Gloria Barrera

Identifying putative Mycobacterium tuberculosis Rv2004c protein sequences that bind specifically to U937 macrophages and A549 epithelial cells.

Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tuberculosis laboratory strain genome has been completely sequenced, and this along with proteomic technology represent powerful tools contributing toward studying the biology of target cell interaction with a facultative bacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosis protein that could have specific mycobacterial functions. This study has revealed that the encoding gene is present in all mycobacterium species belonging to the M. tuberculosis complex. Rv2004c gene transcription was observed in all of this complexs strains except Mycobacterium bovis and Mycobacterium microti. Rv2004c protein expression was confirmed by using antibodies able to recognize a 54‐kDa molecule by immunoblotting, and its location was detected on the M. tuberculosis surface by transmission electron microscopy, suggesting that it is a mycobacterial surface protein. Binding assays led to recognizing high activity binding peptides (HABP); five HABPs specifically bound to U937 cells, and six specifically bound to A549 cells. HABP circular dichroism suggested that they had an α‐helical structure. HABP–target cell interaction was determined to be specific and saturable; some of them also displayed greater affinity for A549 cells than U937 cells. The critical amino acids directly involved in their interaction with U937 cells were also determined. Two probable receptor molecules were found on U937 cells and five on A549 for the two HABPs analyzed. These observations have important biological significance for studying bacillus–target cell interactions and implications for developing strategies for controlling this disease.

Eudragit S100 microparticles containing Spodoptera frugiperda nucleopolyehedrovirus: physicochemical characterization, photostability and in vitro virus release.

The aim of this study was to encapsulate the occlusion bodies (OBs) of Spodoptera frugiperda nucleopolyhedrovirus (SfNPV) in Eudragit® S100 microparticles (MPs), considering this technique as a possible alternative to protect them from deleterious environmental conditions. The MPs were prepared by oil-in-oil emulsion (O/O) solvent evaporation method. Experimental conditions were established according to a previous multi-level experimental design involving the core/polymer ratio as independent variable. The effects of these parameters on particle size and process yield were investigated observing that polymer concentration had a significant effect on particle size. After adequate conditions for MPs formation were determined, virus was encapsulated. The virus microparticles presented a particle size between 50–300 µm and concentration was 2.62 × 109 OBs g−1. Microencapsulation efficiency was 53.43% and virus release adjusted to Higuchi model suggesting diffusion as the release mechanism. Evaluated microencapsulation process protected viral particles of UV-inactivation, suggesting its potential for a biopesticide development.

The Complete Sequence of the First Spodoptera frugiperda Betabaculovirus Genome: A Natural Multiple Recombinant Virus

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a major pest in maize crops in Colombia, and affects several regions in America. A granulovirus isolated from S. frugiperda (SfGV VG008) has potential as an enhancer of insecticidal activity of previously described nucleopolyhedrovirus from the same insect species (SfMNPV). The SfGV VG008 genome was sequenced and analyzed showing circular double stranded DNA of 140,913 bp encoding 146 putative ORFs that include 37 Baculoviridae core genes, 88 shared with betabaculoviruses, two shared only with betabaculoviruses from Noctuide insects, two shared with alphabaculoviruses, three copies of own genes (paralogs) and the other 14 corresponding to unique genes without representation in the other baculovirus species. Particularly, the genome encodes for important virulence factors such as 4 chitinases and 2 enhancins. The sequence analysis revealed the existence of eight homologous regions (hrs) and also suggests processes of gene acquisition by horizontal transfer including the SfGV VG008 ORFs 046/047 (paralogs), 059, 089 and 099. The bioinformatics evidence indicates that the genome donors of mentioned genes could be alpha- and/or betabaculovirus species. The previous reported ability of SfGV VG008 to naturally co-infect the same host with other virus show a possible mechanism to capture genes and thus improve its fitness.

