Sequence analysis of a non-classified, non-occluded DNA virus that causes salivary gland hypertrophy of Musca domestica, MdSGHV

AbstractThe genome of the virus that causes salivary gland hypertrophy in Musca domestica (MdSGHV) was sequenced. This non-classified, enveloped, double stranded, circular DNA virus had a 124,279bp genome. The G + C content was 43.5% with 108 putative methionine-initiated open reading frames (ORFs). Thirty ORFs had homology to database proteins: eleven to proteins coded by both baculoviruses and nudiviruses (p74, pif-1, pif-2, pif-3, odv-e66, rr1, rr2, iap, dUTPase, MMP, and Ac81-like), seven to nudiviruses (mcp, dhfr, ts, tk and three unknown proteins), one to baculovirus (Ac150-like), one to herpesvirus (dna pol), and ten to cellular proteins. Mass spectrum analysis of the viral particles' protein components identified 29 structural ORFs, with only p74 and odv-e66 previously characterized as baculovirus structural proteins. Although most of the homology observed was to nudiviruses, phylogenetic analysis showed that MdSGHV was not closely related to them or to the baculoviruses

Contribution of molecular biology to the improvement of insect viruses as biological control products

A biologia molecular desempenha um importante papel na compreensão das pro-priedades biológicas dos vírus de insetos utilizados no controle biológico de insetos-praga. A biologia molecular pode, também, ser utilizada para melhorar ou manter a eficácia dos produtos utilizados no controle biológico de insetos. Existem descrições de 300 a 500 baculovírus de várias famílias de insetos mas a maioria pertence a Lepidoptera. Poucos destes vírus têm sido utilizados como produtos de controle biológico. O uso de enzimas de restrição para caracterizar o genoma dos baculovírus tem permitido desenvolver um método preciso para a identificação destes diferentes vírus. Uma vez identificados, qualquer vírus contaminante pode ser detectado nas preparações. Estes vírus podem ser melhorados expandindo tanto sua gama de hospedeiros como sua virulência através de recombinação genética. Os eventos de recombinação podem ser gerados e detectados por metodologias moleculares. Técnicas de DNA recombinante podem também ser utilizadas para modificar os baculovírus de maneira que estes passem a produzir toxinas bacterianas ou outras toxinas que afetam insetos. Estes baculovírus poderiam matar seus insetos-alvo de maneira mais rápida. Métodos de biologia molecular são essenciais para a análise de qualidade e identificação de problemas associados com a produção dos vírus de insetos nos quais baculovírus são gerados com inserções de DNA do inseto. Estes transposons de DNA levam o vírus mutante a produzir poucos poliedros. Além disso, genomas virais defeituosos são gerados quando os baculovírus são produzidos em sistemas contínuos de cultura. Biologia molecular é utilizada para detectar estes defeitos e possivelmente seja usada para corrigi-los.Molecular biology has an important role in understanding biological properties of insect viruses used in biological control of insect pests. Molecular biology can also be used to improve or sustain the effectiveness of insect biocontrol products. There have been descriptions of 300 to 500 baculoviruses from various families of insects but mostly from Lepidoptera. Very few of these have been used as biocontrol products. The use of DNA restriction enzymes to characterize the genomes of baculoviruses has allowed for a precise method for identifying these different viruses. Once viruses have been identified, any contaminating virus can be detected in the preparations. These viruses may be improved by expanding the host range and virulence of baculoviruses by genetic recombination. The recombination events can be constructed and detected by molecular methodologies. Recombinant DNA techniques can also be used to modify baculoviruses in a way such that they produce bacterial or insect toxins. Such baculoviruses could kill target insects more quickly. Molecular biology methodologies are essential for quality analysis and detection of problems associated with production of insect viruses in which baculoviruses are generated with insertions of insect DNA. These transposons of DNA cause the mutant viruses to produce few polyhedra. Secondly, defective viral genomes are produced when baculoviruses are grown in continuous culture systems. Molecular biology is used to detect these and hopefully, can be used to correct these defects

Sequence Analysis of the Genome of the Neodiprion sertifer Nucleopolyhedrovirus

The genome of the Neodiprion sertifer nucleopolyhedrovirus (NeseNPV), which infects the European pine sawfly, N. sertifer (Hymenoptera: Diprionidae), was sequenced and analyzed. The genome was 86,462 bp in size. The C+G content of 34% was lower than that of the majority of baculoviruses. A total of 90 methionine-initiated open reading frames (ORFs) with more than 50 amino acids and minimal overlapping were found. From those, 43 ORFs were homologous to other baculovirus ORFs, and 29 of these were from the 30 conserved core genes among all baculoviruses. A NeseNPV homolog to the ld130 gene, which is present in all other baculovirus genomes sequenced to date, could not be identified. Six NeseNPV ORFs were similar to non-baculovirus-related genes, one of which was a trypsin-like gene. Only one iap gene, containing a single BIR motif and a RING finger, was found in NeseNPV. Two NeseNPV ORFs (nese18 and nese19) were duplicates transcribed in opposite orientations from each other. NeseNPV did not have an AcMNPV ORF 2 homolog characterized as the baculovirus repeat ORF (bro). Six homologous regions (hrs) were located within the NeseNPV genome, each containing small palindromes embedded within direct repeats. A phylogenetic analysis was done to root the tree based upon the sequences of DNA polymerase genes of NeseNPV, 23 other baculoviruses, and other phyla. Baculovirus phylogeny was then constructed with 29 conserved genes from 24 baculovirus genomes. Culex nigripalpus nucleopolyhedrovirus (CuniNPV) was the most distantly related baculovirus, branching to the hymenopteran NeseNPV and the lepidopteran nucleopolyhedroviruses and granuloviruses

Control biológico: caracterización del ADN viral mediante mapas de restricción y técnicas de manipulación.

24 Páginas. Series Publicacoes Miscelaneas A4/BR (IICA) no. 88-010.The title of activity of IICA project corresponding to this consultancy is: Cooperation with EMBRAPA on research and applications of genetic resources, biotechnology and biologic control of plagues, diseases and weeds. (MIBA) El título de actividad del proyecto IICA correspondiente a esta consultoría es: Cooperación con EMBRAPA en investigación y aplicaciones de recursos genéticos, biotecnología y control biológico de plagas, enfermedades y malezas

Characterization and biological activity of a Brazilian isolate of Bacillus sphaericus (Neide) highly toxic to mosquito larvae

Primary powders of Bacillus sphaericus strain S2 isolated from soil samples in Brazil, and strain 2362 were produced in a 14 liter fermentor. Growth patterns and sporulation observed in three trials with strains S2 and 2362 in the fermentor were similar. Second-instar larvae of Culex quinquefasciatus, Anopheles albimanus, Anopheles quadrimaculatus, and Aedes aegypti exposed for 48 hr to strain S2 responded with LC50 values of 0.25, 5.95, 12.28 and 140.0 ppb of lyophilized primary powder, respectively. Under the same conditions, strain 2362 resulted in LC50 values of 0.39, 7.16, 16.93 and 307.0 ppb of lyophilized primary powder, respectively, in those mosquito larvae. Statistical analysis of the bioassay data did not show significant differences among LC50 values observed in B. sphaericus strains S2 and 2362, at the 0.05 level. Toxins of strains S2 and 2362 were extracted at pH 12 with NaOH. Electrophoresis of the extracts in polyacrylamide gel under denaturing conditions revealed the 51 and 42 kDa toxins in both S2 and 2362 B. sphaericus strains. The presence of the 42 kDa peptide in the extracts was confirmed by Western blot and Elisa, with anti-42 kDa IgG previously prepared from strain 2362