Identificação e caracterização de sequências repetidas de DNA no genoma do ciclídeo Astronotus ocellatus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ocorrência de rotavírus do grupo D em aves criadas em granjas na mesorregião metropolitana de Belém, Pará

Os rotavírus infectam seres humanos e várias espécies de animais e são constituídos por partículas icosaédricas, não envelopadas, formadas por um genoma de 11 segmentos de dsRNA. São classificados em sete grupos designados de A-G. O rotavírus do grupo D (RVs-D) tem sido documentado em aves, porém existem poucos estudos disponíveis, principalmente com dados de detecção por RT-PCR e obtenção de sequências nucleotídicas. Este estudo objetivou investigar a ocorrência de RVs-D em aves criadas em granjas situadas na Mesorregião Metropolitana de Belém- Pará, no período compreendido entre 2008 a 2011. Foram colhidos 85 pools de amostras fecais provenientes de 37 granjas pertencentes a oito municípios da Mesorregião Metropolitana de Belém. O dsRNA viral foi extraído a partir das suspensões fecais e submetido à eletroforese em gel de poliacrilamida (EGPA) seguido da RT-PCR realizada a partir da construção de iniciadores específicos para os genes VP6 e VP7 do RVs-D. Foi selecionada uma amostra positiva de cada município para o sequenciamento de nucleotídeos dos genes VP6 e VP7, sendo cinco amostras clonadas. A EGPA demonstrou positividade em 14/85 (16,5%) amostras e a RT-PCR em 30/85 (35,3%) amostras. Dos oito municípios estudados, sete apresentaram amostras positivas para RVs-D. Dentre as 37 granjas pertencentes a esses municípios foi observado a presença dessa infecção viral em 17 (45,9%) das granjas estudadas. O intervalo de idade em que o RVs-D foi detectado com maior frequência foi o de aves entre 16 a 30 dias (23/37 – 62%) do total de amostras nesta faixa etária. As sequências nucleotídicas das sete amostras foram classificadas como pertencentes ao RVs-D com bootstrap de 100 corroborando com esse agrupamento. As sequências do gene VP6 apresentaram 90,8-91,3% de similaridade com o protótipo de RVs-D (05V0049) e 98,5-99,9% de similaridade quando comparadas entre si. Para o gene VP7 apresentaram 87-96,1% de similaridade com o protótipo deste grupo e foram 94,1-100% similares quando comparados entre si. A presente análise assume um caráter pioneiro no Brasil e no mundo, permitindo ampliar os conhecimentos acerca do RVs-D.

Diversity and three-dimensional structures of the alpha Mcr of the methanogenic Archaea from the anoxic region of Tucuruí Lake, in Eastern Brazilian Amazonia

ABSTRACT: Methanogenic archaeans are organisms of considerable ecological and biotechnological interest that produce methane through a restricted metabolic pathway, which culminates in the reaction catalyzed by the Methyl-coenzyme M reductase (Mcr) enzyme, and results in the release of methane. Using a metagenomic approach, the gene of the a subunit of mcr (mcrα) was isolated from sediment sample from an anoxic zone, rich in decomposing organic material, obtained from the Tucuruí hydroelectric dam reservoir in eastern Brazilian Amazonia. The partial nucleotide sequences obtained were 83 to 95% similar to those available in databases, indicating a low diversity of archaeans in the reservoir. Two orders were identified -the Methanomicrobiales, and a unique Operational Taxonomic Unit (OTU) forming a clade with the Methanosarcinales according to low bootstrap values. Homology modeling was used to determine the three-dimensional (3D) structures, for this the partial nucleotide sequence of the mcrα were isolated and translated on their partial amino acid sequences. The 3D structures of the archaean mcrα observed in the present study varied little, and presented approximately 70% identity in comparison with the mcrα of Methanopyrus klanderi. The results demonstrated that the community of methanogenic archaeans of the anoxic C1 region of the Tucurui reservoir is relatively homogeneous.

