Development of new microsatellites for the hookworm Ancylostoma caninum and analysis of genetic diversity in Brazilian populations.

Considering the great efforts towards formulating a vaccine against hookworms, and the concerns about the spread of drug resistance through hookworm populations, it is justified to study the molecular diversity and population genetic structure of these nematodes. This work had the aim to develop microsatellite markers to investigate the genetic structure and the molecular diversity of Brazilian populations of Ancylostoma caninum. Seven microsatellites markers were successfully used to characterize five Brazilian populations. These findings may contribute to a better comprehension of the ecology, patterns of transmission, drug resistances and development of immunotherapeutic strategies in hookworms.

Evolutionary interplay between structure, energy and epistasis in the coat protein of the ϕX174 phage family.

Viral capsids are structurally constrained by interactions among the amino acids (AAs) of their constituent proteins. Therefore, epistasis is expected to evolve among physically interacting sites and to influence the rates of substitution. To study the evolution of epistasis, we focused on the major structural protein of the ϕX174 phage family by first reconstructing the ancestral protein sequences of 18 species using a Bayesian statistical framework. The inferred ancestral reconstruction differed at eight AAs, for a total of 256 possible ancestral haplotypes. For each ancestral haplotype and the extant species, we estimated, in silico, the distribution of free energies and epistasis of the capsid structure. We found that free energy has not significantly increased but epistasis has. We decomposed epistasis up to fifth order and found that higher-order epistasis sometimes compensates pairwise interactions making the free energy seem additive. The dN/dS ratio is low, suggesting strong purifying selection, and that structure is under stabilizing selection. We synthesized phages carrying ancestral haplotypes of the coat protein gene and measured their fitness experimentally. Our findings indicate that stabilizing mutations can have higher fitness, and that fitness optima do not necessarily coincide with energy minima.

Evolutionary dynamics of the human NADPH oxidase genes CYBB, CYBA, NCF2, and NCF4: functional implications.

The phagocyte NADPH oxidase catalyzes the reduction of O2 to reactive oxygen species with microbicidal activity. It is composed of two membrane-spanning subunits, gp91-phox and p22-phox (encoded by CYBB and CYBA, respectively), and three cytoplasmic subunits, p40-phox, p47-phox, and p67-phox (encoded by NCF4, NCF1, and NCF2, respectively). Mutations in any of these genes can result in chronic granulomatous disease, a primary immunodeficiency characterized by recurrent infections. Using evolutionary mapping, we determined that episodes of adaptive natural selection have shaped the extracellular portion of gp91-phox during the evolution of mammals, which suggests that this region may have a function in host-pathogen interactions. On the basis of a resequencing analysis of approximately 35 kb of CYBB, CYBA, NCF2, and NCF4 in 102 ethnically diverse individuals (24 of African ancestry, 31 of European ancestry, 24 of Asian/Oceanians, and 23 US Hispanics), we show that the pattern of CYBA diversity is compatible with balancing natural selection, perhaps mediated by catalase-positive pathogens. NCF2 in Asian populations shows a pattern of diversity characterized by a differentiated haplotype structure. Our study provides insight into the role of pathogen-driven natural selection in an innate immune pathway and sheds light on the role of CYBA in endothelial, nonphagocytic NADPH oxidases, which are relevant in the pathogenesis of cardiovascular and other complex diseases.

Complete Genome Sequence of the Novel Phage MG-B1 Infecting Bacillus weihenstephanensis.

Here, we describe a novel virulent bacteriophage that infects Bacillus weihenstephanensis, isolated from soil in Austria. It is the first phage to be discovered that infects this species. Here, we present the complete genome sequence of this podovirus.

Genetic variability in the 5' UTR and NS5A regions of hepatitis C virus RNA isolated from non-responding and responding patients with chronic HCV genotype 1 infection.

Sequence variation among different hepatitis C virus (HCV) isolates has adaptive significance and reflects the modes and intensities of selection mechanisms operating on the virus. In this work, we sought to investigate using classical population genetics parameters, the genetic variability of HCV genotype 1 using the 5' UTR and NS5A regions from treatment non-responding and responding groups of patients. Both regions showed low genetic variability and the 5' UTR showed neutral deviation. No differences were observed in the nonsynonymous/synonymous nucleotide substitution ratio among groups for NS5A. The analysis of molecular variance test of the 5' UTR region showed an 11.94% variation among groups. Phylogenetic analysis showed no correlation between sequence variations and therapeutic responses.

