Evolutionary analysis of apolipoprotein E by Maximum Likelihood and complex network methods.

Apolipoprotein E (apo E) is a human glycoprotein with 299 amino acids, and it is a major component of very low density lipoproteins (VLDL) and a group of high-density lipoproteins (HDL). Phylogenetic studies are important to clarify how various apo E proteins are related in groups of organisms and whether they evolved from a common ancestor. Here, we aimed at performing a phylogenetic study on apo E carrying organisms. We employed a classical and robust method, such as Maximum Likelihood (ML), and compared the results using a more recent approach based on complex networks. Thirty-two apo E amino acid sequences were downloaded from NCBI. A clear separation could be observed among three major groups: mammals, fish and amphibians. The results obtained from ML method, as well as from the constructed networks showed two different groups: one with mammals only (C1) and another with fish (C2), and a single node with the single sequence available for an amphibian. The accordance in results from the different methods shows that the complex networks approach is effective in phylogenetic studies. Furthermore, our results revealed the conservation of apo E among animal groups.

ACUTE GASTROENTERITIS ASSOCIATED WITH NOROVIRUS GII.4 VARIANTS.

BACKGROUND: Norovirus (NoV) is an important etiologic agent of acute gastroenteritis and infects individuals of all ages, especially children in Brazil and worldwide. NoV GII.4 was the most prevalent genotype worldwide because of your extensive genetic diversity. In Brazil, especially in the Northeast, few studies have been developed for identify and molecularly characterize NoV. OBJECTIVE: The present study aimed to detect and describe the molecular epidemiology of NoV associated with acute gastroenteritis. METHODS: The viral RNA extracted from stool samples were subjected to Nested RT-PCR and the genotypes were determined by nucleotide sequences analysis. In total, 278 stool samples assisted at Aliança Hospital in the city of Salvador, with acute gastroenteritis were examined, between March 2009 and July 2012. RESULTS: A high NoV rate (54.2%) was identified in children under 5 years of age. We detected the circulation of different NoV GII.4 variants in Salvador, during the study period as Den Haag 2006b, New Orleans 2009 and Sydney 2012. CONCLUSION: These findings reinforce the need to study the molecular epidemiology of NoV infections in acute gastroenteritis.

The close relationship between South African and Latin American HTLV type 1 strains corroborated in a molecular epidemiological study of the HTLV type 1 isolates from a blood donor cohort.

It has been difficult to explain why all HTLV-1 sequences in Salvador, a city in the northeast of Brazil, belong to the Transcontinental (A) subgroup of the Cosmopolitan (a) subtype, since according to historical data the vast majority of slaves brought to Brazil (through Salvador) came from west Africa, where only the western African subgroup (C) has been found. To shed more light on this subject we conducted a phylogenetic analysis of 23 isolates from blood donors of Salvador. DNA was extracted and submitted to a nested PCR for amplification of the entire LTR region. The PCR products were purified and sequenced on an automated sequencer. Neighbor-joining and maximum likelihood phylogenetic analyses were performed. None of the new sequences from Salvador clustered within the West-African subgroup C. Confirming previous results, all sequences belonged to the Transcontinental subgroup (A) of the Cosmopolitan subtype, and clustered in two Latin American clusters. In addition we showed sequences from southern Africa clustering in both Latin American clusters. One of the new sequences is ancestral to the larger Latin American cluster beta due to a duplication of a 12-bp long fragment, a finding that has not been previously described. These findings support the hypothesis that HTLV-1 isolates circulating in Latin America have a closer relationship to South African compared to West-African HTLV-1 strains. The 12-bp-long duplications in one of the sequences has no obvious clinical or biological implications yet.

Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping.

BACKGROUND: Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes. RESULTS: ITS sequencing and PCR-based methods revealed genetic heterogeneity among the L. amazonensis isolates examined and molecular karyotyping also showed variation in the chromosome size of different isolates. Unrooted genetic trees separated strains into different groups. CONCLUSION: These results indicate that L. amazonensis strains isolated from leishmaniasis patients from northeastern Brazil are genetically diverse, however, no correlation between genetic polymorphism and phenotype were found.

