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Baculoviruses are insect pathogens widely used as biotechnological tools in different fields of life sciences and technologies. The particular biology of these entities (biosafety viruses 1; large circular double-stranded DNA genomes, infective per se; generally of narrow host range on insect larvae; many of the latter being pests in agriculture) and the availability of molecular-biology procedures (e.g., genetic engineering to edit their genomes) and cellular resources (availability of cell lines that grow under in vitro culture conditions) have enabled the application of baculoviruses as active ingredients in pest control, as systems for the expression of recombinant proteins (Baculovirus Expression Vector Systems—BEVS) and as viral vectors for gene delivery in mammals or to display antigenic proteins (Baculoviruses applied on mammals—BacMam). Accordingly, BEVS and BacMam technologies have been introduced in academia because of their availability as commercial systems and ease of use and have also reached the human pharmaceutical industry, as incomparable tools in the development of biological products such as diagnostic kits, vaccines, protein therapies, and—though still in the conceptual stage involving animal models—gene therapies. Among all the baculovirus species, the Autographa californica multiple nucleopolyhedrovirus has been the most highly exploited in the above utilities for the human-biotechnology field. This review highlights the main achievements (in their different stages of development) of the use of BEVS and BacMam technologies for the generation of products for infectious and noninfectious human diseases. Key points • Baculoviruses can assist as biotechnological tools in human health problems. • Vaccines and diagnosis reagents produced in the baculovirus platform are described. • The use of recombinant baculovirus for gene therapy–based treatment is reviewed.

Peripheral arterial disease (PAD) is a progressive, disabling ailment for which no effective treatment exists. Gene therapy-mediated neovascularization has emerged as a potentially useful strategy. We tested the angiogenic and arteriogenic efficacy and safety of a baculovirus (BV) encoding mutant, oxygen-resistant hypoxia-inducible factor 1-alpha (mHIF-1α), in rabbits with PAD.After assessing the transfection efficiency of the BV.mHIF-1α vector and its tubulogenesis potential in vitro, we randomized rabbits with experimental PAD to receive 1 × 10Angiographically visible collaterals were more numerous in BV.mHIF-1α-treated rabbits (8.12 ± 0.42 vs 6.13 ± 1.15 collaterals/cmIn rabbits with PAD, BV.mHIF-1α induced neovascularization and reduced ischemic damage, exhibiting a good safety profile at 14 days post-treatment. Complementary studies to evaluate its potential usefulness in the clinic are needed.

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV), has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Methods: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in a comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative to non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

The adult mammalian cardiomyocyte has a very limited capacity to reenter the cell cycle and advance into mitosis. Therefore, diseases characterized by lost contractile tissue usually evolve into myocardial remodeling and heart failure. Analyzing the cardiac transcriptome at different developmental stages in a large mammal closer to the human than laboratory rodents may serve to disclose positive and negative cardiomyocyte cell cycle regulators potentially targetable to induce cardiac regeneration in the clinical setting. Thus we aimed at characterizing the transcriptomic profiles of the early fetal, late fetal, and adult sheep heart by employing RNA-seq technique and bioinformatic analysis to detect protein-encoding genes that in some of the stages were turned off, turned on, or differentially expressed. Genes earlier proposed as positive cell cycle regulators such as cyclin A, cdk2, meis2, meis3, and PCNA showed higher expression in fetal hearts and lower in AH, as expected. In contrast, genes previously proposed as cell cycle inhibitors, such as meis1, p16, and sav1, tended to be higher in fetal than in adult hearts, suggesting that these genes are involved in cell processes other than cell cycle regulation. Additionally, we described Gene Ontology (GO) enrichment of different sets of genes. GO analysis revealed that differentially expressed gene sets were mainly associated with metabolic and cellular processes. The cell cycle-related genes fam64a, cdc20, and cdk1, and the metabolism-related genes pitx and adipoq showed strong differential expression between fetal and adult hearts, thus being potent candidates to be targeted in human cardiac regeneration strategies. NEW & NOTEWORTHY We characterized the transcriptomic profiles of the fetal and adult sheep hearts employing RNAseq technique and bioinformatic analyses to provide sets of transcripts whose variation in expression level may link them to a specific role in cell cycle regulation. It is important to remark that this study was performed in a large mammal closer to humans than laboratory rodents. In consequence, the results can be used for further translational studies in cardiac regeneration. Fil: Locatelli, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Belaich, Mariano Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentina Fil: López, Ayelén Emilce. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Uranga Vega, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Giménez, Carlos Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Simonin, Jorge Alejandro. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bauza, Maria del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Castillo Velasquez, Martha Giovanna. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Cuniberti, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Crottogini, Alberto José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Cerrudo, Carolina Susana. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

