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

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.

Impacto dos nutrientes N e K e de açúcares solúveis sobre populações de Diabrotica speciosa (Germar) (Coleoptera, Chrysomelidae) e Agrotis ipsilon (Hüfnagel) (Lepidoptera, Noctuidae) na cultura da batata, Solanum tuberosum L. (Solanaceae)

Foi estudada a ocorrência de Diabrotica speciosa (Germar, 1824) (Coleoptera, Chrysomelidae) e de Agrotis ipsilon (Hüfnagel, 1767) (Lepidoptera, Noctuidae) em plantas de batata, cultivares Achat e Monalisa, influenciadas por dosagens de nitrogênio e potássio, e teor mínimo de açúcares solúveis. Os seguintes parâmetros foram avaliados: concentração de nutrientes minerais e açúcar em folha verde, folha senescente, folha em abcisão, haste, tubérculo e planta total usando extratos de infusão em etanol 80%. A maior infestação por larvas de D. speciosa foi na cultivar Monalisa a 150 kg.ha-1 de N + K com 27,03% a P<0,05. Foi observado que o efeito da dosagem de N + K no incremento da concentração de açúcares solúveis aumentou os danos provocados por A. ipsilon nos tubérculos e hastes. A infestação por essas espécies aumentou para 58,82% na cultivar Monalisa, quando a dosagem de nitrogênio foi elevada de 0 para 150 kg.ha-1, na ausência de potássio. Por outro lado, alta dosagem de potássio reduziu os danos causados por A. ipsilon na cultivar Monalisa. Entretanto, não influiu no armazenamento de açúcar solúvel. Os resultados indicaram que na cultivar Achat os teores de açúcares solúveis foram reduzidos, provavelmente sensibilizados pela elevação da dosagem de adubação potássica, diferindo da cultivar Monalisa cuja influência verificada foi pela dosagem do nitrogênio.

Biologia e tabela de vida de fertilidade de Agrotis ipsilon em dieta artificial

O objetivo deste trabalho foi desenvolver uma dieta artificial para criação de Agrotis ipsilon em laboratório com base em parâmetros biológicos e na tabela de vida de fertilidade. A dieta artificial utilizada continha feijão, caseína, proteína de soja, levedura e germe de trigo como fontes protéicas. Os parâmetros biológicos duração e viabilidade das fases larval e pupal, peso de pupas, de ambos os sexos, com 24 horas de idade, razão sexual, longevidade dos adultos, período de pré-oviposição e número de ovos produzidos por fêmea e a tabela de vida de fertilidade foram avaliados. Foram observados seis ínstares larvais com duração de 25,4 dias e viabilidade de 93%. A duração da fase pupal foi de 12,4 dias e viabilidade de 96%. A viabilidade de ciclo total foi 72%. O peso de pupas foi 387 mg (machos) e 484 mg (fêmeas). A razão sexual foi 0,46. O período de pré-oviposição foi de um dia, com 1.806 ovos por fêmea. Na tabela de vida verificou-se que a taxa líquida de reprodução e a razão finita de aumento foram 616,9 e 1,14, respectivamente. A dieta artificial é adequada à manutenção da criação de A. ipsilon, em laboratório.

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 109 infected cells in three liters of culture

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.

Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso

INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5% polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4% of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.

Anti-VP1 and anti-VP2 antibodies detected by immunofluorescence assays in patients with acute human parvovirus B19 infection

Acute human parvovirus B19 infection is followed by an antibody response to the structural proteins of the viral capsid (VP1 and VP2). We used 80 sera collected from 58 erythema infectiosum and 6 transient aplastic crisis patients to test IgM and IgG antibodies against these two proteins in an immunofluorescence assay (IFA) using Sf9 cells infected with recombinant baculovirus expressing either VP1 or VP2 antigen. Although less sensitive than IgM capture enzyme immunoassay using native antigen (MACEIA), we could detect anti-VP1 or anti-VP2 IgM antibodies by IFA in 49 patients with acute infection (76.6%). Detection of IgG anti-VP1 and anti-VP2 by IFA, however, was as sensitive as IgG detection by indirect enzyme immunoassay. By applying IgG avidity IFA to sera of the 15 IgM IFA negative patients we were able to confirm acute infection in further 12 cases by IFA. Overall, acute infection was confirmed by IFA in 61 (95.3%) of the 64 patients.

Avaliação da infestação de insetos-praga associados à batata (Solanum tuberosum L.) sob efeito de nutrientes nitrogenados e potássicos e teores acumulados de aminoácidos livres nas cultivares Achat e Monalisa

Evaluation of insect-pest infestation associated to potato (Solanum tuberosum L.) under effect of nitrogen and potassium fertilizers and the accumulated amount of free aminoacids in Achat and Monalisa cultivars. The objective of this work was to evaluate the occurence of insect-pests on potato plants influenced by dosages of nitrogen and potassium accumulated in plant organs. A total of 169 plants of the Achat and Monalisa cultivars were evaluated to determine the presence-absence of Diabrotica speciosa Germar, 1824 and Agrotis ipsilon Hüfnagel, 1767. The experiment was carried out and executed at the Universidade Federal Fluminense, and the delineation was complete randomized block design, with four replication and nine treatments, using three fertilization level (0; 75 and 150 Kg/ha) with N-urea + KCl. The aminoacid levels were adjusted by the Leucine standard-curve (µg/l), using the Ninhydrin method, at 570 nm. The results showed that the tubercles of Monalisa accumulated high free aminoacid levels with 7,95% in the treatment N1K2 and 7,75% in the N2K1.These treatments, induced the infestation by D. speciosa larvae in 27,03%, when the aminoacid level was 2,01 ± 0,58% (X ± EP), with probability of 0,0196

Lagarta- falsa-medideira, Pseudoplusia includens (WALKER, 1857), nova praga do maracujazeiro no Espírito Santo

A espécie Pseudoplusia includens (Walker) (Lepidoptera: Noctuidae), lagarta-falsa-medideira, ataca diversas culturas de importância econômica, causando, na maioria das vezes, prejuízos consideráveis. Foram realizados levantamentos de todas as fases de desenvolvimento do inseto, no período de abril/2009 a abril/2010, em uma cultura de maracujá-azedo, Passifora edulis f. flavicarpa, no município de Linhares-ES, após ter sido constatada sua presença na área. A lagarta foi observada durante os meses de abril a novembro/2009 e de fevereiro a abril/2010, sendo constatados surtos mais severos nos meses de junho, setembro e novembro/2009, atingindo índices de até 80% de folhas danificadas. A planta invasora Solanum americanum (maria-pretinha), associada à cultura, é também hospedeira do inseto. Como inimigos naturais da lagarta, foram constatados o parasitoide Copidosoma truncatellum (Hymenoptera: Encyrtidae) e o entomopatógeno Baculovirus sp.. Este é o primeiro registro da ocorrência de P. includens na cultura de maracujá-azedo.

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.