Entomopathogenic Viruses in the Neotropics: Current Status and Recently Discovered Species.

The market for biological control of insect pests in the world and in Brazil has grown in recent years due to the unwanted ecological and human health impacts of chemical insecticides. Therefore, research on biological control agents for pest management has also increased. For instance, insect viruses have been used to protect crops and forests around the world for decades. Among insect viruses, the baculoviruses are the most studied and used viral biocontrol agent. More than 700 species of insects have been found to be naturally infected by baculoviruses, with 90% isolated from lepidopteran insects. In this review, some basic aspects of baculovirus infection in vivo and in vitro infection, gene content, viral replication will be discussed. Furthermore, we provide examples of the use of insect viruses for biological pest control and recently characterized baculoviruses in Brazil.

Hematological and biochemical profiles of Mangalarga Marchador mares in the transition period bred on pasture / Perfis hematológico e bioquímico de éguas da raça Mangalarga Marchador no período de transição mantidas a pasto

The present study aimed to evaluate the effects of the transition period on hematological and biochemical constituents in Mangalarga Marchador mares. Forty-eight mares were used to form a maintenance group (MG) and transition group (TG), formed by pregnant mares and, after delivery, infants. Blood samples were collected at the following times: T-60 (60 d pre-delivery), T-30 (30 d pre-delivery), T-15 (15 d pre-delivery), T0 (first 6h post-delivery), T15 (15 d post-delivery), T30 (30 d post-delivery), and T60 (60 d post-delivery). The TG had lower values (P< 0.05) of red blood cells, hematocrit and hemoglobin at T0, T15, T30 and T60 times than MG. The mean corpuscular volume was lower in MG (P< 0.05) than in TG (T0, T15, T30 and T60) and mean corpuscular hemoglobin concentration was higher (P< 0.05) in MG than in TG (T15, T30 and T60). On the other hand, the diameter distribution of red blood cells presented a lower value (P< 0.05) in MG than in TG (T15 and T30). Mares in transition period presented regenerative anemia. The results demonstrate physiological metabolic variations of different intensities during pregnancy, delivery and early lactation.(AU)

O presente estudo teve como objetivo avaliar os efeitos do período de transição em constituintes hematológicos e bioquímicos em éguas Mangalarga Marchador. Foram utilizadas 48 éguas para formar um grupo de manutenção (GM) e um grupo de transição (GT), composto por éguas gestantes e, após o parto, lactentes. Amostras de sangue foram coletadas nos seguintes tempos: T-60 (60 dias pré-parto), T-30 (30 dias pré-parto), T-15 (15 dias pré-parto), T0 (seis primeiras horas pós-parto), T15 (15 dias pós-parto), T30 (30 dias pós-parto) e T60 (60 dias pós-parto). O GT apresentou valores menores (P<0,05) de hemácias, hematócrito e hemoglobina, nos tempos T0, T15, T30 e T60, do que o GM. O volume corpuscular médio foi menor no GM (P<0,05) do que no GT (T0, T15, T30, T60) e a concentração corpórea de hemoglobina corpórea foi maior (P<0,05) no GM do que no GT (T15, T30, T60). Por outro lado, a distribuição do diâmetro dos eritrócitos apresentou um valor menor (P<0,05) no GM do que no GT (T15 e T30). Éguas em período de transição apresentam anemia regenerativa. Os resultados demonstram variações metabólicas de diferentes intensidades durante a gestação, o parto e o início de lactação.(AU)

Biological and molecular characterization of two Anticarsia gemmatalis multiple nucleopolyhedrovirus clones exhibiting contrasting virulence.

