RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

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% variation among groups. Phylogenetic analysis showed no correlation between sequence variations and therapeutic responses.

Prevalence and genotyping of hepatitis C virus in blood donors in the state of Pará, Northern Brazil

Given the scarcity of epidemiological information on hepatitis C virus (HCV) infection in Northern Brazil, we determined the prevalence and genotypic frequency in blood donors in the state of Pará (PA). Blood samples from all of the blood donors at the Fundação HEMOPA (blood bank of PA) from 2004-2006 were screened for the presence of antibodies to anti-HCV and samples seroreactive to anti-HCV were further tested for HCV RNA using real-time PCR. In total, 116 HCV-RNA samples were genotyped, based on maximum likelihood phylogenetic analyses, using BioEdit, Modelgenerator, PHYML and FigTree software. The population consisted of 242,726 volunteers who donated blood from 2004-2006; the most common subgroup was males between the ages of 18-29 years old (37.30%). Within the whole group, 1,112 blood donors (0.46%) had indeterminate or positive serology; among these, 28.78% were males whose ages ranged from 18-29 years. A diagnosis of chronic HCV infection was confirmed for 304 donors (60.20% males; 66.45% were 30-49 years old), resulting in a prevalence of HCV RNA in 0.13% of the samples (304 of 242,726). HCV genotyping revealed a high frequency of genotype 1 (108/116) followed by genotype 3 (8/116). This study found HCV infection to be relatively infrequent in PA; genotype 1 was most commonly isolated. This information can help guide prevention and control policies aimed at efficient diagnosis and control measures.

HIV-1 RNA detection in the amniotic fluid of HIV-infected pregnant women

This study is aimed at evaluating the potential to detect human immunodeficiency virus (HIV) in amniotic fluid (AF) collected at delivery from 40 HIV-positive pregnant women. Thirty patients had a plasma viral load (VL) below 1,000 copies/mL at delivery. VL was positive in three AF samples. No significant association was found between the HIV-1 RNA in AF and the maternal plasma samples. There was no HIV vertical transmission detected.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

The overexpression of the trypanosomatid-exclusive TcRBP19 RNA-binding protein affects cellular infection by Trypanosoma cruzi

To characterise the trypanosomatid-exclusive RNA-binding protein TcRBP19, we analysed the phenotypic changes caused by its overexpression. Although no evident changes were observed when TcRBP19 was ectopically expressed in epimastigotes, the metacyclogenesis process was affected. Notably, TcRBP19 overexpression also led to a decrease in the number of infected mammalian cells. These findings suggest that TcRBP19 may be involved in the life cycle progression of the Trypanosoma cruzi parasite.

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil

Leishmania RNA virus (LRV) has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ), no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V.) guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V.) brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

Circadian clock of Aedes aegypti: effects of blood-feeding, insemination and RNA interference

Mosquitoes are the culprits of some of the most important vector borne diseases. A species’ potential as a vector is directly dependent on their pattern of behaviour, which is known to change according to the female’s physiological status such as whether the female is virgin/mated and unfed/blood-fed. However, the molecular mechanism triggered by and/or responsible for such modulations in behaviour is poorly understood. Clock genes are known to be responsible for the control of circadian behaviour in several species. Here we investigate the impact mating and blood-feeding have upon the expression of these genes in the mosquito Aedes aegypti . We show that blood intake, but not insemination, is responsible for the down-regulation of clock genes. Using RNA interference, we observe a slight reduction in the evening activity peak in the fourth day after dstim injection. These data suggest that, as in Drosophila , clock gene expression, circadian behaviour and environmental light regimens are interconnected in Ae. aegypti .

Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection.

Virtual Guide On Ocular Self-Examination To Support The Self-Care Practice For People With HIV/AIDS

Objetivou-se descrever processo de desenvolvimento da cartilha virtual sobre autoexame ocular para pessoas com HIV/aids. A proposta metodológica seguiu as cinco etapas preconizadas por Falkembach: análise e planejamento, modelagem, implementação, avaliação e distribuição. A adequação da versão impressa para virtual requereu a construção de um vídeo tutorial, agregação de fotos ilustrativas para visualização de possíveis alterações oculares e ferramenta de interatividade com demonstração do resultado do exame ao usuário. Na avaliação inicial do material, foram diagnosticadas falhas no layout. Assim, comandos foram recolocados, unificados, dispostos em local de fácil visualização e foi feita a adequação da linguagem. Considera-se possível promover aproximação do usuário com métodos de prevenção na área da saúde ocular por meio de cartilha virtual, contribuindo para desenvolvimento de habilidades e divulgação do autoexame.


