis of a chitinase from EpapGV, a fast killing betabaculovirus.

The main function of baculoviral chitinase protein (V-CHIA) is to promote the final liquefaction of infected host larvae, facilitating the dispersion of occlusion bodies (OBs) in the environment. In this study, a v-chiA from Epinotia aporema Granulovirus (EpapGV) was identified and characterized. The 1,713 base pairs long open reading frame encodes a protein of 570 amino acids with a predicted molecular weight of 63 kDa. EpapGV V-CHIA sequence alignment resulted 62 % identical to Pieris rapae GV and Blastp search revealed a high conservation among all baculovirus chitinases. Amino acid sequence analysis indicated that the C-terminal KDEL present in most alphabaculovirus chitinases is absent in EpapGV V-CHIA, as well as in the rest of the betabaculoviruses. Phylogenetic analysis was performed with bacterial, lepidopteran, and baculoviral chitinase sequences available in databases. Using an AcMNPV bacmid (bApGOZA) a recombinant Ac-chiAEpapGV was obtained in order to overexpress EpapGV V-CHIA in cell culture. The presence of chitinase was detected in purified AcMNPV-chiAEpapGV OBs. Peritrophic membranes of Anticarsia gemmatalis larvae fed with recombinant OBs showed an altered structure. The results presented in this study show that EpapGV chitinase overexpression in recombinant baculovirus can cause association of this protein with OBs, and suggest that this could be used to evaluate the protein role in early stages of baculoviral infections.

Transcriptomic survey of the midgut of Anthonomus grandis (Coleoptera: Curculionidae).

Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase, and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information.

Functional analysis of Spodoptera frugiperda nucleopolyhedrovirus late expression factors in Sf9 cells.

We used transient expression assays to assess the function of the baculovirus Spodoptera frugiperda M nucleopolyhedrovirus (SfMNPV) homologs of Autographa californica MNPV (AcMNPV) factors involved in late gene expression (lefs), in the Sf9 insect cell-line, which is permissive for both viruses. It is well-established that nineteen AcMNPV lefs support optimal levels of activity from a late promoter-reporter gene cassette in this assay. A subgroup of SfMNPV lefs predicted to function in transcription-specific events substituted the corresponding AcMNPV lefs very efficiently. When all SfMNPV lefs were assayed, including replication lefs, activity was low, but addition of two AcMNPV lefs not encoded in SfMNPV genome, resulted in augmented reporter activity. SfMNPV IE-1 was able to activate an early promoter cis-linked to an hr-derived element from SfMNPV but not from AcMNPV. However, the level of early promoter activation with SfMNPV IE-1 was lower compared to AcMNPV IE-1.

Genome of Epinotia aporema granulovirus (EpapGV), a polyorganotropic fast killing betabaculovirus with a novel thymidylate kinase gene.

BACKGROUND: Epinotia aporema (Lepidoptera: Tortricidae) is an important pest of legume crops in South America. Epinotia aporema granulovirus (EpapGV) is a baculovirus that causes a polyorganotropic infection in the host larva. Its high pathogenicity and host specificity make EpapGV an excellent candidate to be used as a biological control agent. RESULTS: The genome of Epinotia aporema granulovirus (EpapGV) was sequenced and analyzed. Its circular double-stranded DNA genome is 119,082 bp in length and codes for 133 putative genes. It contains the 31 baculovirus core genes and a set of 19 genes that are GV exclusive. Seventeen ORFs were unique to EpapGV in comparison with other baculoviruses. Of these, 16 found no homologues in GenBank, and one encoded a thymidylate kinase. Analysis of nucleotide sequence repeats revealed the presence of 16 homologous regions (hrs) interspersed throughout the genome. Each hr was characterized by the presence of 1 to 3 clustered imperfect palindromes which are similar to previously described palindromes of tortricid-specific GVs. Also, one of the hrs (hr4) has flanking sequences suggestive of a putative non-hr ori. Interestingly, two more complex hrs were found in opposite loci, dividing the circular dsDNA genome in two halves. Gene synteny maps showed the great colinearity of sequenced GVs, being EpapGV the most dissimilar as it has a 20 kb-long gene block inversion. Phylogenetic study performed with 31 core genes of 58 baculoviral genomes suggests that EpapGV is the baculovirus isolate closest to the putative common ancestor of tortricid specific betabaculoviruses. CONCLUSIONS: This study, along with previous characterization of EpapGV infection, is useful for the better understanding of the pathology caused by this virus and its potential utilization as a bioinsecticide.

Analysis of EpapGV gp37 gene reveals a close relationship between granulovirus and entomopoxvirus.

The Epinotia aporema Granulovirus GP37 protein gene has been identified, located, and sequenced. This gene was similar to other baculovirus gp37, to entomopoxvirus fusolin gene, and to the chitin-binding protein gene of bacteria. Sequence analysis indicated that the open reading frame is 669 bp long (the smallest gp37 sequenced at present) and encodes a predicted 222-amino acid protein. This protein is glycosylated and specifically recognized by an entomopoxvirus fusolin antiserum. The pairwise comparison of EpapGV gp37 gene product with all the baculovirus sequences in GenBank yields high similarity values ranging from 45 to 63 % with Cydia pomonella Granulovirus gp37 being the most closely related. The phylogenetic analysis interestingly grouped the granuloviruses in a cluster more closely related to entomopoxviruses than to nucleopolyhedroviruses, suggesting a possible horizontal transfer event between the granulovirus group and the entomopoxvirus group.