Interspecific transmission of small ruminant lentiviruses from goats to sheep.

This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.

Gastroenterite aguda associada a variantes do Norovírus GII. 4 / Acute gastroenteritis associated with norovirus gii. 4 variants

ABSTRACT BACKGROUND: Norovirus (NoV) is an important etiologic agent of acute gastroenteritis and infects individuals of all ages, especially children in Brazil and worldwide. NoV GII.4 was the most prevalent genotype worldwide because of your extensive genetic diversity. In Brazil, especially in the Northeast, few studies have been developed for identify and molecularly characterize NoV. OBJECTIVE: The present study aimed to detect and describe the molecular epidemiology of NoV associated with acute gastroenteritis. METHODS: The viral RNA extracted from stool samples were subjected to Nested RT-PCR and the genotypes were determined by nucleotide sequences analysis. In total, 278 stool samples assisted at Aliança Hospital in the city of Salvador, with acute gastroenteritis were examined, between March 2009 and July 2012. RESULTS: A high NoV rate (54.2%) was identified in children under 5 years of age. We detected the circulation of different NoV GII.4 variants in Salvador, during the study period as Den Haag 2006b, New Orleans 2009 and Sydney 2012. CONCLUSION: These findings reinforce the need to study the molecular epidemiology of NoV infections in acute gastroenteritis.

RESUMO CONTEXTO: Norovírus (NoV) é o agente etiológico mais importante nas gastroenterites agudas e infecta indivíduos de todas as idades, especialmente crianças no Brasil e no mundo. O NoV GII.4 é o genótipo mais prevalente em todo o mundo devido a sua elevada diversidade genética. No Brasil, principalmente no Nordeste, poucos estudos têm sido desenvolvidos a fim de identificar e caracterizar molecularmente o NoV. OBJETIVO: O presente estudo teve como objetivo detectar e descrever a epidemiologia molecular do NoV associado com gastroenterite aguda. MÉTODOS: RNA viral extraído a de amostras de fezes foi submetido a amplificação por Nested-RT-PCR e o genótipo determinado por analise da sequência de nucleotídeos. Um total de 278 amostras de pacientes atendidos no Hospital Aliança, na cidade de Salvador, com gastroenterite aguda foram examinados, entre março de 2009 a julho de 2012. RESULTADOS: Uma alta taxa de NoV (54,2%) foi identificado em crianças de até 5 anos de idade. Detectou-se a circulação de diferentes variantes de NoV GII.4 em Salvador, durante o período do estudo, tais como Den Haag 2006b, New Orleans 2009 e Sydney 2012. CONCLUSÃO: Estes achados reforçam a necessidade de maiores estudos para esclarecer a epidemiologia molecular das infecções por NoV em casos de gastroenterite aguda.

Molecular characterization of long terminal repeat sequences from Brazilian human immunodeficiency virus type 1 isolates.

HIV-1 provirus activation is under control of the long terminal repeat (LTR)-5' viral promoter region, which presents remarkable genetic variation among HIV-1 subtypes. It is possible that molecular features of the LTR contribute to the unusual profile of the subtype C epidemic in the Brazilian Southern region. To characterize the LTR of Brazilian HIV isolates, we analyzed sequences from 21 infected individuals from Porto Alegre and Salvador cities. Sequences were compared with subtype B and C reference strains from different countries. Phylogenetic analysis showed that 17 (81%) samples were subtype B and four (19%) were subtype C. Common patterns of transcription factor binding sites (TFBS) in subtypes B and C sequences were confirmed and other potential TFBS specific for subtype C were found. Brazilian subtype C sequences contained an additional NF-κB biding site, as previously described for the majority of subtype C isolates. The high level of LTR polymorphisms identified in this study might be important for viral fitness.

Acute liver failure associated with rubella virus in a child.