Baculovirus natural populations are known to be genetically heterogeneous and such genotypic diversity could have implications in the performance of biocontrol agents. The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) has been widely used to control the velvetbean caterpillar, Anticarsia gemmatalis, in Brazil. In the present work, morphological and molecular analyses as well as the biological activity of AgMNPV genotypes derived from a Brazilian field isolate (AgMNPV-79) were carried out. The existence of genotypic variants in the population was confirmed by DNA restriction analysis. Although difference in virulence was observed among the variants, the most (Ag79-01) and the least (AgL-16) virulent clones do not show any morphological and cytopathological changes when compared to the most studied isolate (AgMNPV-2D). The complete genome analysis of the two viral clones showed the presence of single open reading frames (ORFs) of the pe-38 and he65 genes, which contrasts with the two split ORFs present in the genome of the AgMNPV-2D isolate. The viral clone AgL-16 has many variations in the ie-2 and pe-38 genes, which are transcription regulatory genes responsible for the regulation of viral early gene expression during insect cell infection. Furthermore, other genes showed alterations like the odv-e56, which have an essential role in the maturation and envelopment of the ODVs, and bro-a and bro-b genes which were fused to form a single ORF. For the Ag79-01, although the total number of single nucleotide variants (SNVs) was more prominent in the pe-38 gene, its genome showed very few modifications in comparison to the AgMNPV-2D genome.

Genome-wide diversity in temporal and regional populations of the betabaculovirus Erinnyis ello granulovirus (ErelGV).

BACKGROUND: Erinnyis ello granulovirus (ErelGV) is a betabaculovirus infecting caterpillars of the sphingid moth E. ello ello (cassava hornworm), an important pest of cassava crops (Manihot esculenta). In this study, the genome of seven field isolates of the virus ErelGV were deep sequenced and their inter- and intrapopulational sequence diversity were analyzed. RESULTS: No events of gene gain/loss or translocations were observed, and indels were mainly found within highly repetitive regions (direct repeats, drs). A naturally occurring isolate from Northern Brazil (Acre State, an Amazonian region) has shown to be the most diverse population, with a unique pattern of polymorphisms. Overall, non-synonymous substitutions were found all over the seven genomes, with no specific gathering of mutations on hotspot regions. Independently of their sizes, some ORFs have shown higher levels of non-synonymous changes than others. Non-core genes of known functions and structural genes were among the most diverse ones; and as expected, core genes were the least variable genes. We observed remarkable differences on diversity of paralogous genes, as in multiple copies of p10, fgf, and pep. Another important contrast on sequence diversity was found on genes encoding complex subunits and/or involved in the same biological processes, as late expression factors (lefs) and per os infectivity factors (pifs). Interestingly, several polymorphisms in coding regions lie on sequences encoding specific protein domains. CONCLUSIONS: By comparing and integrating information about inter- and intrapopulational diversity of viral isolates, we provide a detailed description on how evolution operates on field isolates of a betabaculovirus. Our results revealed that 35-41% of the SNPs of ErelGV lead to amino acid changes (non-synonymous substitutions). Some genes, especially non-core genes of unknown functions, tend to accumulate more mutations, while core genes evolve slowly and are more conserved. Additional studies would be necessary to understand the actual effects of such gene variations on viral infection and fitness.

5-ALA-mediated photodynamic therapy reduces the parasite load in mice infected with Leishmania braziliensis.

Photodynamic therapy (PDT) has proven to be an effective alternative for the treatment of cutaneous leishmaniasis. Skin lesions consist of ulcers with well-defined raised edges, and granular floor. Th1 immune response is the protective profile in patients infected with Leishmania. In this study, the photodynamic therapy with 5-aminolevulinic acid, the parasitic load, and the modulation of the immune response was evaluated in mice infected with Leishmania braziliensis. Balb/c mice were infected with L. braziliensis and subsequently treated with three sections of PDT. The parasite load and mRNA expression of cytokines (IFN-γ, IL-4, IL-17, IL-22, IL-27, IL-10) and transcription factors (GATA-3, Foxp3 and T-bet) were analysed by quantitative PCR. The parasite load in the treated group was significantly lower than in the untreated group (P<.0001); in PDT treated animals, we observed an increase in IFN-γ and T-bet mRNA (P=.012 and P=.0071). There was a significant reduction in mRNA expression of IL-22 associated with an increased expression of IL-27 mRNA in the animals treated with light only (P=.0001). 5-ALA associated with photodynamic therapy promotes a reduction in parasite load and an increased expression of IFN-γ and T-bet mRNA.

The complete genome of a baculovirus isolated from an insect of medical interest: Lonomia obliqua (Lepidoptera: Saturniidae).

