RESUMO
Baculoviruses have been applied for biocontrol of agricultural pests, such as velvetbean caterpillar (Anticarsia gemmatalis) and fall armyworm (Spodoptera frugiperda). Cell culture is an interesting approach for large-scale production of these viruses. Co-infection of a host cell with two distinct viruses can contribute to reduce costs due to saving cell culture media, bioreactor space and the resulting co-occluded polyhedra may help to reduce final biopesticide costs. The baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) and Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) were chosen to test a model for in vitro co-infection in SF21 cells. Different proportions of SfMNPV/AgMNPV were evaluated along three in vitro passages by optical microscopy analysis of cells and real-time PCR (qPCR) of DNA obtained from budded viruses (BVs) and occlusion bodies (OBs). The kinetics of viral protein synthesis was carried out for analysis of the co-infection in first passage and bioassays with the resulting OBs were performed against A. gemmatalis and S. frugiperda larvae. The results demonstrated successful co-infection in these cells. The quantity of SfMNPV and AgMNPV in supernatants and sediments tends to be maintained stable during the three passages, although the amount of AgMNPV was higher than SfMPNV in most of the experiments. Analysis of the kinetics of radiolabed proteins showed that the cell protein synthesis was shut off and two distinct bands of about 30 kDa, regarded to be the polyhedrin of each virus, were strongly detected at 48 and 72 hp.i. Although the pathogenicity of the produced viruses was not completely satisfactory, the bioassays confirmed occurrence of co-infected larvae with disproportional amount of each virus.
Assuntos
Microbiologia Industrial , Nucleopoliedrovírus , Spodoptera , Virologia , Animais , Microbiologia Industrial/métodos , Microbiologia Industrial/tendências , Larva/virologia , Nucleopoliedrovírus/fisiologia , Células Sf9 , Spodoptera/virologia , Virologia/métodos , Virologia/tendênciasRESUMO
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. METHOD: 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.
Assuntos
Terapia Genética , Vetores Genéticos/genética , Nucleopoliedrovírus/genética , Proteínas Recombinantes/genética , Baculoviridae/genética , Técnicas de Transferência de Genes , Genoma Viral/genética , Recombinação Homóloga/genética , Humanos , Vírion/genéticaRESUMO
Polyhedron envelope protein (PEP) is the major component of the calyx that surrounds the baculovirus occlusion body (OB). PEP has been associated with the stabilization and resistance of polyhedra in the environment. Due to the abundant levels of PEP in OBs, we decided to use this protein as a fusion partner to redirect foreign proteins to baculovirus polyhedra. In this study we developed a strategy that involves the generation of a monoclonal transformed insect cell line expressing a protein of interest fused to the the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) N-terminus of PEP that enables the packaging of foreign proteins into the OBs without generating a recombinant baculovirus. This proved to be an efficient platform that could be exploited to improve wild type baculovirus for their use as bioinsecticides without facing the concerns of releasing genetically modified DNA to the environment and bypassing the associated regulatory issues. We demonstrated, using immunological, proteomic and microscopy techniques, that the envelope of AgMNPV OBs can effectively trap chimeric proteins in an infected insect cell line expressing AgMNPV PEP fused to the enhanced green fluorescent protein (eGFP). Furthermore, packaging of chimeric PEP also took place with heterologous OBs such as those of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), another group I alphabaculovirus.
Assuntos
Baculoviridae/metabolismo , Nucleopoliedrovírus/metabolismo , Corpos de Oclusão Virais/metabolismo , Proteínas de Matriz de Corpos de Inclusão/metabolismo , Proteômica , Animais , Baculoviridae/genética , Linhagem Celular , Genes Reporter , Insetos , Nucleopoliedrovírus/genética , Corpos de Oclusão Virais/genética , Proteínas de Matriz de Corpos de Inclusão/genética , Proteínas RecombinantesRESUMO
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 (0.5-2 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.