Deletion Genotypes Reduce Occlusion Body Potency but Increase Occlusion Body Production in a Colombian Spodoptera frugiperda Nucleopolyhedrovirus Population

A Colombian field isolate (SfCOL-wt) of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a mixture of different genotypes. To evaluate the insecticidal properties of the different genotypic variants, 83 plaque purified virus were characterized. Ten distinct genotypes were identified (named A through J). SfCOL-A was the most prevalent (71±2%; mean ± SE) showing a PstI restriction profile indistinguishable to that of SfCOL-wt. The remaining nine genotypes presented genomic deletions of 3.8 - 21.8 Kb located mainly between nucleotides 11,436 and 33,883 in the reference genome SfMNPV-B, affecting the region between open reading frames (ORFs) sf20 and sf33. The insecticidal activity of each genotype from SfCOL-wt and several mixtures of genotypes was compared to that of SfCOL-wt. The potency of SfCOL-A occlusion bodies (OBs) was 4.4-fold higher than SfCOL-wt OBs, whereas the speed of kill of SfCOL-A was similar to that of SfCOL-wt. Deletion genotype OBs were similarly or less potent than SfCOL-wt but six deletion genotypes were faster killing than SfCOL-wt. The potency of genotype mixtures co-occluded within OBs were consistently reduced in two-genotype mixtures involving equal proportions of SfCOL-A and one of three deletion genotypes (SfCOL-C, -D or -F). Speed of kill and OB production were improved only when the certain genotype mixtures were co-occluded, although OB production was higher in the SfCOL-wt isolate than in any of the component genotypes, or mixtures thereof. Deleted genotypes reduced OB potency but increased OB production of the SfCOL-wt population, which is structured to maximize the production of OBs in each infected host.

Characterisation of a Colombian granulovirus (Baculoviridae: Betabaculovirus) isolated from Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae

A strategy for biological control of the fall armyworm, Spodoptera frugiperda, has included the use of baculoviruses principally the nucleopolyhedrovirus SfMNPV, which have been extensively characterised. In contrast, the granulovirus of S. frugiperda (SfGV) has been poorly studied even though it is able to enhance the infectivity and virulence of NPVs. In this work, a Colombian SfGV isolate (VG008) was characterised in comparison with a reference isolate from Brazil (VG014). The viral morphology was characterised by ovoidal-shaped occlusion bodies (OB) that contained one single internal virion. Median lethal concentrations (LC50) and mean times to death (MTD) were 4.5 × 105 OBs/mL and 29 days for VG008 and 1.6 × 105 OBs/mL and 33 days for VG014. Both isolates reduced their insecticidal activity by 94%, after one hour of direct irradiation with ultraviolet light type B. The most prominent protein had an apparent molecular mass of 27 kDa and corresponded with the Granulin. Genomic comparison among isolates from Colombia and Brazil generated by restriction profiles showed differences in the number and size of fragments. Partial sequences of lef-8 and lef-9 genes and complete sequence of gran gene of Colombian SfGV isolate (VG008) showed high similarity values with VG014 and SfGV A12-4 homologous sequences, showing genetic distance lower than 0.015 (Kimura 2-parameter model), which confirmed that the three isolates belong to the same viral species. The characterisation of VG008 isolate demonstrated its high genomic and biological similarity with the Brazilian isolate.

The sf32 Unique Gene of Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) Is a Non-Essential Gene That Could Be Involved in Nucleocapsid Organization in Occlusion-Derived Virions

A recombinant virus lacking the sf32 gene (Sf32null), unique to the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), was generated by homologous recombination from a bacmid comprising the complete viral genome (Sfbac). Transcriptional analysis revealed that sf32 is an early gene. Occlusion bodies (OBs) of Sf32null contained 62% more genomic DNA than viruses containing the sf32 gene, Sfbac and Sf32null-repair, although Sf32null DNA was three-fold less infective when injected in vivo. Sf32null OBs were 18% larger in diameter and contained 17% more nucleocapsids within ODVs than those of Sfbac. No significant differences were detected in OB pathogenicity (50% lethal concentration), speed-of-kill or budded virus production in vivo. In contrast, the production of OBs/larva was reduced by 39% in insects infected by Sf32null compared to those infected by Sfbac. The SF32 predicted protein sequence showed homology (25% identity, 44% similarity) to two adhesion proteins from Streptococcus pyogenes and a single N-mirystoylation site was predicted. We conclude that SF32 is a non-essential protein that could be involved in nucleocapsid organization during ODV assembly and occlusion, resulting in increased numbers of nucleocapsids within ODVs.