Caracterização genética, correlação antigênica e ecoepidemiológica dos vírus do grupo C (Bunyaviridae, orthobunyavirus) isolados nas américas

Até o presente momento, estudos moleculares para os vírus do grupo C (Bunyaviridae, Orthobunyavirus) não foram publicados. O presente trabalho determinou as seqüências nucleotídicas completas para os segmento ARN pequenos (P-ARN) e a seqüencias parciais para os segmentos de ARN médio (M-ARN) dos vírus do grupo C. A seqüencia completa do segmento P-ARNvariou de 915 a 926 nucleotídeos, e revelou organização genômica semelhante em comparação aos demais ortobunyavírus. Baseado nos 705 nt do gene N, os membros do grupo C foram distribuídos em três grupos filogenéticos principais, com exceção do vírus Madrid que foi posicionado fora destes três grupos. A análise da cepa BeH 5546 do vírus Caraparu revelou que o mesmo apresenta seu segmento P-ARN semelhante ao do vírus Oriboca , sendo um vírus rearranjado em natureza. Em adição, a análise dos 345 nt do gene Gn para sete vírus do grupo C e para a cepa BeH 5546, revelou uma diferente topologia filogenética, sugerindo um padrão de rearranjo genético entre estes vírus. Estes achados representam as primeiras evidências de rearranjo genético em natureza entre os vírus do grupo C, dos quais vários são patógenos humanos. Finalmente, nossos dados genéticos corroboraram dados de relacionamento antigênico entre esses vírus determinados utilizando métodos sorológicos (testes de fixação de complemento, inibição da hemaglutinação e neutralização), sugerindo que a associação de dados informativos aos níveis molecular, sorológico e eco-epidemiológico podem contribuir para o melhor entendimento da epidemiologia molecular dos ortobunyavírus.

Caracterização molecular de cepas do Vírus da raiva (Lyssavirus; Rhabdoviridae) isoladas no Estado do Pará

A raiva é uma das zoonoses mais antigas e temidas pelo homem devido a seu desfecho fatal. Os cães ainda são considerados os principais responsáveis pela manutenção e transmissão da raiva para o homem. Porém, nos últimos anos os morcegos hematófagos têm ganhado destaque como potenciais transmissores de raiva para animais e humanos nas Américas. Recentemente, várias epidemias de raiva humana transmitida por morcegos hematófagos foram relatados no estado do Pará, o que mostra uma grande alteração no ambiente natural destes animais. A amplificação parcial do gene N pela técnica de RT-PCR foi aplicada em 62 amostras positivas para o Vírus da raiva, pela imunofluorescência direta e prova biológica. As seqüências nucleotídicas obtidas foram comparadas entre si e com outras amostras de vírus rábico isoladas no Brasil, utilizando os métodos de análise filogenética máxima verossimilhança e Bayesiano. Estas análises permitiram traçar o perfil epidemiológico molecular das variantes virais circulantes no estado do Pará, observando a emergência da transmissão de casos associados à variante antigênica 3 (VAg3), comumente encontrada em morcegos hematófagos Desmodus rotundus em detrimento dos casos relacionados à variante antigênica 2 (VAg2) associada a cães domésticos, bem como a identificação de três linhagens genéticas relacionadas a VAg3 e uma relacionada a VAg2 e uma possível nova variante isolada de morcego frugívoro Uroderma bilobatum.

Infecção experimental em hamster dourados (Mesocricetus auratus) e caracterização genética parcial de cinco Phlebovirus (Bunyaviridae) do Complexo Candiru isolados na Amazônia brasileira

