Bergmann Morais Ribeiro

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt)) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTD-lacZ). Bioassays with thirdinstar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTD-lacZ compared to the wild-type virus. A mean of 20:4 � 10 8 OBs/g/larva and 40:7 � 10 8 OBs/g/larva was produced from vAgEGTD-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC50) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTD-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTD-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV. 2003 Elsevier Science (USA). All rights reserved.

Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putative receptors for the Cry1B protein of Bacillus thuringiensis

Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. They interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in midgut epithelial cells lysis. In this work we had cloned, sequenced and expressed a cry1Ba toxin gene from the B thuringiensis S601 strain which was previously shown to be toxic to Anthonomus grandis, a cotton pest. The Cry1Ba6 protein expressed in an acrystaliferous B. thuringiensis strain was toxic to A. grandis in bioassays. The binding of Cry1Ba6 toxin to proteins located in the midgut brush border membrane of A. grandis was analyzed and we found that Cry1Ba6 binds to two proteins (62 and 65kDa) that showed alkaline phosphatase (ALP) activity. This work is the first report that shows the localization of Cry toxin receptors in the midgut cells of A. grandis.

Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

BackgroundBaculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae.ResultsBioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill.ConclusionsBaculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs.

Recombinant Cry1Ia protein is highly toxic to cotton boll weevil (Anthonomus grandis Boheman) and fall armyworm (Spodoptera frugiperda).

Aims:  To evaluate the activity of cry1Ia gene against cotton pests, Spodoptera frugiperda and Anthonomus grandis.

The major leucyl aminopeptidase of Trypanosoma cruzi (LAPTc) assembles into a homohexamer and belongs to the M17 family of metallopeptidases

BackgroundPathogens depend on peptidase activities to accomplish many physiological processes, including interaction with their hosts, highlighting parasitic peptidases as potential drug targets. In this study, a major leucyl aminopeptidolytic activity was identified in Trypanosoma cruzi, the aetiological agent of Chagas disease.ResultsThe enzyme was isolated from epimastigote forms of the parasite by a two-step chromatographic procedure and associated with a single 330-kDa homohexameric protein as determined by sedimentation velocity and light scattering experiments. Peptide mass fingerprinting identified the enzyme as the predicted T. cruzi aminopeptidase EAN97960. Molecular and enzymatic analysis indicated that this leucyl aminopeptidase of T. cruzi (LAPTc) belongs to the peptidase family M17 or leucyl aminopeptidase family. LAPTc has a strong dependence on neutral pH, is mesophilic and retains its oligomeric form up to 80°C. Conversely, its recombinant form is thermophilic and requires alkaline pH.ConclusionsLAPTc is a 330-kDa homohexameric metalloaminopeptidase expressed by all T. cruzi forms and mediates the major parasite leucyl aminopeptidolytic activity. Since biosynthetic pathways for essential amino acids, including leucine, are lacking in T. cruzi, LAPTc could have a function in nutritional supply.

Characterization of the Ecdysteroid UDP-Glucosyltransferase (egt) Gene of Anticarsia gemmatalis Nucleopolyhedrovirus

The Anticarsia gemmatalis nucelopolyhedrovirus (AgMNPV) egt gene was cloned, sequenced and its expression characterized by RT-PCR and western blot analysis. Sequence analysis of the gene indicated the presence of an open reading frame (ORF) of 1482 nucleotides, which codes for a polypeptide of 494 amino acids. A TATA box and a conserved regulatory sequence (CATT) found in other baculovirus early genes were present in the promoter region of the egt gene. A poly-A consensus sequence was present in the 3′ untranslated region (3′-UTR) of the gene. Homology comparisons showed that the EGT protein of AgMNPV is most closely related (95.9% amino acid sequence identity) to the EGT from the Choristoneura fumiferana DEF nucleopolyhedrovirus (CfDEF). Transcriptional analysis of the AgMNPV egt gene showed that egt-specific transcripts can be detected both early and late in infection. The EGT protein was detected, by western blot analysis, in the intra- (from 12 to 48 h post-infection) and extra-cellular (from 12 to 96 h post-infection) fractions of infected insect cells. The AgMNPV Bgl II-F fragment, which has homology to the AcMNPV ie-1 gene, was cloned and used to cotransfect SF21 cells with the cloned AgMNPV egt gene. EGT activity was observed, suggesting that AgMNPV ie-1 can transactivate egt expression.

