RESUMO
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.
Assuntos
Nucleopoliedrovírus/química , Nucleopoliedrovírus/fisiologia , Spodoptera/virologia , Proteínas do Envelope Viral/química , Motivos de Aminoácidos , Animais , Sistemas CRISPR-Cas , Teste de Complementação Genética , Larva/virologia , Mariposas/virologia , Nucleopoliedrovírus/genética , Filogenia , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
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
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
The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac12 gene, which is conserved in ten other baculovirus, codes a predicted 217 amino acid protein of unknown function. In this study, we investigated the role of ac12 during baculovirus infection, by generating an ac12 knockout virus. The transfection of the recombinant genome in insect cells resulted in unaltered viral dispersion and occlusion body production when compared to the control bacmid. This finding demonstrates that ac12 is a non-essential gene. Transmission and scanning electron microscopy (SEM) analyses showed that ac12 knockout virus produced occlusion bodies morphologically similar to those obtained with the control and capable to occlude virions. However, a slight but significant size difference was detected by SEM observation of purified occlusion bodies. This difference suggests that ac12 may be involved in regulatory pathways of polyhedrin production or occlusion body assembly without affecting either viral occlusion or oral infectivity in Rachiplusia nu larvae. This was evidenced by bioassays that showed no significant differences in the conditions tested. A qPCR analysis of viral gene expression during infection evidenced regulatory effects of ac12 over some representative genes of different stages of the viral cycle. In this study, we also showed that ac12 is transcribed at early times after infection and remains detectable up to 72 hours post-infection. The mRNA is translated during the infection and results in a protein that encodes an F-box domain that interacts in vivo and in vitro with S phase kinase associated protein 1 (SKP1) adaptor protein, which is potentially involved in protein ubiquitination pathways.
Assuntos
Interações Hospedeiro-Patógeno , Nucleopoliedrovírus/fisiologia , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Corpos de Inclusão Viral/ultraestrutura , Larva/virologia , Lepidópteros/virologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Ligação Proteica , Proteínas Virais/genética , Replicação ViralRESUMO
The Chrysodeixis includens nucleopolyhedrovirus (ChinNPV: Baculoviridae: Alphabaculovirus) is a registered insecticide for the management of soybean looper, Chrysodeixis includens (Walker, [1858]) in Brazil. We conducted studies of baseline susceptibility of Brazilian populations of C. includens to the ChinNPV (Chrysogen, AgBiTech, Fort Worth, TX) as valuable knowledge in support of Integrated Pest Management and Insect Resistance Management programs. In bioassays, neonates were infected with different concentrations of ChinNPV using the droplet feeding bioassay method. Larvae were then transferred to artificial diet and mortality was assessed at 7 d. Results confirm that neonates from Brazilian populations of C. includens are susceptible to ChinNPV. Concentrations from 1.0 × 103 to 1.0 × 108 occlusion bodies (OBs) per ml caused mortality from 1.5 to 99%, respectively. The LC50 ranged from 1.4 × 105 to 7.7 × 105 OBs per ml for populations of C. includens (5.5-fold variation). Similar variation was detected for the LC90 which ranged from 1.6 × 107 to 7.7 × 107 OBs per ml (4.8-fold variation). Importantly, the field-collected populations showed equivalent susceptibility to the reference susceptible population. This indicates a low interpopulation variation in susceptibility of Brazilian populations of C. includens to ChinNPV, representing natural geographic variation and not variation caused by previous selection pressure. The candidate diagnostic concentration of 2.9 × 108 OBs per ml was estimated based on the pooled data and caused mortality ranging from 98.6 to 100%. This concentration will be used in proactive resistance monitoring programs. The Chrysogen will be a valuable tool as a new mode of action in C. includens resistance management in Brazil.
Assuntos
Interações Hospedeiro-Patógeno , Mariposas/virologia , Nucleopoliedrovírus/fisiologia , Controle Biológico de Vetores , AnimaisRESUMO
The retroviral Gag protein is frequently used to generate 'virus-like particles' (VLPs) for a variety of applications. Retroviral Gag proteins self-assemble and bud at the plasma membrane to form enveloped VLPs that resemble natural retrovirus virions, but contain no viral genome. The baculovirus expression vector system has been used to express high levels of the retroviral Gag protein to produce VLPs. However, VLP preparations produced from baculovirus-infected insect cells typically contain relatively large concentrations of baculovirus budded virus (BV) particles, which are similar in size and density to VLPs, and thus may be difficult to separate when purifying VLPs. Additionally, these enveloped VLPs may have substantial quantities of the baculovirus-encoded GP64 envelope protein in the VLP envelope. Since VLPs are frequently produced for vaccine development, the presence of the GP64 envelope protein in VLPs, and the presence of Autographa californica multicapsid nucleopolyhedrovirus BVs in VLP preparations, is undesirable. In the current studies, we developed a strategy for reducing BVs and eliminating GP64 in the production of VLPs, by expressing the human immunodeficiency virus type 1 gag gene in the absence of the baculovirus gp64 gene. Using a GP64null recombinant baculovirus, we demonstrate Gag-mediated VLP production and an absence of GP64 in VLPs, in the context of reduced BV production. Thus, this approach represents a substantially improved method for producing VLPs in insect cells.