Os flebovírus (família Bunyaviridae; gênero Phlebovirus), possuem importância considerável em saúde publica, pois podem causar uma variedade de síndromes clínicas. Na região amazônica brasileira até o momento já foram isolados 23 flebovírus sendo que quatro se destacam por terem sido isolados de humanos: Virus Alenquer, Virus Candiru, Virus Morumbi e Virus Serra Norte. Estes mais o Virus Itaituba, isolado de Didelphis marsupialis, fazem parte do Complexo Candiru (grupo Candiru) e foram utilizados para estudos de caracterização genética e de infecção experimental em hamsters dourados (Mesocricetus auratus). Os Hamsters mostraram-se suscetíveis a infecção por esses flebovírus, apesar de não terem apresentado sinais de doença. A análise histopatológica demonstrou aspectos lesionais no fígado, nos rins, no baço, nos pulmões e no sistema nervoso central, sendo as lesões mais intensas no fígado comprovadas pela presença dos antígenos virais empregando a técnica de imunohistoquímica. A análise das seqüências nucleotídicas parciais dos segmentos PRNA e MRNA obtidas para os cinco flebovírus estudados mostraram maior similaridade genética entre si do que com outros membros do gênero Phlebovirus. Sendo as mesmas geneticamente mais relacionadas ao Virus Punta Toro. Filogeneticamente, independentemente do segmento genômico analisado, os cinco flebovírus constituem um grupo monofilético, sendo que a análise pelo método de máxima verossimilhança demonstrou diferentes origens evolutivas para os segmentos de RNA o que sugere a ocorrência de rearranjo genético em natureza entre estes cinco flebovírus amazônicos.

Caracterização molecular de espécies da família Anaplasmataceae em leucócitos e plaquetas de cães de Jaboticabal-SP e de Campo Grande-MS

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Sequenciamento de DNA e imunoistoquímica renal para detecção de Leishmania sp em cães

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise ampla do genoma para detecção de erros de montagem no genoma de referência bovino e para detecção de locos relacionados a características de produção e reprodução da raça GIR

Pós-graduação em Genética e Melhoramento Animal - FCAV

Genetic Variability of Beauveria bassiana and a DNA Marker for Environmental Monitoring of a Highly Virulent Isolate Against Cosmopolites sordidus

The banana weevil Cosmopolites sordidus (Germar) is one of a number of pests that attack banana crops. The use of the entomopathogenic fungus Beauveria bassiana as a biological control agent for this pest may contribute towards reducing the application of chemical insecticides on banana crops. In this study, the genetic variability of a collection of Brazilian isolates of B. bassiana was evaluated. Samples were obtained from various geographic regions of Brazil, and from different hosts of the Curculionidae family. Based on the DNA fingerprints generated by RAPD and AFLP, we found that 92 and 88 % of the loci were polymorphic, respectively. The B. bassiana isolates were attributed to two genotypic clusters based on the RAPD data, and to three genotypic clusters, when analyzed with AFLP. The nucleotide sequences of nuclear ribosomal DNA intergenic spacers confirmed that all isolates are in fact B. bassiana. Analysis of molecular variance showed that variability among the isolates was not correlated with geographic origin or hosts. A RAPD-specific marker for isolate CG 1024, which is highly virulent to C. sordidus, was cloned and sequenced. Based on the sequences obtained, specific PCR primers BbasCG1024F (5'-TGC GGC TGA GGA GGA CT-3') and BbasCG1024R (5'-TGC GGC TGA GTG TAG AAC-3') were designed for detecting and monitoring this isolate in the field.

Diversity and Adaptation of Human Respiratory Syncytial Virus Genotypes Circulating in Two Distinct Communities: Public Hospital and Day Care Center

HRSV is one of the most important pathogens causing acute respiratory tract diseases as bronchiolitis and pneumonia among infants. HRSV was isolated from two distinct communities, a public day care center and a public hospital in Sao Jose do Rio Preto - SP, Brazil. We obtained partial sequences from G gene that were used on phylogenetic and selection pressure analysis. HRSV accounted for 29% of respiratory infections in hospitalized children and 7.7% in day care center children. On phylogenetic analysis of 60 HRSV strains, 48 (80%) clustered within or adjacent to the GA1 genotype; GA5, NA1, NA2, BA-IV and SAB1 were also observed. SJRP GA1 strains presented variations among deduced amino acids composition and lost the potential O-glycosilation site at amino acid position 295, nevertheless this resulted in an insertion of two potential O-glycosilation sites at positions 296 and 297. Furthermore, a potential O-glycosilation site insertion, at position 293, was only observed for hospital strains. Using SLAC and MEME methods, only amino acid 274 was identified to be under positive selection. This is the first report on HRSV circulation and genotypes classification derived from a day care center community in Brazil.

