Molecular biology of baculovirus and its use in biological control in Brazil

Os baculovírus são vírus patogênicos a insetos, encontrados principalmente na ordem Lepidoptera. A família Baculoviridae é taxonomicamente dividida em dois gêneros: Nucleopolyhedrovirus e Granulovirus, que diferem pela morfologia do corpo de oclusão. Os nucleopoliedrovírus (NPV) possuem corpos de inclusão poliédrica (PIB), contendo múltiplas partículas virais, enquanto os granulovírus (GV) contêm, em geral, partículas únicas, ocluídas em corpos protéicos de forma ovóide. Durante o ciclo de vida são produzidos dois tipos de progênies virais: BV ("Budded Virus") e PDV ("Polyhedra Derived Virus"), que são essenciais para o processo de infecção e propagação do vírus. Os baculovírus têm sido empregados no controle de pragas e, por serem específicos e restritos a invertebrados, são considerados agentes seguros de controle biológico. Recentemente têm sido amplamente utilizados como vetor de expressão de genes heterólogos por produzirem e processarem, em grande quantidade, proteínas de procariotos e eucariotos. Além disso, técnicas de DNA recombinante têm permitido a produção de inseticidas virais geneticamente modificados. Este trabalho constitui uma revisão sobre a taxonomia, estrutura, replicação e biologia molecular de baculovírus e sobre seu uso como bioinseticida no Brasil.Baculoviruses are insect viruses found mainly in Lepidoptera. The family Baculoviridae is taxonomically divided in two genera, Nucleopolyhedrovirus and Granulovirus, which differ by occlusion body morphology. NPVs (Nucleopolyhedroviruses) have polyhedrical inclusion bodies (PIBs) containing multiple viral particles, while GVs (Granuloviruses) appear to be generally single particles occluded in oval shaped occlusion bodies. During the life cycle, two different viral progenies are produced: BV (Budded Virus) and PDV (Polyhedra Derived Virus), which are essential for the infectious process and virus propagation in host cells. Baculoviruses are being used for pest control and they are especially safe due to their specificity and invertebrate-restricted host range. Baculoviruses have been used as vectors for high level protein expression ofheterologous genes from prokaryotic and eukaryotic organisms. Also, recombinant DNA techniques have allowed the production of genetically modified viral insecticides. This study is a review on the taxonomy, structure, replication and molecular biology of baculoviruses, as well as their use as bioinsecticides in Brazil

Baculovirus biopesticides – a safe alternative to chemical protection of plants

ABSTRACT Chemical pest control agents, though extensively used in all countries of the world, have been widely regarded as ecologically unacceptable. Therefore, there is the increased social pressure to replace them gradually with biopesticides which are safe to humans and non-target organisms. Viruses of a few families infect invertebrates but only those belonging to the family Baculoviridae have been used as biopesticides because they are safe to wildlife and their specificity is very narrow. Until recently, the application as bioinsectides was limited because of their slow killing action and technical problems for in vitro commercial production. However, successful protection of large area of soybean fields in Brazil revived the interest in baculoviruses as effective agents for biocontrol and the wider application for pest control is very likely to occur in future. To improve baculovirus killing properties, two approaches can be foreseen: i) in countries where use of genetically modified organisms is restricted, changes in biopesticide formulations and the improvements of the in vitro production are to be expected, ii) in countries with more relaxed attitude towards genetically modified organisms, the killing activity of baculoviruses will be improved by genetic modifications of the baculovirus genome

Infectividade in vitro de Spodoptera frugiperda multiple nucleopolyhedrovirus a diferentes linhagens celulares de insetos