To understand the mechanism of replication used by baculoviruses, it is essential to describe all the factors involved, including virus and host proteins and the sequences where DNA synthesis starts. A lot of work on this topic has been done, but there is still confusion in defining what sequence/s act in such functions, and the mechanism of replication is not very well understood. In this work, we performed an AgMNPV replication kinetics into the susceptible UFL-Ag-286 cells to estimate viral genome synthesis rates. We found that the viral DNA exponentially increases in two different phases that are temporally separated by an interval of 5 h, probably suggesting the occurrence of two different mechanisms of replication. Then, we prepared a plasmid library containing virus fragments (0.5&ndash, 2 kbp), which were transfected and infected with AgMNPV in UFL-Ag-286 cells. We identified 12 virus fragments which acted as origins of replication (ORI). Those fragments are in close proximity to core genes. This association to the core genome would ensure vertical transmission of ORIs. We also predict the presence of common structures on those fragments that probably recruit the replication machinery, a structure also present in previously reported ORIs in baculoviruses.

Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numerous biotechnological applications, such as the biological control of agricultural pests, the expression of recombinant proteins or the gene delivery of therapeutic sequences in mammals, among others. Their genomes encode between 80 and 200 proteins, of which 38 are shared by all reported species. Thanks to multi-omic studies, there is remarkable information about the baculoviral proteome and the temporality in the virus gene expression. This allows some functional elements of the genome to be very well described, such as promoters and open reading frames. However, less information is available about the transcription termination signals and, consequently, there are still imprecisions about what are the limits of the transcriptional units present in the baculovirus genomes and how is the processing of the 3′ end of viral mRNA. Regarding to this, in this review we provide an update about the characteristics of DNA signals involved in this process and we contribute to their correct prediction through an exhaustive analysis that involves bibliography information, data mining, RNA structure and a comprehensive study of the core gene 3′ ends from 180 baculovirus genomes.

Beauveria bassiana chitinases are involved in degrading the chitin in insects’ exoskeletons and internal structures, and thus are important virulence factors as they participate in initial to final steps of infection. In this work, the B. bassiana (Bv062 isolate) open reading frame (ORF) encoding a chitinase identified as Chit37 (orthologous Bvchit1) was molecularly cloned and expressed in Escherichia coli, and the potential of the recombinant protein (rChit37) to enhance the insecticidal activity of Bv062 conidia against second instar Diatraea saccharalis larvae was studied. rChit37 was produced in both soluble and insoluble fractions of an E. coli culture. Both fractions expressing endo- (90 mU/µL) and exochitinase (170 mU/µL) enzymatic activity, with optimum conditions for enzyme activity of 45 °C and pH 5.0. His-tag affinity chromatography was used to purify the rChit37 from the soluble fraction. Purified rChit37 was then diluted to 200 and 300 µg/mL for use as Bv062 conidia additive (1x106con/mL) in a laboratory bioassay against D. saccharalis larvae. No significant differences were observed between the efficacy of Bv062 conidia applied alone or mixed with 200 µg/mL purified rChit37. However, 300 µg/mL rChit37 increased BV062 conidia insecticidal activity, achieving 96.7% efficacy (14 days post-infection) and 6.2 days median lethal time (LT50), compared to 60% efficacy and 8.9 days for conidia alone. rChit37 addition did not affect conidial viability in terms of germination (96.6% after 24 h) or vigour estimated as germ-tube elongation rate. This work provides proof of concept about soluble recombinant chitinase as an additive to enhance B. bassiana virulence against D. saccharalis. © 2020 The Authors

El presente libro recoge los desarrollos mas relevantes a nivel mundial, las experiencias de Corpoica (hoy agrosavia) y el trabajo de decadas de los coautores nacionales e internacionales que hacen parte del mismo. La documentacion sobre los avances y las perspectivas en la materia tiene la intencion de acelerar los nuevos desarrollos en aspectos aun no estudiados del control biologico y estimular el progreso en su implementacion. Esta parte comprende informacion relacionada con la Implementacion del control biologico, ademas de reflexiones y recomendaciones de los autores sobre el futuro del control biologico.