Molecular confirmation of ovine herpesvirus 2-induced malignant catarrhal fever lesions in cattle from Rio Grande do Norte, Brazil

Molecular findings that confirmed the participation of ovine herpesvirus 2 (OVH-2) in the lesions that were consistent with those observed in malignant catarrhal fever of cattle are described. Three mixed-breed cattle from Rio Grande do Norte state demonstrated clinical manifestations that included mucopurulent nasal discharge, corneal opacity and motor incoordination. Routine necropsy examination demonstrated ulcerations and hemorrhage of the oral cavity, corneal opacity, and lymph node enlargement. Significant histopathological findings included widespread necrotizing vasculitis, non-suppurative meningoencephalitis, lymphocytic interstitial nephritis and hepatitis, and thrombosis. PCR assay performed on DNA extracted from kidney and mesenteric lymph node of one animal amplified a product of 423 base pairs corresponding to a target sequence within the ovine herpesvirus 2 (OVH-2) tegument protein gene. Direct sequencing of the PCR products, from extracted DNA of the kidney and mesenteric lymph node of one cow, amplified the partial nucleotide sequences (423 base pairs) of OVH-2 tegument protein gene. Blast analysis confirmed that these sequences have 98-100% identity with similar OVH-2 sequences deposited in GenBank. Phylogenetic analyses, based on the deduced amino acid sequences, demonstrated that the strain of OVH-2 circulating in ruminants from the Brazilian states of Rio Grande do Norte and Minas Gerais are similar to that identified in other geographical locations. These findings confirmed the active participation of OVH-2 in the classical manifestations of sheep associated malignant catarrhal fever.

Molecular Characterization and Physical Mapping of Two Classes of 5S rDNA in the Genomes of Gymnotus sylvius and G. inaequilabiatus (Gymnotiformes, Gymnotidae)

The nucleotide sequences of the 5S rRNA multigene family and their distribution across the karyotypes in 2 species of Gymnotiformes, genus Gymnotus (G. sylvius and G. inaequilabiatus) were investigated by means of fluorescence in situ hybridization (FISH). The results showed the existence of 2 distinct classes of 5S rDNA sequences in both species: class I and class II. A high conservative pattern of the codifying region of the 5S rRNA gene was identified, contrasting with significant alterations detected in the nontranscribed spacer (NTS). The presence of TATA-like sequences along the NTS of both species was an expected occurrence, since such sequences have been associated with the regulation of the gene expression. FISH using 5S rDNA class I and class II probes revealed that both gene classes were collocated in the same chromosome pair in the genome of G. sylvius, while in that of G. inaequilabiatus, class II appeared more disperse than class I. Copyright (C) 2012 S. Karger AG, Basel

A possible new mechanism for the control of miRNA expression in neurons

The control of gene expression by miRNAs has been widely investigated in different species and cell types. Following a probabilistic rather than a deterministic regimen, the action of these short nucleotide sequences on specific genes depends on intracellular concentration,which in turn reflects the balance between biosynthesis and degradation. Recent studies have described the involvement of XRN2, an exoribonuclease, in miRNA degradation and PAPD4, an atypical poly(A) polymerase, in miRNA stability. Herein, we examined the expression of XRN2 and PAPD4 in developing and adult rat hippocampi. Combining bioinformatics and real-time PCR,we demonstrated that XRN2 and PAPD4 expression is regulated by the uncorrelated action of transcription factors, resulting in distinct gene expression profiles during development. Analyses of nuclei position and nestin labeling revealed that both proteins progressively accumulated during neuronal differentiation, and that they are weakly expressed in immature neurons and absent in glial and endothelial cells. Despite the differences in subcellular localization, both genes were concurrently identified within identical neuronal subpopulations, including specific inhibitory interneurons. Thus, we cope with a singular circumstance in biology: an almost complete intersected expression of functional-opposed genes, reinforcing that their antagonistically driven actions on miRNAs “make sense” if simultaneously present at the same cells. Considering that the transcriptome in the nervous system is finely tuned to physiological processes, it was remarkable that miRNA stability-related genes were oncurrently identified in neurons that play essential roles in cognitive functions such as memory and learning. In summary, this study reveals a possible new mechanism for the control of miRNA expression.

