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

The genome sequence of Pseudoplusia includens single nucleopolyhedrovirus and an analysis of p26 gene evolution in the baculoviruses

Background: Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IE) is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae) (Walker, 1858). In Brazil, the C. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. The PsinSNPV genome was determined and the phylogeny of the p26 gene within the family Baculoviridae was investigated. Results: The complete genome of PsinSNPV was sequenced (Roche 454 GS FLX – Titanium platform), annotated and compared with other Alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. The circular double stranded DNA genome is 139,132 bp in length, with a GC content of 39.3 % and contains 141 open reading frames (ORFs). PsinSNPV possesses the 37 conserved baculovirus core genes, 102 genes found in other baculoviruses and 2 unique ORFs. Two baculovirus repeat ORFs (bro) homologs, bro-a (Psin33) and bro-b (Psin69), were identified and compared with Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. The homologous repeats (hrs) are absent from the PsinSNPV genome, which is also the case in ChchNPV and TnSNPV. Two p26 gene homologs (p26a and p26b) were found in the PsinSNPV genome. P26 is thought to be required for optimal virion occlusion in the occlusion bodies (OBs), but its function is not well characterized. The P26 phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the Baculoviridae family. The presence of a signal peptide only in the PsinSNPV p26a/ORF-20 homolog indicates distinct function between the two P26 proteins. Conclusions: PsinSNPV has a genomic sequence apparently different from other baculoviruses sequenced so far. The complete genome sequence of PsinSNPV will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide

Schematic representation of the <i>polh</i> locus in AgMNPV and two recombinant viruses.

<p>The figure shows the orientation of <i>v-cath</i> and <i>chi</i>A genes, as well as the promoter and the <i>polh</i> gene and flanking ORFs (ORF 1629 and ORF2).</p

Chitinase activity analysis.

<p>(A) Chitinase activity detected in 100 micrograms of total protein from purified polyhedra of AgMNPV and vAgp2100Cf.chiA/v-cath using the DNS method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074592#pone.0074592-Miller1" target="_blank">[34]</a>. (B) Chitinase activity in wild type and recombinant-virus infected insect cell extracts (100 micrograms of total protein) measured using the [4MU-(GlcNAc) 2] substrate. (C) After electrophoresis in a native polyacrylamide gel the chitinase activity was measured using chitin glycol (1%) substrate. In both assays, the recombinant-virus infected insect cell extracts showed higher chitinase activity when compared with mock infected and wild type infected insect cells extracts. It is possible to see a faint band in all lanes of the gel shown in C (large arrow), which could explain the endogenous chitinase activity detected in mock and wild type-infected insect cells extracts. The arrow heads shows the chitinase activity detected only in recombinant virus infected insect cells extracts. All assays carried out in triplicate. Tukey Test (<i>P</i><0.05). Abs – Absorbance (550 nm).</p

Effect caused by the wild type (AgMNPV) and recombinant (vAgp2100Cf.chiA/<i>v-cath</i>) viruses in <i>A. gemmatalis</i> larvae.

<p>(A) Structural analysis of internal tissues of larvae infected with AgMNPV and vAgp2100Cf.chiA/v-cath at different times post infection. MG – midgut, CT – cuticle; FT – fat tissue. (B) L. Larvae infected with wild type and recombinant virus at 168 h p.i., showing body liquefaction and melanized cuticle only in recombinant virus-infected larvae (vAgp2100Cf.chiA/v-cath).</p

Cysteine protease activity analysis.

<p>(A) Proteolytic activity detected in 100 micrograms of total protein from hemolymph of uninfected- (mock), infected AgMNPV- and vAgp2100Cf.chiA/v-cath–insect larvae using keratin blue as substrate. The hemolymph of recombinant virus infected larvae showed an increased proteolytic activity when compared to wild type and uninfected insects (B) Proteolytic activity detected detected in 100 micrograms of total protein from purified polyhedra of AgMNPV and vAgp2100Cf.chiA/v-cath using keratin blue as substrate. Polyhedra from vAgp2100Cf.chiA/v-cath showed increased porteolytic activity when compared to AgMNPV polyhedra (C) The cysteine protease activity of uninfected UFL-AG-286 cells extract (mock), AgMNPV and vAgp2100Cf.chiA/v-cath-infected UFL-AG-286 cells extract (40 h p.i.) were measured in the presence or absence of a cysteine protease inhibitor (E-64). All extracts showed a reduction in the protease activity level, indicating the presence of cysteine proteases in all samples. However, the level of activity in the absence of E-64 was significantly higher in vAgp2100Cf.chiA/v-cath-infected UFL-AG-286 cell extracts when compared to uninfected UFL-AG-286 cells and AgMNPV-infected insect cell extracts. All assays were carried out in triplicate. Tukey Test (<i>P</i><0.05). Abs – Absorbance.</p