Adult mammalian cardiomyocytes (CMs) exhibit limited proliferative capacity, as cell cycle activity leads to an increase in DNA content, but mitosis and cytokinesis are infrequent. This makes the heart highly inefficient in replacing with neoformed cardiomyocytes lost contractile cells as occurs in diseases such as myocardial infarction and dilated cardiomyopathy. Regenerative therapies based on the implant of stem cells of diverse origin do not warrant engraftment and electromechanical connection of the new cells with the resident ones, a fundamental condition to restore the physiology of the cardiac syncytium. Consequently, there is a growing interest in identifying factors playing relevant roles in the regulation of the CM cell cycle to be targeted in order to induce the resident cardiomyocytes to divide into daughter cells and thus achieve myocardial regeneration with preservation of physiologic syncytial performance. Despite the scientific progress achieved over the last decades, many questions remain unanswered, including how cardiomyocyte proliferation is regulated during heart development in gestation and neonatal life. This can reveal unknown cell cycle regulation mechanisms and molecules that may be manipulated to achieve cardiac self-regeneration. We hereby revise updated data on CM cell cycle regulation, participating molecules and pathways recently linked with the cell cycle, as well as experimental therapies involving them. Fil: Locatelli, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina. Universidad Favaloro; Argentina Fil: Giménez, Carlos Sebastián. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Uranga Vega, Martin. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina Fil: Crottogini, Alberto José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina. Universidad Favaloro; Argentina Fil: Belaich, Mariano Nicolas. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The tomato leafminer Tuta absoluta (Meyrick 1917) (Lepidoptera:Gelechiidae) is a devastating pest, causing losses of up to 100%. An interesting tool for its control is the use of the insect specific viruses of Baculoviridae family. Despite its high potential, its use on T. absoluta larvae has been poorly studied. In this work, Colombian granuloviruses VG013 and VG003 isolated from T. absoluta and Tecia solanivora (Lepidoptera:Gelechiidae) larvae sampled in tomato and potato crops, respectively, were morphologically, biologically and molecularly characterized. Occlusion bodies showed ovoid shape containing one nucleocapsid. Restriction endonuclease analysis revealed a pattern similar to Phthorimaea operculella granulovirus and bioinformatics studies showed that both isolates are variants of that baculovirus specie. Similar mean lethal concentrations (LC50) on T. absoluta larvae were estimated for both viruses, although VG013 exhibited shorter mean time to death than VG003. The latter reached a higher OBs yield in comparison with VG013. These results demonstrated an interesting potential of evaluated betabaculoviruses to control T. absoluta populations and pointed key features to its use under field conditions. Fil: Gómez Valderrama, J. A.. Corporacion Colombiana de Investigacion Agropecuaria; Colombia Fil: Barrera, G.. Corporacion Colombiana de Investigacion Agropecuaria; Colombia Fil: López Ferber, M.. Ecole Nationale Supérieure Des Mines Dalès; Francia Fil: Belaich, Mariano Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; Argentina Fil: Ghiringhelli, Pablo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; Argentina Fil: Villamizar, L.. Corporacion Colombiana de Investigacion Agropecuaria; Colombia

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. Fil: Barrera, Gloria Patricia. Corporación Colombiana de Investigación Agropecuaria; Colombia Fil: Villamizar, Laura Fernanda. Corporación Colombiana de Investigación Agropecuaria; Colombia. AgResearch Ltd. Lincoln Research Centre; Nueva Zelanda Fil: Espinel, Carlos. Corporación Colombiana de Investigación Agropecuaria; Colombia Fil: Quintero, Edgar Mauricio. Centro de Investigación de la Caña de Azúcar de Colombia; Colombia Fil: Belaich, Mariano Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular. Área de Virosis Emergentes y Zoonótica; Argentina Fil: Toloza, Deisy Liseth. Corporación Colombiana de Investigación Agropecuaria; Colombia Fil: Ghiringhelli, Pablo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular. Área de Virosis Emergentes y Zoonótica; Argentina Fil: Vargas, Germán. Centro de Investigación de la Caña de Azúcar de Colombia; Colombia