Assuntos
HIV-1/genética , Nucleopoliedrovírus/fisiologia , Vírion/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Animais , Células Cultivadas , Microscopia Eletrônica de Transmissão , Nucleopoliedrovírus/genética , Recombinação Genética , Spodoptera/virologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Vírion/genética , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
Baculovirus infection follows a transcriptionally controlled sequence of gene expression that occurs by activation of different viral gene promoter sequences during infection. This sequence of promoter activation may be disrupted by cellular defenses against viral infection, which might interfere with viral progeny formation. In this work, the activity of the ie1, gp64, lef-1, vp39, p6.9 and polh promoters of the Anticarsia gemmatalis multiple nucleopolyhedrovirus was assessed during infection of permissive, semipermissive and nonpermissive cell lines by a novel methodology that detects reporter protein luminescence in real-time. This technique allowed us to characterize in rich detail the AgMNPV promoters in permissive cell lines and revealed differential profiles of expression in cells with limited permissivity that correlate well with limitations in viral DNA replication. Semipermissive and nonpermissive cell lines presented delays and restrictions in late and very late promoter expression. Cells undergoing apoptosis did not inhibit late gene expression; however, viral progeny formation is severely affected. This work demonstrates the application of the real-time luminescence detection methodology and how the promoter expression profile may be used to diagnose cellular permissivity to baculovirus infection.
Assuntos
Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/fisiologia , Regiões Promotoras Genéticas , Replicação Viral , Animais , Linhagem Celular , Genes Reporter , Medições Luminescentes , Proteínas Luminescentes/análiseRESUMO
This study describes four multiple nucleocapsid nucleopolyhedrovirus isolates recovered from infected larvae of beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), on crops in two different geographical regions of Mexico. Molecular and biological characterization was compared with characterized S. exigua multiple nucleopolyhedrovirus (SeMNPV) isolates from the United States (SeUS1 and SeUS2) and Spain (SeSP2). Restriction endonuclease analysis of viral DNA confirmed that all Mexican isolates were SeMNPV isolates, but molecular differences between the Mexican and the reference isolates were detected using PCR combined with restriction fragment length polymorphism (RFLP). Amplification of the variable region V01 combined with RFLP distinguished the two Mexican isolates, SeSLP6 and SeSIN6. BglII digestions showed that the majority of the isolates contained submolar bands, indicating the presence of genetic heterogeneity. Amplification of the variable regions V04 and V05 distinguished between American and the Spanish isolates. Biological characterization was performed against two laboratory colonies of S. exigua, one from Mexico, and another from Switzerland. Insects from the Mexican colony were less susceptible to infection than insects from Se-Swiss colony. In the Se-Mex colony, SeSP2 was the most pathogenic isolate followed by SeSIN6, although their virulence was similar to most of the isolates tested. In Se-Swiss colony, similar LD50 values were observed for the five isolates, although the virulence was higher for the SeSLP6 isolate, which also had the highest OB (occlusion body) yield. We conclude that the Mexican isolates SeSIN6 and SeSLP6 possess insecticidal traits of value for the development of biopesticides for the control of populations of S. exigua.
Assuntos
DNA Viral/genética , Nucleopoliedrovírus/fisiologia , Controle Biológico de Vetores , Spodoptera/virologia , Animais , Larva/crescimento & desenvolvimento , Larva/virologia , México , Nucleopoliedrovírus/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Spodoptera/crescimento & desenvolvimentoRESUMO
Baculoviruses are able to enter into mammalian cells, where they can express a transgene that is placed under an appropriate promoter, without producing infectious progeny. ORF109 encodes an essential baculovirus protein that participates in the interaction of the baculovirus with mammalian cells. To date, the mechanisms underlying this interaction are not yet known. We demonstrated that although a Ac109 knock out virus maintained its ability to enter into BHK-21 cells, there was a marked reduction in the expression efficiency of the nuclear transgene. Moreover, the amount of free cytoplasmic viral DNA, which was detected by transcription of a reporter gene, was severely diminished. These results suggest Ac109 could be involved in maintaining the integrity of the viral nucleic acid.
Assuntos
Deleção de Genes , Nucleopoliedrovírus/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Cricetinae , Técnicas de Inativação de Genes , Genes Reporter , Nucleopoliedrovírus/isolamento & purificação , Nucleopoliedrovírus/fisiologia , Cultura de Vírus , Replicação ViralRESUMO
This work studied 17 insecticides belonging to nucleopolyhedrovirus (NPV), Bacillus thuringiensis (Bt kurstaki and Bt aizawai), benzoylureas (insect growth regulators [IGRs]), carbamates, organophosphates, spinosyns, and diamides against larvae of Helicoverpa armigera (Hübner), invasive species in the South American continent. Larvae of different instars were fed for 7 d with untreated or insecticide-treated diets. Mortality was recorded daily for 7 d, and surviving larvae were individually weighed on the seventh day. The NPV and Bt insecticides caused 100% mortality of first-instar larvae and first-instar and second-instar larvae, respectively. However, both NPV and Bt-based products caused low mortality of third-instar larvae and did not kill older larvae. The IGR lufenuron was highly effective against all three ages of larvae tested, whereas teflubenzuron and triflumuron produced maximum 60% mortality of second-instar larvae and lower than 50% to older larvae. Thiodicarb, chlorantraniliprole, indoxacarb, chlorpyrifos, and chlorfenapyr, irrespective of tested age, caused 100% mortality of larvae, with the last two insecticides reaching 100% mortality within 2 d of feeding on the treated diet. Flubendiamide caused lower mortality but significantly affected the weight of surviving larvae, whereas neither spinosad nor methomyl produced significant mortality or affected the weight of larvae. Based on the results, the age of H. armigera larvae plays an important role in the recommendation of NPV and Bt insecticides. Furthermore, there are potential options between biological and synthetic insecticides tested against H. armigera, and recording larval size during monitoring, in addition to the infestation level, should be considered when recommending biological-based insecticides to control this pest.