Identification of nucleopolyhedrovirus that infect Nymphalid butterflies Agraulis vanillae and Dione juno.

Dione juno and Agraulis vanillae are very common butterflies in natural gardens in South America, and also bred worldwide. In addition, larvae of these butterflies are considered as pests in crops of Passiflora spp. For these reasons, it is important to identify and describe pathogens of these species, both for preservation purposes and for use in pest control. Baculoviridae is a family of insect viruses that predominantly infect species of Lepidoptera and are used as bioinsecticides. Larvae of D. juno and A. vanillae exhibiting symptoms of baculovirus infection were examined for the presence of baculoviruses by PCR and transmission electron microscopy. Degenerate primers were designed and used to amplify partial sequences from the baculovirus p74, cathepsin, and chitinase genes, along with previously designed primers for amplification of lef-8, lef-9, and polh. Sequence data from these six loci, along with ultrastructural observations on occlusion bodies isolated from the larvae, confirmed that the larvae were infected with nucleopolyhedroviruses from genus Alphabaculovirus. The NPVs from the two different larval hosts appear to be variants of the same, previously undescribed baculovirus species. Phylogenetic analysis of the sequence data placed these NPVs in Alphabaculovirus group I/clade 1b.

Genomic analysis of an Argentinean isolate of Spodoptera frugiperda granulovirus reveals that various baculoviruses code for Lef-7 proteins with three F-box domains.

A new isolate of the Spodoptera frugiperda granulovirus, SfGV ARG, was completely sequenced and analyzed. The SfGV ARG genome is 139,812 bp long and encodes 151 putative open reading frames. Of these ORFs, 56 were found in betabaculoviruses, 19 of which are present only in GVs closely related to SfGV. Seven ORFs found homologs in this small GV group and also in noctuid NPVs. ORF066 codes a 74 amino acid protein, overlapped with nudix gene, with several homologs in baculovirus, found by tblastn search. Comparison with the genome of the Colombian isolate SfGV VG008 resulted in SfGV being 1101 bp smaller and lacking a homologue of VG008 ORF084, which codes for Lef-7. However, we found that ORF051 shows remote homology to Lef-7 proteins. Moreover, analysis of ORF051 along with Lef-7 proteins coded by a group of noctuid specific GVs and NPVs indicated that Lef-7 proteins coded by these viruses include three F-box domains in contrast to the single one reported for AcMNPV Lef-7. SfGV ARG genome also contains a split photolyase as a distinct feature not found in VG008. BlastX analysis revealed that a complete photolyase is coded considering a putative frameshift in a poly-A tract, which resembles known slippery sequences involved in programmed ribosome frameshifting.

Baculovirus insecticides in Latin America: historical overview, current status and future perspectives.

Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries) will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway.

Development of a recombination system for the generation of occlusion positive genetically modified Anticarsia gemmatalis multiple nucleopolyhedrovirus.

Anticarsia gemmatalis is an important pest in legume crops in South America and it has been successfully controlled using Anticarsia gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV) in subtropical climate zones. Nevertheless, in temperate climates its speed of kill is too slow. Taking this into account, genetic modification of AgMNPV could lead to improvements of its biopesticidal properties. Here we report the generation of a two-component system that allows the production of recombinant AgMNPV. This system is based on a parental AgMNPV in which the polyhedrin gene (polh) was replaced by a bacterial ß-galactosidase (lacZ) gene flanked by two target sites for the homing endonuclease I-PpoI. Co-transfection of insect cells with linearized (I-PpoI-digested) parental genome and a transfer vector allowed the restitution of polh and the expression of a heterologous gene upon homologous recombination, with a low background of non-recombinant AgMNPV. The system was validated by constructing a recombinant occlusion-positive (polh+) AgMNPV expressing the green fluorescent protein gene (gfp). This recombinant virus infected larvae normally per os and led to the expression of GFP in cell culture as well as in A. gemmatalis larvae. These results demonstrate that the system is an efficient method for the generation of recombinant AgMNPV expressing heterologous genes, which can be used for manifold purposes, including biotechnological and pharmaceutical applications and the production of orally infectious recombinants with improved biopesticidal properties.

An immunochemical method for quantitation of Epinotia aporema granulovirus (EpapGV).