Entomopathogenic nematodes (EPN) belonging to the Heterorhabditidae family are lethal parasites of soil-dwelling insects. Two species were reported in Argentina: Heterorhabditis argentinensis and Heterorhabditis bacteriophora characterized mainly by morphometric features. In this work a comparative and phylogenetic study between five Heterorhabditis populations from Argentina was conducted to analyze the variability between strains and to evaluate the taxonomic position of Heterorhabditis argentinensis. The PCA analyses of morphometric characters separated the larger juvenile, female and male H. argentinensis from H. bacteriophora populations. The juvenile (IJs) stage provided the clearest separation of Heterorhabditis populations presenting the least variability between strains. The variable L and MBW were highly related to H. argentinensis IJs. Three groups were separated by this stage considering PC1 and PC2: one formed by H. bacteriophora OLI, RIV and RN strains, (isolates from Córdoba and Río Negro province), one for H. bacteriophora VELI strain (Buenos Aires province) and one for H. argentinensis (Santa Fe province). Heterorhabditis bacteriophora VELI and H. argentinensis isolated from regions with more rainfalls and humidity presented larger values for morphometric features. Molecular analyses showed the Argentinian populations (H. bacteriophora VELI strain and H. argentinensis), forming a same clade, with six other H. bacteriophora populations (not from Argentina) with a genetic similarity between them of 99%. Heterorhabditis argentinensis presented one unique nucleotide that was not present in any of the other species of the clade. Considering the results of this study H. argentinensis would be conspecific to H. bacteriophora, constituting a strain with a great morphometric variation where the host and climatic conditions could have influenced on the measurements., Nematóides entomopatogênicos (EPN) pertencentes á família Heterorhabditidae são parasitas letais de insetos que vivem no solo. Duas espécies foram relatados na Argentina: Heterorhabditis argentinensis e Heterorhabditis bacteriophora, caracterizada principalmente por características morfométricas. Neste trabalho um estudo comparativo e filogenética entre cinco populações do Heterorhabditis da Argentina foi conduzido para analisar a variabilidade entre as linhagens e avaliar a posição taxonômica das Heterorhabditis argentinensis. Características morfométricas de Heterorhabditis bacteriophora VELI e H. argentinensis isoladas de regiões com mais chuvas e umidade apresentaram dimensões maiores. Analisa o PCA de personagens morfométricas separou a maior juvenil, feminino e masculino H. argentinensis de H. bacteriophora populações. A fase juvenil (JIs) fornece a mais clara separação de populações Heterorhabditis apresentando a menor variação entre as cepas. A L variável e MBW foram altamente relacionada com H. argentinensis JIs. Três grupos foram separados por esta fase considerando PC1 e PC2: um formado por H. bacteriophora OLI, RIV e estirpes RN, (isolados de Córdoba e província de Rio Negro), um para a estirpe H. bacteriophora VELI (província de Buenos Aires) e um para H. argentinensis (província de Santa Fe). Heterorhabditis bacteriophora VELI e H. argentinensis isolado a partir de regiões com mais chuvas e umidade apresentaram maiores valores para as características morfométricas. A análise molecular mostrou as populações da Argentina (estirpe H. bacteriophora VELI e H. argentinensis), formando um mesmo subtipo, com seis outras populações H. bacteriophora (não da Argentina), com uma similaridade genética entre eles de 99%. Heterorhabditis argentinensis apresentado um único nucleótido que não estava presente em nenhum dos outros espécies do clado. Considerando os resultados deste estudo H. argentinensis seria conspecific a H. bacteriophora, constituindo uma estirpe com uma grande variação morfométrica onde o anfitrião e as condições climáticas podem ter influenciado nas medições., Centro de Estudios Parasitológicos y de Vectores

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.

Background: Baculoviruses 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. Results: Genome 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. Conclusions: The 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. Fil: Barrera, Gloria Patricia. Corporación Colombiana de Investigación Agropecuaria; Colombia Fil: Belaich, Mariano Nicolas. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Patarroyo, Manuel Alfonso. Universidad del Rosario; Colombia. Instituto de Inmunología de Colombia; Colombia Fil: Villamizar, Laura Fernanda. Corporación Colombiana de Investigación Agropecuaria; Colombia Fil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

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. Fil: Cuartas, Paola E.. Corporación Colombiana de Investigación Agropecuaria; Colombia Fil: Barrera, Gloria P.. Corporación Colombiana de Investigación Agropecuaria; Colombia Fil: Belaich, Mariano Nicolas. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Barreto, Emiliano. Universidad Nacional de Colombia; Colombia Fil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Villamizar, Laura F.. Corporación Colombiana de Investigación Agropecuaria; Colombia

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.