Acute liver failure is a syndrome with a wide range of etiologic possibilities in children, but in up to 50% of the cases in the literature no diagnosis is established. This case report adds rubella virus to the list of possible causes of acute liver failure. This association was made by serologic, cell culture, molecular, histopathologic, and immunohistochemical methods.

Interspecific transmission of small ruminant lentiviruses from goats to sheep

This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.

Characterization and genetic variability of Fusarium verticillioides strains isolated from corn and sorghum in Brazil based on fumonisins production, microsatellites, mating type locus, and mating crosses.

Fusarium verticillioides (Gibberella fujikuroi mating population A) is a producer of fumonisins and one of the main contaminants of corn grains. In Brazil, some studies analyzing strains isolated from corn have demonstrated high levels of fumonisins, whereas the levels for strains isolated from sorghum have been found to be low. In the present study, we investigated the genetic diversity of 22 F. verticillioides strains isolated from corn and 21 strains isolated from sorghum cultivated in the State of São Paulo, Brazil. Differences in the genetic profile were observed between the strains isolated from the two substrates using single primer amplification reaction by polymerase chain reaction (SPAR-PCR). Fumonisins levels were higher in strains isolated from corn than in those isolated from sorghum. The MAT-1 and MAT-2 alleles were identified by PCR, and the isolates were subsequently crossed with Fusarium thapsinum (G. fujikuroi mating population F) reference strains because this species is morphologically similar to F. verticillioides and produces low levels of fumonisins. The SPAR haplotypes of some strains isolated from sorghum were similar to the F. thapsinum reference strain haplotypes, but there was no fertile mating between the strains isolated from the two substrates and the F. thapsinum references strains. The MAT-1:MAT-2 proportion was 5:17 and 14:7 for isolates from corn and sorghum, respectively.

Genetic variability of porcine parvovirus isolates revealed by analysis of partial sequences of the structural coding gene VP2.

The 3'-terminal 853 nt (and the putative 283 aa) sequence of the VP2-encoding gene from 29 field strains of porcine parvovirus (PPV) were determined and compared both to each other and with other published sequences. Sequences were examined using maximum-parsimony and statistical analyses for nucleotide diversity and sequence variability. Among the nucleotide sequences of the PPV field strains, 26 polymorphic sites were encountered; 22 polymorphic sites were detected in the putative amino acid sequence. Mapping polymorphic sites of protein data onto the three-dimensional (3D) structure of PPV VP2 revealed that almost all substitutions were located on the external surface of the viral capsid. Mapping amino acid substitutions to the alignment between PPV VP2 sequences and the 3D structure of canine parvovirus (CPV) capsid, many PPV substitutions were observed to map to regions of recognized antigenicity and/or to contain phenotypically important residues for CPV and other parvoviruses. In spite of the high sequence similarity, genetic analysis has shown the existence of at least two virus lineages among the samples. In conclusion, these results highlight the need for close surveillance on PPV genetic drift, with an assessment of its potential ability to modify the antigenic make-up of the virus.

Molecular cloning, sequence analysis and expression of the snake follicle-stimulating hormone receptor.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control gonadal function in mammalian and many non-mammalian vertebrates through the interaction with their receptors, FSHR and LHR. Although the same is true for some reptilian species, in Squamata (lizards and snakes) there is no definitive evidence for the presence of either two distinct gonadotropins or two distinct gonadotropin receptors. Our aim was to characterize the gonadotropin receptor(s) of the Bothrops jararaca snake. Using a cDNA library from snake testis and amplification of the 5'-cDNA ending, we cloned a cDNA related to FSHR. Attempts to clone a cDNA more closely related to LHR were unsuccessful. Expression of FSHR mRNA was restricted to gonadal tissues. The snake FSHR is a G protein-coupled receptor with 673 amino acids, and the aminoterminal domain with 346 amino acids consists of a nine leucine-rich repeat-containing subdomain (LRR) flanked by two cysteine-rich subdomains. The beta-strands in the LRR are conserved with exception of the third, a region that may be important for FSH binding. In contrast with mammalian, avian and amphibian FSHRs, the snake FSHR presents amino acid deletions in the carboxyterminal region of the extracellular domain which are also seen in fish and lizard FSHRs. cAMP assays with the recombinant protein transiently expressed in HEK-293 cells showed that the snake FSHR is more sensitive to human FSH (hFSH) than to human chorionic gonadotropin. Phylogenetic analysis indicated that the squamate FSHRs group separately from mammalian FSHRs. Our data are consistent with the apparently unique gonadotropin-receptor system in Squamata reptilian subgroup. Knowledge about the snake FSHR structure may help identify structural determinants for receptor function.