Genetic variability in the 5' UTR and NS5A regions of hepatitis C virus RNA isolated from non-responding and responding patients with chronic HCV genotype 1 infection

Sequence variation among different hepatitis C virus (HCV) isolates has adaptive significance and reflects the modes and intensities of selection mechanisms operating on the virus. In this work, we sought to investigate using classical population genetics parameters, the genetic variability of HCV genotype 1 using the 5' UTR and NS5A regions from treatment non-responding and responding groups of patients. Both regions showed low genetic varia-bility and the 5' UTR showed neutral deviation. No differences were observed in the nonsynonymous/synonymous nucleotide substitution ratio among groups for NS5A. The analysis of molecular variance test of the 5' UTR region showed an 11.94 percent variation among groups. Phylogenetic analysis showed no correlation between sequence variations and therapeutic responses.

Molecular systematics of the genus Artibeus (Chiroptera: Phyllostomidae).

A molecular phylogeny of the genus Artibeus using 19 of the 20 recognized species, many with samples from a broad geographic range, is presented. The analysis shows a clear distinction between the two subgenera (or genera), the 'large'Artibeus and the 'small'Dermanura, in both mitochondrial and nuclear genes. The placement and status of A. concolor remains inconclusive and is presented as the third subgenus Koopmania. The phylogenies and divergence time estimates show a marked influence of the Andes in the formation of the subgenera and the main lineages inside each subgenus. Nuclear genes showed a highly incomplete lineage sorting among species inside subgenera Artibeus and Dermanura. Indeed, shared alleles were also found between Artibeus and Koopmania, which are presumed to have split apart during the Miocene, showing that great care should be taken in using these markers. Cytochrome-b gene divergences and monophyly analyses suggest that A. lituratus and A. intermedius are indeed conspecifics. These analyses also suggested the existence of at least four 'new' species revealing a significant cryptic diversity inside the genus.

Prospection, structural analysis and phylogenetic relationships of endogenous gamma-phospholipase A(2) inhibitors in Brazilian Bothrops snakes (Viperidae, Crotalinae).

During the last 20 years, there have been an increasing number of reports on endogenous phospholipase A(2) inhibitors (PLIs) in the sera of snakes. These studies have demonstrated the existence of three different structural classes of PLIs (alpha, beta and gamma). The gamma class members are potent inhibitors of phospholipases A(2) (PLA(2)) from the venom of Viperidae snakes. These enzymes, together with the mammalian pro-inflammatory PLA(2), belong to the IIA class of the PLA(2)-superfamily. Although coming from distinct sources, these phospholipases A(2) share main structural features. For this reason, gammaPLIs have been considered as potential models for the development of selective inhibitors of pro-inflammatory PLA(2) in humans. In spite of the rich diversity of the ophidian fauna in Brazil, only two gammaPLI representatives, from Crotalus durissus terrificus and Lachesis muta, have been described in Brazilian snakes so far. Here we investigated the presence of transcripts of novel gammaPLIs in six Bothrops species (Viperidae, Crotalinae) commonly found in our country: Bothrops alternatus, Bothrops erythromelas, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi. gammaPLI transcripts were present in every species analysed. The deduced mature proteins possessed 181 amino acid residues following a 19-residue signal peptide, similar to the gammaPLIs from C. d. terrificus taken as our model, with the exception of the deduced proteins from B. erythromelas and B. neuwiedi snakes. In these particular cases, an insertion of 4-amino acid residues was consistently present. A Bayesian tree was obtained for the Brazilian Bothrops gammaPLIs, showing four clusters: (1) L. muta and B. jararacussu, (2) B. alternatus, (3) B. erythromelas and B. neuwiedi, (4) B. jararaca and B. moojeni. Detailed structural analysis and further comparisons of these novel Bothrops inhibitors with gammaPLIs from New and Old World snakes are provided.

CYBB, an NADPH-oxidase gene: restricted diversity in humans and evidence for differential long-term purifying selection on transmembrane and cytosolic domains.

CYBB encodes the gp91-phox protein of the phagocytic NADPH oxidase; the innate immunity-related enzymatic complex responsible for the respiratory burst. Mutations in CYBB can cause chronic granulomatous disease (CGD), a primary immunodeficiency characterized by ineffective microbicidal activity, for which over 150 family-specific mutations have been described. It is also plausible that common SNPs in CYBB alter the expression or function of gp91-phox, determining differences in susceptibility to complex disorders such as autoimmune or infectious diseases. We have resequenced the exons, UTRs, and intronic regions of CYBB in 102 ethnically diverse individuals and genotyped nine tag-SNPs in 942 individuals from 52 worldwide populations. The 28 observed SNPs (none of which nonsynonymous) reside on 28 haplotypes that can be collapsed into five clades. CYBB shows lower diversity than other X-chromosome genes and most of the between-population genetic variance was observed among Africans and non-Africans. The African population shows the highest diversity and the lowest linkage disequilibrium (LD). Because there is extensive shared LD among non-Africans, tag-SNPs can be effectively employed in gene-centric association studies and are portable across Eurasian and Native American populations. Comparison of CYBB coding sequences among mammals evidences the action of long-term purifying selection, which is stronger on the C-terminal cytosolic domain than on the N-terminal transmembrane domain of gp91-phox.