The gp64 locus of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate Santa Fe (AgMNPV-SF) was characterised molecularly in our laboratory. To this end, we have located and cloned a AgMNPV-SF genomic DNA fragment containing the gp64 gene and sequenced the complete gp64 locus. Nucleotide sequence analysis indicated that the AgMNPV gp64 gene consists of a 1500 nucleotide open reading frame (ORF), encoding a protein of 499 amino acids. Of the seven gp64 homologues identified to date, the AgMNPV gp64 ORF shared most sequence similarity with the gp64 gene of Orgyia pseudotsugata MNPV. The GP64 from AgMNPV is the smallest baculoviral envelope glycoprotein found to date, differing in 10 or more residues from the other group I nucleopolyhedroviruses. The biological activity of AgMNPV GP64 protein was assessed by cell fusion assays in UFL-AG-286 cells using the obtained recombinant plasmids. In the upstream and downstream regions, relative to the gp64 ORF, we found different conserved transcriptional and post-transcriptional regulatory elements, respectively.

The pampean region in the Buenos Aires province of Argentina is a major horticultural area. Biological pesticides to control pest insects available in the market are scarce in this country and nonexistent based on entomonematodes. In this paper, we characterized a native entomopathogenic nematode isolated from soil in the pampas, and evaluated its infectivity and reproductive capacity in the strawberry sap beetle Lobiopa insularis. Morphological and molecular-genetic analysis determined this isolate to be a nematode of Heterorhabditis bacteriophora, thereafter designated as the VEli strain. A 901-bp genomic-DNA fragment was isolated (GenBank Accesion No KJ575524). The new strain was pathogenic against L. insularis. In 200 ml containers with autoclaved clay soil (100 ml), at concentrations of 1,000; 5,000 and 10, 000 IJs, mortality in larvae ranged from 25 to 77 %. At a concentration of 10,000 IJs, infectivity was higher in larvae and pupae (77 ± 20% and 64 ± 25 %, respectively) than in adults (2.2 ± 1.1 %). The lethal concentration required to kill 50 and 90 % of the larvae (LC50 and LC90) over 10 days was 2,840 and 23,743 IJs. Differences were observed in the parasitism levels and emergence time of IJs between concentrations and stages, although not for production. Heterorhabditis bacteriophora strain VEli is the first entomopathogenic nematode found to infect and kill L. insularis, capable of completing its life cycle in all strawberry sap beetle stages. This availability would enable the establishment and subsequent dispersal of the strain in strawberry fields. Finally, this report constitutes the first presentation of data on the morphological, morphometric, and molecular-genetic characterization of a native entomopathogenic nematode strain from the Buenos Aires province, Argentina., En la región pampeana, provincia de Buenos Aires, Argentina, la horticultura es una de las principales actividades agrícolas. Los plaguicidas biológicos disponibles en el mercado son escasos en el país e inexistentes en base a nematodos. En este trabajo, se caracterizó un nematodo entomopatógeno nativo aislado de la pampa y se evaluó su infectividad y capacidad reproductiva en el insecto de las fresas, Lobiopa insularis. El análisis morfológico y genéticomolecular determinó este aislado como el nematodo Heterorhabditis bacteriophora, designado cepa-VEli. Se aisló un fragmento del ADN genómico de 901 pb (acceso GenBank No KJ575524). La nueva cepa fue patógena contra L. insularis. En recipientes de 200 ml con suelo arcilloso autoclavado (100 ml), a concentraciones de 1.000; 5.000 y 10.000 JIs, la mortalidad en larvas varió de 25 a 77 %. A una concentración de 10,000 JIs, la infección fue mayor en larvas y pupas (77 ± 20 % y 64 ± 25 %, respectivamente) que en adultos (2,2 ± 1,1 %). La concentración letal requerida para matar 50 y 90 % de las larvas (CL50 y CL90) después de 10 días fue 2.840 y 23.743 IJs. Se observaron diferencias en los niveles de parasitismo y en el tiempo de emergencia de los JIs entre concentraciones y estadios aunque no en la producción. Heterorhabditis bacteriophora VEli es el primer nematodo entomopatógeno que infecta y mata L. insularis, capaz de completar su ciclo de vida en todas las etapas del escarabajo de la savia de la fresa. Esta disponibilidad permitiría el establecimiento y posterior dispersión de la cepa en los campos de fresa. Por último, este informe constituye la primera presentación de datos sobre la caracterización morfológica, morfométrica y genética molecular de una cepa nativa de nematodos entomopatógenos para la provincia de Buenos Aires, Argentina., Centro de Estudios Parasitológicos y de Vectores, Facultad de Ciencias Naturales y Museo