Design and implementation of bioinformatics tools for large scale genome annotation

The continuous increase of genome sequencing projects produced a huge amount of data in the last 10 years: currently more than 600 prokaryotic and 80 eukaryotic genomes are fully sequenced and publically available. However the sole sequencing process of a genome is able to determine just raw nucleotide sequences. This is only the first step of the genome annotation process that will deal with the issue of assigning biological information to each sequence. The annotation process is done at each different level of the biological information processing mechanism, from DNA to protein, and cannot be accomplished only by in vitro analysis procedures resulting extremely expensive and time consuming when applied at a this large scale level. Thus, in silico methods need to be used to accomplish the task. The aim of this work was the implementation of predictive computational methods to allow a fast, reliable, and automated annotation of genomes and proteins starting from aminoacidic sequences. The first part of the work was focused on the implementation of a new machine learning based method for the prediction of the subcellular localization of soluble eukaryotic proteins. The method is called BaCelLo, and was developed in 2006. The main peculiarity of the method is to be independent from biases present in the training dataset, which causes the over‐prediction of the most represented examples in all the other available predictors developed so far. This important result was achieved by a modification, made by myself, to the standard Support Vector Machine (SVM) algorithm with the creation of the so called Balanced SVM. BaCelLo is able to predict the most important subcellular localizations in eukaryotic cells and three, kingdom‐specific, predictors were implemented. In two extensive comparisons, carried out in 2006 and 2008, BaCelLo reported to outperform all the currently available state‐of‐the‐art methods for this prediction task. BaCelLo was subsequently used to completely annotate 5 eukaryotic genomes, by integrating it in a pipeline of predictors developed at the Bologna Biocomputing group by Dr. Pier Luigi Martelli and Dr. Piero Fariselli. An online database, called eSLDB, was developed by integrating, for each aminoacidic sequence extracted from the genome, the predicted subcellular localization merged with experimental and similarity‐based annotations. In the second part of the work a new, machine learning based, method was implemented for the prediction of GPI‐anchored proteins. Basically the method is able to efficiently predict from the raw aminoacidic sequence both the presence of the GPI‐anchor (by means of an SVM), and the position in the sequence of the post‐translational modification event, the so called ω‐site (by means of an Hidden Markov Model (HMM)). The method is called GPIPE and reported to greatly enhance the prediction performances of GPI‐anchored proteins over all the previously developed methods. GPIPE was able to predict up to 88% of the experimentally annotated GPI‐anchored proteins by maintaining a rate of false positive prediction as low as 0.1%. GPIPE was used to completely annotate 81 eukaryotic genomes, and more than 15000 putative GPI‐anchored proteins were predicted, 561 of which are found in H. sapiens. In average 1% of a proteome is predicted as GPI‐anchored. A statistical analysis was performed onto the composition of the regions surrounding the ω‐site that allowed the definition of specific aminoacidic abundances in the different considered regions. Furthermore the hypothesis that compositional biases are present among the four major eukaryotic kingdoms, proposed in literature, was tested and rejected. All the developed predictors and databases are freely available at: BaCelLo http://gpcr.biocomp.unibo.it/bacello eSLDB http://gpcr.biocomp.unibo.it/esldb GPIPE http://gpcr.biocomp.unibo.it/gpipe