A silencing suppressor protein (NSs) of a tospovirus enhances baculovirus replication in permissive and semipermissive insect cell lines.

The nonstructural protein (NSs) of the Tomato spotted wilt virus (TSWV) has been identified as an RNAi suppressor in plant cells. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) designated vAcNSs, containing the NSs gene under the control of the viral polyhedrin (polh) gene promoter, was constructed and the effects of NSs in permissive, semipermissive and nonpermissive insect cells to vAcNSs infection were evaluated. vAcNSs produced more budded virus when compared to wild type in semipermissive cells. Co-infection of vAcNSs with wild type baculoviruses clearly enhanced polyhedra production in all host cells. Confocal microscopy analysis showed that NSs accumulated in abundance in the cytoplasm of permissive and semipermissive cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Co-infection of vAcNSs with a recombinant AcMNPV containing the enhanced green fluorescent protein (egfp) gene, significantly increased EGFP expression in semipermissive cells and in Anticarsia gemmatalis-hemocytes. Absence of small RNA molecules of egfp transcripts in this cell line and in a permissive cell line indicates the suppression of gene silencing activity. On the other hand, vAcNSs was not able to suppress RNAi in a nonpermissive cell line. Our data showed that NSs protein of TSWV facilitates baculovirus replication in different lepidopteran cell lines, and these results indicate that NSs could play a similar role during TSWV-infection in its thrips vector.

Construction of a recombinant Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV-2D) harbouring the β-galactosidase gene

Summary. We have constructed a transfer vector (pAgGal) containing the β-galactosidase gene under control of the Escherichia coli gpt and AgMNPV polyhedrin (polh) promoters. The transfer vector was cotransfected with wild type Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) DNA into A. gemmatalis (UFL-AG-286) cells and a recombinant baculovirus (vAgGalA2) was isolated. The β-galactosidase gene insertion was checked by polymerase chain reaction (PCR) using DNA from AgMNPV and vAgGalA2 and primers specific for regions upstream and downstream of the polh gene. Insect cells (UFL-AG-286) were infected with the recombinant vAgGalA2 and wild type AgMNPV viruses and the production of the heterologous protein analyzed by SDS-PAGE and Pulse-Chase. β-galactosidase was expressed at high levels late on infection as expected for a gene under the control of the polh promoter. The highly expressed β-galactosidase protein was also shown to be biologically active by a β-galactosidase assay.

Structural and ultrastructural studies of Anticarsia gemmatalis midgut cells infected with the baculovirus A. gemmatalis nucleopolyhedrovirus

Anticarsia gemmatalis is a lepidopteran insect susceptible to A. gemmatalis nucleopolyhedrovirus (AgNPV), which is being used in a large scale, in Brazil, as a biological control agent against this serious soybean pest. Baculovirus usually infects its insect host through the midgut epithelium. In the midgut, it replicates in the nuclei of epithelial cells, producing progeny virus and establishing systemic infection. The AgNPV infection of A. gemmatalis midgut was studied using light and electron microscopy. It was observed that AgNPV enters the midgut mainly through columnar cells. Although the virus was not found in the nuclei of columnar cells until late on infection, it is believed that these cells are the primary sites of infection and replication. This fact can be explained by the continuous regeneration of the midgut epithelium. Besides, the infection may be occurring in isolated cells, making it more difficult to be visualized by electron microscopy. At 48 h post infection, hemocytes and tracheoblasts are infected and polyhedra are formed later in these cells, which are the secondary sites of infection.

Comparative analysis of American Dengue virus type 1 full-genome sequences

Dengue virus (DENV; Genus Flavivirus, Family Flaviviridae) has been circulating in Brazil since at least the mid-1980s and continues to be responsible for sporadic cases of Dengue fever and Dengue hemorrhagic fever throughout this country. Here, we describe the full genomes of two new Brazilian DENV-serotype 1 (DENV-1) variants and analyze these together with all other available American DENV-1 full-genome sequences. Besides confirming the existence of various country-specific DENV-1 founder effects that have produced a high degree of geographical structure in the American DENV-1 population, we also identify that one of the new viruses is one of only three detectable intra-American DENV-1 recombinants. Although such obvious evidence of genetic exchange among epidemiologically unlinked Latin American DENV-1 sequences is relatively rare, we find that at the population-scale there exists substantial evidence of pervasive recombination that most likely occurs between viruses that are so genetically similar that it is not possible to reliably distinguish and characterize individual recombination events.