Assuntos
Replicação do DNA , DNA Viral/genética , Genoma Viral , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/fisiologia , Animais , Linhagem Celular , Cinética , Mariposas/virologia , Origem de Replicação , Replicação Viral/genéticaRESUMO
Baculovirus natural populations are known to be genetically heterogeneous and such genotypic diversity could have implications in the performance of biocontrol agents. The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) has been widely used to control the velvetbean caterpillar, Anticarsia gemmatalis, in Brazil. In the present work, morphological and molecular analyses as well as the biological activity of AgMNPV genotypes derived from a Brazilian field isolate (AgMNPV-79) were carried out. The existence of genotypic variants in the population was confirmed by DNA restriction analysis. Although difference in virulence was observed among the variants, the most (Ag79-01) and the least (AgL-16) virulent clones do not show any morphological and cytopathological changes when compared to the most studied isolate (AgMNPV-2D). The complete genome analysis of the two viral clones showed the presence of single open reading frames (ORFs) of the pe-38 and he65 genes, which contrasts with the two split ORFs present in the genome of the AgMNPV-2D isolate. The viral clone AgL-16 has many variations in the ie-2 and pe-38 genes, which are transcription regulatory genes responsible for the regulation of viral early gene expression during insect cell infection. Furthermore, other genes showed alterations like the odv-e56, which have an essential role in the maturation and envelopment of the ODVs, and bro-a and bro-b genes which were fused to form a single ORF. For the Ag79-01, although the total number of single nucleotide variants (SNVs) was more prominent in the pe-38 gene, its genome showed very few modifications in comparison to the AgMNPV-2D genome.
Assuntos
Lepidópteros/virologia , Nucleopoliedrovírus , Virulência/genética , Animais , Bioensaio , Linhagem Celular , Genes Virais , Variação Genética , Genoma Viral , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/patogenicidade , Nucleopoliedrovírus/ultraestrutura , Controle Biológico de Vetores , Filogenia , Células Sf9RESUMO
Abstract Chrysodeixis includens has become the major Lepidopteran pest of soybean crops, especially in the Brazilian Cerrado (savanna) region. A native isolate of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) from this region, Buritis, MG, was assessed for its biological and molecular features. In addition, in vitro co-infection with Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), another virus of an important soybean pest, was tested. The ChinNPV-Buritis isolate presented an average LC50 of 7,750 occlusion bodies (OBs)/ml of diet in C. includens larvae. Analysis of restriction endonuclease profiles of viral DNA revealed similarities with previously described ChinNPV isolates IE, IF, and IG from Brazil, although the presence of submolar bands indicates genetic heterogeneity. Optical microscopy analysis in conjunction with quantitative PCR (qPCR) demonstrated in vitro infection of this isolate in IPLB-SF-21AE, Sf9, and BTI-Tn-5B1-4 cell lines, but the amount of ChinNPV tends to decrease through serial passages. The qPCR method developed in this study successfully detected both AgMNPV and ChinNPV from cell culture and from infected larvae. The cell line Tn-5B1-4 is indicated for future development of in vitro production and co-infection studies.
Assuntos
Bombyx , Nucleopoliedrovírus , Agentes de Controle Biológico , LarvaRESUMO
Baculoviruses are highly specific and only capable of replication in arthropod hosts. The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most studied baculovirus at the molecular level and the Anticarsia gemnatalis multiple nucleopolyhedrovirus (AgMNPV) is the most used viral insecticide worldwide. AcMNPV have also been shown to stimulate the mammalian immune response acting as an adjuvant. In order to evaluate the effects of AgMNPV in modulating macrophage and lymphocyte activation, we have stimulated these cells in vitro and inoculated BALB/c mice intranasally with the two viral phenotypes (PIBs and BVs) and compared with the response induced by the same phenotypes of AcMNPV. Our results showed that baculoviruses are able to modulate mammalian immune response; in vitro they increase phagocytosis, NO2 production and Th1 cells response. In vivo, AgMNPV BVs or PIBs do not induce an inflammatory reaction in normal lung but during a fungal lung infection they can change the type of adaptive response developed. Considering our data, AgMNPV can be considered more useful as a vaccine vector or immune adjuvant than AcMNPV.