The objective of this work was to evaluate the response of an in vitro host range to Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), a pathogenic virus to the fall armyworm (Spodoptera frugiperda, Lepidoptera: Noctuidae), for the further development of a biopesticide based on cell culture systems. The cell lines from Bombyx mori (BM-5), Lymantria dispar (IPLB-LD-625Y), Trichoplusia ni (BTI-Tn-5B1-4), Anticarsia gemmatalis (UFL-AG-286), and S. frugiperda (IPLB-SF-21AE and Sf9) were tested for their susceptibility to a highly-virulent Brazilian isolate of SfMNPV. The cytopathic effects induced by the virus, the production of viral particles, and the synthesis of viral polypeptides were examined and compared. Both S. frugiperda cell lines showed hypertrophy of cell nuclei and production of many polyhedra. The SDS-Page of radiolabed proteins showed that the cell protein synthesis was shutoff, while an intense band of about 30 kDa, recognized as polyhedrin, was synthesized. The other cell lines did not show polyhedra production, although some of them underwent morphological changes and protein synthesis shutdown in response to virus infection. The SF-21 and Sf9 cell lines are recommended for further in vitro production of SfMNPV.O objetivo deste trabalho foi avaliar uma gama de hospedeiros, in vitro, quanto à resposta a Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), um vírus patogênico à lagarta-do-cartucho (Spodoptera frugiperda; Lepidoptera: Noctuidae), para o posterior desenvolvimento de um biopesticida baseado em sistema de cultura de células. As linhagens celulares de Bombyx mori (BM-5), Lymantria dispar (IPLB-LD-625Y), Trichoplusia ni (BTI-Tn-5B1-4), Anticarsia gemmatalis (UFL-AG-286) e S. frugiperda (IPLB-SF-21AE e Sf9) foram testadas quanto a sua suscetibilidade a um isolado brasileiro de SfMNPV altamente virulento. Os efeitos citopáticos induzidos pelo vírus, a produção de partículas virais e a síntese de polipeptídeos virais foram examinados e comparados. Ambas as células de S. frugiperda apresentaram hipertrofia dos núcleos celulares e produção de muitos poliedros. O SDS-Page de proteínas com radiomarcação mostrou que as proteínas celulares apresentaram inibição de síntese, enquanto uma intensa banda de cerca de 30 kDa, reconhecida como poliedrina, era sintetizada. As outras linhagens celulares não apresentaram produção de poliedros, apesar de algumas terem apresentado alterações morfológicas e inibição de síntese proteica em resposta à infecção viral. As linhagens celulares SF-21 e Sf9 são recomendadas para a posterior produção in vitro de SfMNPV.

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Multitemperature Single Stranded Conformational Polymorphism

Baculoviruses have been used as biopesticides for decades. Recently, due to the excessive use of chemical pesticides there is a need for finding new agents that may be useful in biological protection. Sometimes few isolates or species are discovered in one host. In the past few years, many new baculovirus species have been isolated from environmental samples, thoroughly characterized and thanks to next generation sequencing methods their genomes are being deposited in the GenBank database. Next generation sequencing (NGS) methodology is the most certain way of detection, but it has many disadvantages. During our studies, we have developed a method based on Polymerase chain reaction (PCR) followed by Multitemperature Single Stranded Conformational Polymorphism (MSSCP) which allows for distinguishing new granulovirus isolates in only a few hours and at low-cost. On the basis of phylogenetic analysis of betabaculoviruses, representative species have been chosen. The alignment of highly conserved genes—granulin and late expression factor-9, was performed and the degenerate primers were designed to amplify the most variable, short DNA fragments flanked with the most conserved sequences. Afterwards, products of PCR reaction were analysed by MSSCP technique. In our opinion, the proposed method may be used for screening of new isolates derived from environmental samples

Erinnyis ello granulovirus for cassava hornworm control in the Acre State of Brazil

The hornworm (Erinnyis ello) is an important pest of cassava and rubber tree with high capacity of migration and can be controlled with biological control agents such as baculoviruses. Baculoviruses are rod-shaped enveloped viruses with circular double-stranded DNA genomes infecting insects of the order Lepidoptera. Prior to large-scale use of certain baculovirus, it is essential to assess its genetic and morphological information. This work aimed to characterize the baculovirus isolate occurring in Erinnyis ello populations at small farms in Cruzeiro do Sul, Acre State, Brazil and to develop parameters for field management using this isolate. Viral particles were purified from infected larvae through ultracentrifugation in sucrose gradient. After treatment and negative staining with uranyl acetate 2%, viral particles were visualized using transmission electron microscope. The virus with ovicylindrical occlusion body presenting only one virion per envelope and size less than 0.5 μm was identified as a member of the genus Betabaculovirus. Viral DNA was extracted through phenol/chloroform cycles, digested with different restriction enzymes and separated with agarose gel electrophoresis. The restriction enzyme patterns obtained with the enzymes Bam HI and Hind III revealed seven and three DNA fragments, respectively; cleavage with Pst I, generated eight molar fragments and three submolar fragments and Eco RI digestion resulted in 21 fragments, with some submolar bands. Comparison of the DNA restriction pattern from ErelGV isolated in Acre with that described for ErelGV isolated from SC State showed high similarity. This isolate named as ErelGV-Acre presented potential for control of E. ello populations