Epinotia aporema granulovirus (EpapGV) is a baculovirus that affects E. aporema larvae and has proven to be a good candidate for the biocontrol of this important pest in South America. As part of the quality control of the production of a bioinsecticide based on EpapGV, a sensitive method was developed for the detection and quantitation of the virus. To this end, we used the major occlusion body (OB) protein (granulin) to generate polyclonal antibodies in rabbits. Purified IgG fractions from hyperimmune sera were labeled with biotin and used as detecting antibodies in a double antibody sandwich enzyme linked immunosorbent assays (ELISA). No cross-reactivity was detected with any of the nucleopolyhedroviruses (NPV) tested in this study, while a minor degree of reactivity was observed with the closely related Cydia pomonella granulovirus (CpGV). The performance of the ELISA was satisfactory in terms of sensitivity, detecting as little as 0.53 ng/ml of EpapGV granulin in suspensions of purified virus OB. This represented 2.0x10(4) OB/ml. Granulin was also detected in complex and highly diluted bioinsecticidal formulate mixtures. In time course experiments, the virus was detected as early as 24 h post infection (p.i.). The results of the studies demonstrate that this method is a convenient, rapid and inexpensive alternative for routine detection and quantitation of EpapGV.

Multiplex PCR and quality control of Epinotia aporema granulovirus production.

A specific multiplex PCR was developed for the rapid and highly sensitive quality control of the viral DNA during Epinotia aporema granulovirus (EpapGV) production. At the beginning of this work only 2.3% of the EpapGV genomic sequence was known. In order to increase the availability of specific information, the terminal sequences of the inserts of several selected clones of EpapGV genomic libraries were determined. These data comprised 8.4% of the total DNA sequence and corresponded to regions distributed throughout the genome. Based on the small fraction of known sequence available a set of 32 primers was designed, using information theory to set the basis for this study. Each pair of designed primers was initially tested in individual PCRs to assess the correct size of the expected product and the sensitivity of the amplification. The specificity was verified in multiplex PCRs, using alternatively 1-3 sets of selected 5-6 primer pairs and EpapGV DNA preparations from different sources and degrees of purity. The results indicate that the multiplex PCR could be used for quality control in the bioinsecticide production, as well as in other applications such as the detection of latent infections in E. aporema colonies, and studies related to virus distribution, vertical transmission, host range, or persistence in the field.

Sequencing and characterisation of p74 gene in two isolates of Anticarsia gemmatalis MNPV.

P74 is a protein encoded in the genome of baculoviruses, associated with the envelopes of occluded virus. Its presence proved to be essential for per os infection. In first place, in this work we designed two universal primers to amplify a sequence region of the p74 ORF in baculoviruses from different classification groups. Then, by the use of these amplicons we obtained the complete sequence of the p74 locus from two isolates of AgMNPV, 2D (Brazil) and SF (Argentina). In the flanking regions we determined the complete sequence of p10 gene and a portion of p26 gene. Comparing both p74 sequence data (ORFs of 1935 bp) we found fifteen nucleotide changes that result in six amino acid changes. Comparisons of AgMNPV p74s with other baculovirus homologous genes indicate a close relationship with other group I Nucleopolyhedrovirus, in particular CfDEFNPV. These results were based on ORF sequence, amino acid sequence and gene order. The predictive studies about secondary structure and hydrophobic index point at six regions potentially associated to its function or native conformation. Finally, the detection of p74 mRNA after virus DNA replication confirms a late expression pattern.

Identification and characterization of the ecdysteroid UDP-glycosyltransferase gene of Epinotia aporema granulovirus.

The ecdysteroid UDP-glycosyltransferase (egt) gene of Epinotia aporema granulovirus (EpapGV) was cloned sequenced and its biological activity was assessed. It encodes a protein of 446 amino acids. Direct evidence that the cloned gene encodes an active EGT protein was obtained by transient expression assays in insect cells. The upstream untranslated region of the egt gene exhibits several consensus early promoter elements. Accordingly, the gene is expressed early upon infection of Epinotia aporema larvae and the EGT activity remains high until later times post infection. Sequence analyses indicate the presence of clusters of amino acid residues conserved among all the baculoviral EGTs, although their relation with proper protein folding, ligand binding and catalytic activity remain to be assessed. Phylogenetic trees consistently cluster the granulovirus EGTs separating them clearly from the nucleopolyhedroviruses.

Physical and genetic map of Epinotia aporema granulovirus genome.

The bean shoot borer, Epinotia aporema (Lep. Tortricidae), is an economically important pest of legume crops in South America. Recently, a granulovirus (EpapGV) was isolated from E. aporema larvae, and evaluated as a potential biological control agent. In order to generate a restriction map and to investigate the gene organisation of EpapGV genome, DNA isolated from occlusion bodies as well as a set of cloned genomic fragments were analysed using combinations of restriction endonucleases and Southern blot analyses that lead to a first version of the physical map. It was subsequently confirmed and refined by sequencing the termini of the cloned fragments and assessing their contiguity by comparing the sequences with databases to identify putative ORFs spanning neighbour fragments. This was also aided by PCR amplifications with primers that pointed outwards of the cloned viral DNA. The granulin gene was positioned on the physical map, cloned and sequenced. Its 747-nucleotide-long ORF encodes a predicted protein of 29 kDa and the core of the baculovirus very late promoter ATAAG was found 29 nucleotides upstream the initiation codon. In addition, 27 putative ORFs were located on the map and used to explore the genome organisation by GeneParityPlot against the fully sequenced granulovirus genomes. These data, taken together with the phylogenetic tree generated by alignment of the major occlusion proteins, indicate that EpapGV is closely related to CpGV, but has a distinct gene organisation.