Lonomia obliqua (Lepidoptera: Saturniidae) is a species of medical importance due to the severity of reactions caused by accidental contact with the caterpillar bristles. Several natural pathogens have been identified in L. obliqua, and among them the baculovirus Lonomia obliqua multiple nucleopolyhedrovirus (LoobMNPV). The complete genome of LoobMNPV was sequenced and shown to have 120,022 bp long with 134 putative open reading frames (ORFs). Phylogenetic analysis of the LoobMNPV genome showed that it belongs to Alphabaculovirus group I (lepidopteran-infective NPV). A total of 12 unique ORFs were identified with no homologs in other sequenced baculovirus genomes. One of these, the predicted protein encoded by loob035, showed significant identity to an eukaryotic transcription terminator factor (TTF2) from the Lepidoptera Danaus plexippus, suggesting an independent acquisition through horizontal gene transfer. Homologs of cathepsin and chitinase genes, which are involved in host integument liquefaction and viral spread, were not found in this genome. As L. obliqua presents a gregarious behavior during the larvae stage the impact of this deletion might be neglectable.

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.

Molecular analysis of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) shows an interruption of an inhibitor of apoptosis gene (iap-3) by a new class-II piggyBac-related insect transposon.

A new piggyBac-related transposable element (TE) was found in the genome of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus interrupting an inhibitor of apoptosis gene. This mutant virus induces apoptosis upon infection of an Anticarsia gemmatalis cell line, but not in a Trichoplusia ni cell line. The sequence of the new TE (which was named IDT for iap disruptor transposon) has 2531 bp with two DNA sequences flanking a putative Transposase (Tpase) ORF of 1719 bp coding for a protein with 572 amino acids. These structural features are similar to the piggyBac TE, also reported for the first time in the genome of a baculovirus. We have also isolated variants of this new TE from different lepidopteran insect cells and compared their Tpase sequences.

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. METHODS AND RESULTS: Had isolated and characterized a toxin gene from the Bacillus thuringiensis S1451 strain which have been previously shown to be toxic to S. frugiperda and A. grandis. The toxin gene (cry1Ia) was amplified by PCR, sequenced, and cloned into the genome of a baculovirus. The Cry1Ia protein was expressed in baculovirus infected insect cells, producing protein inclusions in infected cells. The Cry1Ia protein has used in bioassays against to S. frugiperda and A. grandis. CONCLUSIONS: Bioassays using the purified recombinant protein showed high toxicity to S. frugiperda and A. grandis larvae. Molecular modelling of the Cry1Ia protein translated from the DNA sequence obtained in this work, showed that this protein possibly posses a similar structure to the Cry3A protein. Ultrastructural analysis of midgut cells from A. grandis incubated with the Cry1Ia toxin, showed loss of microvilli integrity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the cry1Ia is a good candidate for the construction of transgenic plants resistant to these important cotton pests.

Morphological characterization of Anticarsia gemmatalis M nucleopolyhedrovirus infection in haemocytes from its natural larval host, the velvet bean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae).

For a better understanding of virus x host interactions, transmission electron microscopy was used to characterize the intrahaemocoelic infection of Anticarsia gemmatalis larval haemocytes by A. gemmatalis M nucleopolyhedrovirus (AgMNPV). At 12 h post-infection (h p.i.), we observed nuclear hypertrophy, budded virus assembling, and protrusion towards the cytoplasm, virion envelopment, and accumulation of fibrillar aggregates in the cytoplasm. Around 24 h p.i., fibrillar aggregates also appeared inside nuclei of infected cells. By 48 h p.i., virogenic stroma and polyhedra were visualised in nuclei and at 72 h p.i., widespread infection in haemocytes was observed. Cell remnants and free polyhedra were phagocytosed by granular haemocyte 1 and plasmatocytes. Entire cells were phagocytosed only by plasmatocytes. Necrosis of infected cells was quite common, suggesting a putative cytotoxic response. Granular haemocyte 1 presented a more exuberant protrusion of budded viruses in comparison to other haemocytes. All types of haemocytes were shown to be infected, and the intense virus replication in some of these cells reveals the importance of haemolymph for AgMNPV spread in its natural host, a critical factor for permissiveness.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis.