The family Baculoviridae is a large group of insect viruses containing circular double-stranded DNA genomes of 80 to 180 kbp, which have broad biotechnological applications. A key feature to understand and manipulate them is the recognition of orthology. However, the differences in gene contents and evolutionary distances among the known members of this family make it difficult to assign sequence orthology. In this study, the genome sequences of 58 baculoviruses were analyzed, with the aim to detect previously undescribed core genes because of their remote homology. A routine based on Multi PSI-Blast/tBlastN and Multi HaMStR allowed us to detect 31 of 33 accepted core genes and 4 orthologous sequences in the Baculoviridae which were not described previously. Our results show that the ac53 , ac78 , ac101 ( p40 ), and ac103 ( p48 ) genes have orthologs in all genomes and should be considered core genes. Accordingly, there are 37 orthologous genes in the family Baculoviridae .

The mosquito iridescent viruses (MIVs) are large icosahedral DNA viruses that replicate and assemble in the cytoplasm of the host. Paracrystalline arrangements of virions that accumulate in the cytoplasm produce an iridescent color that is symptomatic of acute infections. In August 2010, we found larvae of Culex pipiens with these symptoms in suburban ditches around the city of La Plata, Argentina. Electron microscope studies, DNA sequencing, and phylogenetic analysis of the major capsid protein confirmed this as the first record of an MIV in C. Pipiens.

M. Leticia Ferrelli1, Marcelo F. Berretta2, Mariano N. Belaich3, P. Daniel Ghiringhelli3, Alicia Sciocco-Cap2 and Victor Romanowski1 1Instituto de Biotecnologia y Biologia Molecular, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, CONICET 2Laboratorio de Ingenieria Genetica y Biologia Celular y Molecular Area Virosis de Insectos, Departamento de Ciencia y Tecnologia, Universidad Nacional de Quilmes 3Instituto de Microbiologia y Zoologia Agricola, INTA Castelar Argentina

Integrated Pest Management (IPM) can be defined as “an ecologically based pest control strategy that relies heavily on natural mortality factors and seeks out control tactics that disrupt these factors as little as possible” (Flint & Bosch, 1981). IPM began to be applied because the extensive use of chemical insecticides show different types of environmental damages, as development of resistant insects, the appearance of new pests, injury to bird and mammals populations and human health damage due to the release of toxic waste on environment and food contamination. The aims of IPM are to protect crops, with minimum cost and risk for humans, animals and ecosystems. The development and application of IPM requires the knowledge of how the ecosystem influences on pest insects and its natural control agents, and how to modify this environment to control particularly pest insects and avoid the related chemical agent's problems.