Cytogenetic characterization of the strongly electric Amazonian eel, Electrophorus electricus (Teleostei, Gymnotiformes), from the Brazilian rivers Amazon and Araguaia

A karyotype analysis of the electric eel, Electrophorus electricus (Teleostei, Gymnotiformes), a strongly electric fish from northern South America, is presented. Two female specimens were analyzed, one from the Amazon River and one from the Araguaia River. The specimens had a chromosomal number of 2n = 52 (42M-SM + 10A). C-bands were present in a centromeric and pericentromeric position on part of the chromosomes; some interstitial C-bands were also present. Heteromorphic nucleolus organizer regions (NORs) were detected in two chromosome pairs of the specimen from the Amazon River. The chromosome number and karyotype characteristics are similar to those of other Gymnotidae species. The genera Electrophorus and Gymnotus are positioned as the basal lineages in the Gymnotiformes phylogeny.

Molecular subtyping of Campylobacter jejuni subsp. jejuni strains isolated from different animal species in the state of São Paulo, Brazil

O objetivo do presente trabalho foi caracterizar geneticamente estirpes de Campylobacter jejuni subsp. jejuni isoladas de humanos e de diferentes origens animais (bovinas, suínas, cães, primatas, javalis, suínos e aves de corte). Um total de 828 amostras (fezes, carcaças, fetos abortados e útero histerectomizado) foram analisadas por métodos de rotina bacteriológica e 36 estirpes de C. jejuni foram isoladas. Trinta estirpes de origem fecal humana foram obtidas de laboratórios de análises clínicas da cidade de São Paulo. As 66 estirpes de C. jejuni isoladas foram submetidas à caracterização genética. Oligonucleotídeos baseados no gene fla A foram usados na reação de polimerase em cadeia (PCR) e amplificou um fragmento de 702 pb. Os produtos obtidos pela PCR foram avaliados pelas técnicas de seqüenciamento e análise genealógica. Análise da variabilidade genética das 66 estirpes revelou 44 diferentes subtipos de C. jejuni. Um subtipo de origem humana apresentou seqüência idêntica à de C. jejuni depositada no GenBank (GENBANK acesso número AF050186). A subtipagem das estirpes de C. jejuni baseadas no seqüenciamento da região variável do gene fla A e na análise do alinhamento das seqüências pelo método da Máxima Parcimônia, mostraram-se altamente discriminatórios fornecendo melhores condições para a correta diferenciação entre estirpes originárias de surto e as isoladas esporadicamente. Este foi o primeiro estudo de subtipagem molecular de estirpes de C. jejuni de origem humana e animal utilizando a técnica do seqüenciamento com análise genealógica realizado no Estado de São Paulo, Brasil.

Molecular markers in Chinese carps and their interspecific hybrids

A sample consisting of 2 Ctenopharyngodon idella (grass carp) females, 2 Aristichthys nobilis (bighead carp) males, and 10 interspecific hybrids was analyzed using a multidisciplinary approach. The 10 hybrids were the only surviving specimens of a crossbreeding experiment. The cytological approach, involving the measurement of the largest axis of the erythrocytes, revealed that the parental species were diploid, whereas, among the hybrids, eight individuals were triploid, and two were diploid. Two of the triploid individuals were analyzed cytogenetically and showed 2n = 3X = 72 chromosomes. Using the biochemical and molecular approaches, it was possible to confirm that one of the C. idella specimens was the true mother of the hybrids. Those data also allowed to rule out both males from parenthood. The molecular analyses also indicated the absence of gynogenetic individuals among the hybrids. We discuss the use of a multidisciplinary approach as an efficient tool in the monitoring of aquaculture programs.