Characterization of field bovine herpesvirus samples using random amplified polymorphic DNA (RAPD).

Bovine herpesvirus 5 (BoHV-5) and Bovine herpesvirus 1 (BoHV-1) are members of the family Herpesviridae, subfamily Alphaherpesvirinae. Although BoHV-1 and BoHV-5 are closely related viruses (their genomes share 85% similarity), they exhibit some important differences at the genetic level which may explain the differences in their pathogenicity and epidemiological characteristics. Bovine herpesvirus 5 is a neurovirulent virus that causes fatal meningoencephalitis in calves and has been described in Australia, Argentina, United States and Brazil. BoHV-1 causes infectious bovine rhinotracheitis (IBR) and is reported around the world. The aim of this study was to establish the use of random amplified polymorphic DNA (RAPD) followed by phylogenetic analysis to characterize field samples of bovine herpesviruses. This technique was used to study the relationship of Brazilian samples of bovine encephalitis virus isolated in semen and the central nervous system (CNS), including BoHV-1.1 and BoHV-1.2 reference subtype virus for comparison. The samples tested could be clearly separated into BoHV-1.1, BoHV-1.2 and BoHV-5 by RAPD using two different primers. The results showed that RAPD analysis can be used as a diagnostic as well as a molecular epidemiological tool for herpesviruses.

Evolutionary studies on an alpha-amylase gene segment in bats and other mammals.

Comparative studies of salivary glands showed that they maybe related to the adaptive radiation of bats, especially in the family Phylostomidae. In this study we have been searching for a likely relationship between different feeding habits found in bats and possible adaptive changes in a coding segment of the alpha-amylase enzyme. We have also tested some hypothesis about the phylogenetic relationship of bats and other mammals. A 663 bp segment of the alpha-amylase gene, corresponding to the exon 4 and part of the intron c, was sequenced in nine bat species. The exon 4 was also sequenced in further ten mammalian species. The phylogenetic trees generated with different methods produced the same results. When the intron c and the exon 4 were independently analyzed, they showed distinct topologies involving the bat species Sturnira lilium, different from the traditional bat phylogeny. Phylogenetic analysis of bats, primates and rodents supports the Euarchontoglires-Laurasiatheria hypothesis about the relationship among these groups. Selection tests showed that the alpha-amylase exon 4 is under strong purifying selection, probably caused by functional constraints. The conflicting bat phylogenies could not be explained by evolutionary convergence due to adaptive forces, and the different topologies may be likely due to the retention of plesiomorphic characters or the independent acquisition by evolutionary parallelism.

Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae).

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae)

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Dominant culturable bacterial microbiota in the digestive tract of the American black vulture (Coragyps atratus Bechstein 1793) and search for antagonistic substances

As bactérias anaeróbias estritas e facultativas cultiváveis do trato digestivo de seis urubus (Coragyps atratus Bechstein 1793) foram isoladas e identificadas. Após a captura, as aves receberam uma alimentacão de baixa contaminacão durante uma semana para eliminar possíveis microorganismos alóctonos. A seguir, amostras colhidas na língua, estomago e intestinos foram pesadas, submetidas a diluicões decimais numa câmara anaeróbia, inoculadas em meios de cultura e incubadas em aerobiose e anaerobiose a 37ºC para enumeracão, isolamento e identificacão. As bactérias isoladas foram usadas posteriormente como produtoras e reveladoras para detectar possíveis fenômenos de antagonismo. A populacão bacteriana total ao longo do trato digestivo variou de 3,46 n 0,39 log UFC/g no estômago até 10,75 n 0,37 log UFC/g no intestino distal. Algumas bactérias foram isoladas pela primeira vez do trato digestivo de C. atratus: Actinomyces bovis, Lactobacillus cellobiosus, Micrococcus luteus, Neisseria sicca, Clostridium bifermentans, Enterobacter agglomerans, Peptostreptococcus sp., Sarcina sp., Serratia odorifera, and Shigella flexneri. Associacões entre microorganismos foram observadas durante o isolamento em dois casos, um envolvendo A. bovis e N. sicca, e o outro envolvendo A. bovis e um bastonete Gram-negativo. Hetero-, iso- e autoantagonismos foram observados, sugerindo um papel ecológico para esses microorganismos em termos de autocontrole populacional e de barreira ambiental no trato digestivo dessas aves.