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.

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

We have constructed a transfer vector (pAgGal) containing the beta-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 beta-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. Beta-galactosidase was expressed at high levels late on infection as expected for a gene under the control of the polh promoter. The highly expressed beta-galactosidase protein was also shown to be biologically active by a beta-galactosidase assay.

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. ATATA 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.

Production of viral progeny in insect cells undergoing apoptosis induced by a mutant Anticarsia gemmatalis nucleopolyhedrovirus.

The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is the most successful viral biopesticide in use worldwide. We have demonstrated that despite widespread apoptosis and no protein synthesis at 48 h p.i., UFL-AG-286 cells infected with a mutant of AgMNPV (vApAg), produced significant amounts of budded virus (BVs) and viral DNA late in infection. However, a different susceptible cell line (BTI-Tn5B 1-4) showed no signs of apoptosis and produced 3.5 times more budded virus when infected with vApAg. A comparison of DNA from AgMNPV and vApAg digested with the same restriction enzymes showed differences in the restriction pattern, indicating that the vApAg phenotype might be due to a mutation in a gene or genes responsible for directly or indirectly inhibiting apoptosis in UFL-AG-286 cells.

Screening and characterization of Bacillus thuringiensis isolates from Brazil for the presence of coleoptera-specific cry genes.

Forty-three Bacillus thuringiensis isolates from Brazil and 3 from Argentina were screened, using the polymerase chain reaction (PCR), for various coleoptera-specific cry genes. Seven isolates produced specific and/or nonspecific DNA fragments in a PCR reaction with primers specific for two coleopteran cry genes and 4 of these produced DNA fragments with primers specific for 7 known coleopteran cry genes. These isolates showed, by electron microscopy, the presence of spherical crystals. They also showed proteins of around 70 kDa which were immunologically similar to the Cry3Aa protein from B. thuringiensis subsp. tenebrionis. The 3 isolates from Argentina were toxic to T. molitor, and although no isolate from Brazil showed toxicity, they might show toxicity to another insect species.

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). Sinceper 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.

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

A mutant baculovirus with a temperature-sensitive IE-1 transregulatory protein.

We have mapped the mutation responsible for the temperature-sensitive (ts) phenotype of tsB821, a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus (H. H. Lee and L. K. Miller, J. Virol. 31:240-252, 1979), to a single nucleotide which changes alanine 432 of the multifunctional regulatory protein IE-1 to a valine. Mapping was done with a combination of marker rescue and transient expression assays, hybrid gene construction by overlap PCR gene splicing, and nucleotide sequence analysis. Cells infected with tsB821 at high multiplicities of infection showed a spectrum of responses from severe cytopathic effects, including apoptosis, to a lack of obvious signs of infection. Protein synthesis in tsB821-infected cells at the restrictive temperature appeared similar to uninfected cell protein synthesis, but viral DNA replication and budded virus production were observed, albeit in a delayed manner. The dependence of early and late promoter activity on the wild-type IE-1 gene, ie-1, was observed in transient expression assays. However, the dependence of early promoter activity on ie-1 was strongest in the absence of other viral genes. Thus, other viral genes appear to be able to compensate, at least in part, for the lack, or low levels, of ie-1 in transient expression assays using early promoters. The mutant should prove useful in further defining the function(s) of IE-1.

Expression of full-length and truncated forms of crystal protein genes from Bacillus thuringiensis subsp. kurstaki in a baculovirus and pathogenicity of the recombinant viruses.

Full-length and truncated forms of the crystal protein gene cryIA(b) derived from Bacillus thuringiensis subsp. kurstaki HD-1 and full-length cryIA(c) gene of B. thuringiensis subsp. kurstaki HD-73 were introduced into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus, in place of the polyhedrin gene. All gene constructs were expressed at high levels in insect cells and insects upon infection with the recombinant viruses. The protein products were shown to be biologically and immunologically similar to the natural crystal protein. The expressed proteins formed crystals (in insects) up to 10 times bigger (in length) than their bacterial counterpart. The LT50 values for recombinant viruses were not significantly shorter than wild-type virus.