Diversification of Ramphastinae (Aves, Ramphastidae) prior to the Cretaceous/Tertiary boundary as shown by molecular clock of mtDNA sequences

Partial cytochrome b and 12S rDNA mitochondrial DNA sequences of eight representatives of the Ramphastidae family were analyzed. We applied the linearized tree method to identify sequences evolving at similar rates and estimated the divergence times among some of the taxa analyzed. After excluding Ramphastos tucanus and Capito dayi from our data set, the remaining taxa presented a constant rate of DNA substitution, and branch lengths could be re-estimated with a clock constraint using the maximum likelihood method. Branch lengths were calibrated assuming that Galliformes and Piciformes split around 100 million years ago (mya). Our results indicate that Ramphastinae, and probably Capitoninae, diverged from other Piciformes in the Late Cretaceous (82 mya), suggesting that Piciformes is another avian order that survived the mass extinction event occurred 65 mya at the Cretaceous/Tertiary (K/T) boundary. The divergence times estimated within the Ramphastinae genera cover the period from the Middle Eocene (around 47 mya) through the Late Miocene (9.5 mya). Our estimate of divergence time is coincidental with the split of the African and the South American continents and other intense geologic activities and modifications of the areas which correspond to the current Neotropics. These events might have influenced the diversification of Ramphastinae in South America.

Biologia molecular e evolução

O progresso no conhecimento sobre a estrutura do material genético, de sua função no desenvolvimento e na fisiologia dos organismos e de seu papel nos processos evolutivos tem ocorrido num ritmo impressionante e acelerado. Seria espantoso, para alguémformado na década de 1940, por exemplo, comparar o que se sabia então nessa área com o que se sabe hoje em dia. Felizmente, a ciência brasileira, graças a algumas Universidades e a órgãos nacionais e estrangeiros financiadores da atividade científica, está conseguindo acompanhar e participar desse processo. Este livro é uma demonstração dessas considerações. Todos os autores deste livro ensinam e pesquisam no Brasil e tratam em seus capítulos com muita clareza e atualidade de muitos aspectos da evolução e daadaptação dos organismos no nível molecular. Em uma apresentação do livro como um todo, não seria justo salientar este ou aquele aspecto do texto, pois o importante é seu conjunto, próprio de um livro em padrão internacional. Mais justo a fazer é recomendar a sua leitura e o seu estudo a todos os interessados em evolução e, especialmente, no nível molecular.

Biologia molecular e evolução / Molecular biology and evolution

O progresso no conhecimento sobre a estrutura do material genético, de sua função no desenvolvimento e na fisiologia dos organismos e de seu papel nos processos evolutivos tem ocorrido num ritmo impressionante e acelerado. Seria espantoso, para alguémformado na década de 1940, por exemplo, comparar o que se sabia então nessa área com o que se sabe hoje em dia. Felizmente, a ciência brasileira, graças a algumas Universidades e a órgãos nacionais e estrangeiros financiadores da atividade científica, está conseguindo acompanhar e participar desse processo. Este livro é uma demonstração dessas considerações. Todos os autores deste livro ensinam e pesquisam no Brasil e tratam em seus capítulos com muita clareza e atualidade de muitos aspectos da evolução e daadaptação dos organismos no nível molecular. Em uma apresentação do livro como um todo, não seria justo salientar este ou aquele aspecto do texto, pois o importante é seu conjunto, próprio de um livro em padrão internacional. Mais justo a fazer é recomendar a sua leitura e o seu estudo a todos os interessados em evolução e, especialmente, no nível molecular.