Hemograma e sobrevivência de camarões marinhos após silenciamento do WSSV por RNA de interferência

O objetivo deste trabalho foi avaliar a proteção antiviral específica via RNA de interferência (RNAi) contra o vírus da síndrome da mancha-branca (WSSV), em camarões marinhos (Litopenaeus vannamei). Os camarões foram injetados com uma sequência dsRNA específica (vp28 do envelope viral), seguida por desafio com WSSV após 48 horas. Avaliaram-se o hemograma às 0, 3, 6, 24, 48 e 72 horas após o desafio, e a taxa de mortalidade durante 30 dias. Nos animais tratados com dsRNA vp28, a infecção viral foi limitada, e a sobrevivência (73%) e a "clearance" viral (80%) foram maiores do que nos camarões infectados, não tratados, que apresentaram 100% de mortalidade em cinco dias. Nos camarões tratados com dsRNA, o hemograma diminuiu até 6 horas após o desafio, seguido por aumento, tendo atingido o nível normal em 72 horas. O tratamento com dsRNA vp28 limita a infecção nos camarões por WSSV, restaura as suas condições imunológicas e promove "clearance" viral na maioria dos sobreviventes. Esses resultados são indicativos de que dsRNA vp28 pode servir como ferramenta molecular para combater o WSSV e que o RNAi representa abordagem promissora para controlar doenças virais em camarões cultivados.

COMPARISON OF RNA EXTRACTION METHODS FOR Passiflora edulis SIMS LEAVES

ABSTRACT Functional genomic analyses require intact RNA; however, Passiflora edulis leaves are rich in secondary metabolites that interfere with RNA extraction primarily by promoting oxidative processes and by precipitating with nucleic acids. This study aimed to analyse three RNA extraction methods, Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), TRIzol® Reagent (Invitrogen) and TRIzol® Reagent (Invitrogen)/ice -commercial products specifically designed to extract RNA, and to determine which method is the most effective for extracting RNA from the leaves of passion fruit plants. In contrast to the RNA extracted using the other 2 methods, the RNA extracted using TRIzol® Reagent (Invitrogen) did not have acceptable A260/A280 and A260/A230 ratios and did not have ideal concentrations. Agarose gel electrophoresis showed a strong DNA band for all of the Concert™ method extractions but not for the TRIzol® and TRIzol®/ice methods. The TRIzol® method resulted in smears during electrophoresis. Due to its low levels of DNA contamination, ideal A260/A280 and A260/A230 ratios and superior sample integrity, RNA from the TRIzol®/ice method was used for reverse transcription-polymerase chain reaction (RT-PCR), and the resulting amplicons were highly similar. We conclude that TRIzol®/ice is the preferred method for RNA extraction for P. edulis leaves.

Transgenic passionfruit expressing RNA derived from Cowpea aphid-borne mosaic virus is resistant to passionfruit woodiness disease

Sixteen transgenic yellow passionfruit (Passiflora spp.) plants (R0) were obtained which express a non-translatable transgenic RNA corresponding to the 3' region of the NIb gene and the 5' region of the CP gene, derived from the genome of a Brazilian isolate of Cowpea aphid-borne mosaic virus (CABMV). The transgenic plants were propagated by stem cuttings and challenged by sap inoculation with isolates CABMV-MG1 and CABMV-PE1. One transgenic plant (TE5-10) was resistant to the isolate CABMV-MG1, but susceptible to CABMV-PE1. The remaining transgenic plants developed systemic symptoms, equal to non-transformed plants, when inoculated with either isolate. The absence of virus in TE5-10 plants was confirmed by indirect ELISA. Transcription analysis of the transgene demonstrated that the TE5-10 plant did not accumulate transgenic mRNA, even before inoculation. After inoculation, viral RNA was only detected in plants inoculated with CABMV-PE1. These results confirm that the transgenic plant TE5-10 is resistant to isolate CABMV-MG1, and suggest that the resistance mechanism is post-transcriptional gene silencing, which is already activated in the transgenic plants before virus inoculation.

Viróides e virusóides: relíquias do mundo de RNA

Até meados do século XX, os vírus eram considerados os representantes mais simples da escala biológica. A descoberta dos RNAs satélites e dos viróides por volta de 1970 foi surpreendente, pois comprovou-se a existência de uma nova classe de moléculas auto-replicativas ainda mais simples, denominada agentes sub-virais. Há indícios de que os viróides e virusóides (que formam uma classe de RNAs satélites), teriam feito parte do "Mundo de RNA" (que precedeu o mundo atual baseado no DNA e proteínas), podendo ser considerados fósseis moleculares dessa era antiga. A simplicidade desses agentes sub-virais e o fato de que a molécula de RNA deve interagir diretamente com fatores do hospedeiro para o desenvolvimento do seu ciclo infeccioso colocam esses patógenos como um modelo para o estudo de processos metabólicos celulares. Nos últimos anos, tem-se observado um volume grande de publicações visando elucidar aspectos da interação viróide/hospedeiro, como os mecanismos da patogênese, movimento dos viróides nas plantas hospedeiras, silenciamento gênico e atividades das ribozimas. Mudanças recentes ocorridas na taxonomia desses patógenos com a criação de famílias, gêneros e espécies, além da descoberta de novos viróides, também têm sido verificadas. A presente revisão visa atualizar o leitor quanto aos recentes avanços nas pesquisas com viróides, principalmente na taxonomia, filogenia e em vários aspectos moleculares da interação viróide/hospedeiro. Estão incluídas também algumas características dos virusóides e sua relação evolutiva com os viróides.