Agriculture is a discipline that has accompanied human beings since the beginning of civilization. The cultivation of different vegetables for centuries has allowed selecting varieties that far exceed the capabilities of many wild type plants originally used as a food source. That situation derived in the manipulation of natural ecosystems, transforming them into spaces where they can only grow and develop the desired species. In our world, plants are the staple diet of many organisms including invertebrates like Lepidoptera. During the larval stage, these insects can consume a large amount of leaf tissue causing serious damage to the plant. If we think that most vegetables have insect predators, agricultural crops can be transformed into an inviting habitat, allowing the development of these animals. In conclusion, all crops have pests that threaten their productivity. Given this scenario, many pest control strategies have been used by human beings to protect the health of their crops: treatment with chemical insecticides, development of transgenic plants and biological control applications (Christou et al, 2006; Gilligan, 2008). Baculovirus is a large family of insect pathogens that infect and kill different species of Lepidoptera, Hymenoptera and Diptera (Theilmann et al, 2005). In particular, many lepidopteron are pests in agriculture transforming these viruses in an important biocontrol tools for their natural hosts (Entwistle, 1998; Moscardi, 1999; Szewczyk et al, 2006). Baculoviruses have double-stranded circular DNA genomes of 80,000-180,000 bp, containing between 80 to 180 genes depending on the specie (van Oers & Vlak, 2007; Miele et al, 2011). In early stages of virus cycle, this pathogen is produced as Budded Viruses (BVs): the genome contained in a protein capsid (nucleocapsid), which is surrounded by a lipid membrane. In change, in the last phase of multiplication processes appear the Occluded Bodies (OBs): protein crystals (forming polyhedra or granules) containing nucleocapsids wrapped by a lipid membrane with a different composition (ODVs or Occluded Derived Viruses, with single or multiple nucleocapsids depending on the specie) (Rohrman, 2008). These two virus phenotypes have different biological properties; while OBs are specialists (infecting larvae by per os route with a narrow host range; responsible of primary infection in midgut cells), the BVs are generalists (infecting a wide range of different insect cells triggering their death; responsible for secondary infection). In the pest control strategies, baculoviruses (OBs) are introduced on the crops to infect and kill larvae through the production of an epizooty.

The Baculoviridae is a large group of insect viruses containing circular double-stranded DNA genomes of 80 to 180 kbp. In this study, genome sequences from 57 baculoviruses were analyzed to reevaluate the number and identity of core genes and to understand the distribution of the remaining coding sequences. Thirty one core genes with orthologs in all genomes were identified along with other 895 genes differing in their degrees of representation among reported genomes. Many of these latter genes are common to well-defined lineages, whereas others are unique to one or a few of the viruses. Phylogenetic analyses based on core gene sequences and the gene composition of the genomes supported the current division of the Baculoviridae into 4 genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus.

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the -492 and -357 versions contains sequence motifs important for the level of the lacZ reporter gene expression.

The gp64 locus of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate Santa Fe (AgMNPV-SF) was characterised molecularly in our laboratory. To this end, we have located and cloned a AgMNPV-SF genomic DNA fragment containing the gp64 gene and sequenced the complete gp64 locus. Nucleotide sequence analysis indicated that the AgMNPV gp64 gene consists of a 1500 nucleotide open reading frame (ORF), encoding a protein of 499 amino acids. Of the seven gp64 homologues identified to date, the AgMNPV gp64 ORF shared most sequence similarity with the gp64 gene of Orgyia pseudotsugata MNPV. The GP64 from AgMNPV is the smallest baculoviral envelope glycoprotein found to date, differing in 10 or more residues from the other group I nucleopolyhedroviruses. The biological activity of AgMNPV GP64 protein was assessed by cell fusion assays in UFL-AG-286 cells using the obtained recombinant plasmids. In the upstream and downstream regions, relative to the gp64 ORF, we found different conserved transcriptional and post-transcriptional regulatory elements, respectively.

Background Anticarsia gemmatalis is a pest in South America's soybean crops, which could be controlled by the Multinucleopolyhedrovirus of A. gemmatalis (AgMNPV). Currently, its commercial production is based on infected larvae. However, the possibility of using modified baculoviruses in Integrated Pest Management programs has stimulated an interest to develop alternative multiplication processes. This study evaluated the AgMNPV production in UFL-Ag-286 cells previously deprived Fetal Bovine Serum. Results Culture media containing 1% FBS during the previous 48 hours achieved a synchronized condition where 90% of cells were found in G0/G1 stage, showing the presence of non-filamentous actin. All characteristics were estimated from cellular viability tests, cell actin detection trials and flow cytometer cell cycle analysis. AgMNPV production was tested by transcript studies and budded viruses (BVs) and occlusion bodies (OBs) yield quantitation. Results showed that the productivity in FBS deprived cells was 9.8 times more in BVs and 3.8 times more in OBs with respect to non-treated cells. Conclusions UFL-Ag-286 cells previously deprived in FBS shown to be a better host for AgMNPV propagation, increasing the useful for both in vitro bioinsecticide production and applications such as recombinant protein expression or gene delivery.