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Real-Time Polymerase Chain Reaction (Real-Time PCR)

Baculoviridae is a highly diverse family of rod-shaped viruses with double-stranded DNA. To date, almost 100 species have had their complete genomic sequences deposited in the GenBank database, a quarter of which comprises granuloviruses (GVs). Many of the genomes are sequenced using next-generation sequencing, which is currently considered the best method for characterizing new species, but it is time-consuming and expensive. Baculoviruses form a safe alternative to overused chemical pesticides and therefore there is a constant need for identifying new species that can be active components of novel biological insecticides. In this study, we have described a fast and reliable method for the detection of new and differentiation of previously analyzed granulovirus species based on a real-time polymerase chain reaction (PCR) technique with melting point curve analysis. The sequences of highly conserved baculovirus genes, such as granulin and late expression factors 8 and 9 (lef-8 and lef-9), derived from GVs available to date have been analyzed and used for degenerate primer design. The developed method was tested on a representative group of eight betabaculoviruses with comparisons of melting temperatures to allow for quick and preliminary granulovirus detection. The proposed real-time PCR procedure may be a very useful tool as an easily accessible screening method in a majority of laboratories

In vitro infectivity of Spodoptera frugiperda multiple nucleopolyhedrovirus to different insect cell lines

<div><p>Abstract: The objective of this work was to evaluate the response of an in vitro host range to Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), a pathogenic virus to the fall armyworm (Spodoptera frugiperda, Lepidoptera: Noctuidae), for the further development of a biopesticide based on cell culture systems. The cell lines from Bombyx mori (BM-5), Lymantria dispar (IPLB-LD-625Y), Trichoplusia ni (BTI-Tn-5B1-4), Anticarsia gemmatalis (UFL-AG-286), and S. frugiperda (IPLB-SF-21AE and Sf9) were tested for their susceptibility to a highly-virulent Brazilian isolate of SfMNPV. The cytopathic effects induced by the virus, the production of viral particles, and the synthesis of viral polypeptides were examined and compared. Both S. frugiperda cell lines showed hypertrophy of cell nuclei and production of many polyhedra. The SDS-Page of radiolabed proteins showed that the cell protein synthesis was shutoff, while an intense band of about 30 kDa, recognized as polyhedrin, was synthesized. The other cell lines did not show polyhedra production, although some of them underwent morphological changes and protein synthesis shutdown in response to virus infection. The SF-21 and Sf9 cell lines are recommended for further in vitro production of SfMNPV.</p></div

Sonoprocessing is an effective strategy to encapsulate fisetin into Saccharomyces cerevisiae cells

International audienceThe encapsulation of fisetin into S. cerevisiae cells through sonoporation coupled with drying is reported for the first time in the literature. To establish the best conditions to maximize the amount of internalized fisetin, the cell density (5-10% w/v), fisetin concentration (1-3 mg/mL), acoustic energy density (0-333.3 W/L), and drying method (freeze-drying and spray drying) were analyzed through a Box-Behnken experimental design (BBD) coupled with response surface methodology (RSM). Higher encapsulation efficiency (EE) was achieved with a cell density of 10% w/v, while fisetin concentration of 3 mg/mL favored the encapsulation yield (EY) and antioxidant activity (AA). Higher EE (67.7%), EY (25.7 mg/g), and AA (90%) were registered when an acoustic density of 333.3 W/L was used. Furthermore, both drying protocols promoted fisetin encapsulation, but through spray drying, the EE, EY, and AA were 11.5%, 11.1%, and 26.6% higher than via freeze-drying, respectively. This work proved that fully filled biocapsules were produced through sonoprocessing, and their morphology was influenced by the acoustic energy and drying process. Overall, these results open new perspectives for the application of sonoprocessing-assisted encapsulation, paving the way for developing innovative yeast-based delivery systems for lipophilic compounds such as fisetin. KEY POINTS: • Sonoprocessing improves the encapsulation of fisetin into S. cerevisiae cells • Spray drying promotes fisetin loading into yeasts' intracellular space and cavities • Fisetin binding with yeast extracellular agents are favored by freeze-drying