Evidence of recent interspecies horizontal gene transfer regarding nucleopolyhedrovirus infection of Spodoptera frugiperda

BackgroundBaculoviruses are insect-associated viruses carrying large, circular double-stranded-DNA genomes with significant biotechnological applications such as biological pest control, recombinant protein production, gene delivery in mammals and as a model of DNA genome evolution. These pathogens infect insects from the orders Lepidoptera, Hymenoptera and Diptera, and have high species diversity which is expressed in their diverse biological properties including morphology, virulence or pathogenicity. Spodoptera frugiperda (Lepidoptera: Noctuidae), the fall armyworm, represents a significant pest for agriculture in America; it is a host for baculoviruses such as the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) (Colombia strain, genotype A) having been classified as a Group II alphabaculovirus making it a very attractive target for bioinsecticidal use.ResultsGenome analysis by pyrosequencing revealed that SfMNPV ColA has 145 ORFs, 2 of which were not present in the other sequenced genotypes of the virus (SfMNPV-NicB, SfMNPV-NicG, SfMNPV-19 and SfMNPV-3AP2). An in-depth bioinformatics study showed that ORF023 and ORF024 were acquired by a recent homologous recombination process between Spodoptera frugiperda and Spodoptera litura (the Oriental leafworm moth) nucleopolyhedroviruses. Auxiliary genes are numerous in the affected locus which has a homologous region (hr3), a repetitive sequence associated with genome replication which became lost in SfColA along with 1 ORF. Besides, the mRNAs associated with two acquired genes appeared in the virus’ life-cycle during the larval stage. Predictive studies concerning the theoretical proteins identified that ORF023 protein would be a phosphatase involved in DNA repair and that the ORF024 protein would be a membrane polypeptide associated with cell transport.ConclusionsThe SfColA genome was thus revealed to be a natural recombinant virus showing evidence of recent horizontal gene transfer between different baculovirus species occurring in nature. This feature could be the cause of its high insecticidal power and therefore SfColA becomes a great candidate for bioinsecticide formulations.

Identification of Diatraea spp. (Lepidoptera: Crambidae) based on cytochrome oxidase II

Diatraea spp. (Lepidoptera: Crambidae) are a group of insects that are agriculture pests in many economically relevant crops such as sugarcane, sorghum, corn and rice. Recognized species for this genus respond differentially to natural enemies used in their biological control, emphasizing the importance of species in a regional approach. Currently, identification is based on the male genitalia. However, the availability of specimens collected from field and subjectivity based on the character recognition can seriously hamper species identification, and therefore result in inadequate pest management. To overcome this, individuals of Diatraea spp. preliminarily classified male genitalia and obtained from reared conditions and the field (both derived from natural populations occurring in Colombia) were analyzed using genitalic morphometry and molecular biology specifically using a fragment of the cytochrome oxidase subunit II (CO II) mitochondrial gene. Although morphometric analysis did not show any overriding results regarding genitalia morphology, the bioinformatics analyses of CO II sequences resulted in an adequate classification of the individuals within the recognized species. It also, revealed that the occurrence of clades associated with geographical distribution may be associated with cryptic species. The latter was also confirmed by a Single-Strand Conformation Polymorphism (SSCP) methodology evaluating the same fragment of CO II. This experimental approach allows properly recognizing each species and in consequence is proposed as an effective tool in Diatraea species identification.

Cotesia flavipes (Hymenoptera: Braconidae) as a Biological Control Agent of Sugarcane Stem Borers in Colombia's Cauca River Valley

Summary Releases of the parasitoid Cotesia flavipes have been carried out since late 2014 in Colombias Cauca River Valley to complement other biological control on sugarcane stem borers (Diatraea spp.). To confirm the identity of the species being released as well as those recovered in the field, including samples from the department of Santander in western Colombia, specimens were examined using a fragment of cytocrome oxydase I (COI). Results confirmed that the genetic identity of the specimens sequenced corresponded to C. flavipes, with a genetic divergence of 0.00 to 0.03 as compared with GenBank registers. In the Cauca River Valley, parasitoid distribution currently extends from La Virginia (Risaralda) to Palmira (Valle del Cauca), and is mainly associated to the geographical distribution of the new Diatraea species reported in the region, D. busckella and D. tabernella.