Quantitation of HIV-1 RNA Viral Load Using NASBA Methodology and Comparison with other Surrogate Markers for Disease Progression

In this study, HIV-1 viral load quantitation determined by Nucleic Acid Sequence Based Amplification (NASBA) was compared with other surrogate disease progression markers (antigen p24, CD4/CD8 cell counts and b-2 microglobulin) in 540 patients followed up at São Paulo, SP, Brazil. HIV-1 RNA detection was statistically associated with the presence of antigen p24, but the viral RNA was also detected in 68% of the antigen p24 negative samples, confirming that NASBA is much more sensitive than the determination of antigen p24. Regarding other surrogate markers, no statistically significant association with the detection of viral RNA was found. The reproducibility of this viral load assay was assessed by 14 runs of the same sample, using different reagents batches. Viral load values in this sample ranged from 5.83 to 6.27 log (CV = 36 %), less than the range (0.5 log) established to the determination of significant viral load changes.

Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of internal transcriber spacer of the RNA ribosomal gene

Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana.

Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay.

Identification of Biomphalaria havanensis and Biomphalaria obstructa populations from Cuba using polymerase chain reaction and restriction fragment length polymorphism of the ribosomal RNA intergenic spacer

In Cuba, several Biomphalaria species have been reported such as B. orbignyi, B. schrammi, B. helophila, B. havanensis and B. peregrina; only the latter three are considered as potential hosts of Schistosoma mansoni. The specific identification of Biomphalaria species is based on anatomical and morphological characters of genital organs and shells. The correct identification of these snails is complicated by the high variation in these characters, similarity among species and in some cases by the small size of the snails. In this paper, we reported the classical morphological identification, the use of PCR and RFLP analysis of the internal transcribed spacer region of the ribosomal RNA genes for molecular identification of seven snail populations from different localities in Cuba. Using morphological and molecular analysis, we showed that among the studied Cuban Biomphalaria populations only B. havanensis and B. obstructa species were found.

Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania)

To establish the relationships of the lizard- and mammal-infecting Leishmania, we characterized the intergenic spacer region of ribosomal RNA genes from L. tarentolae and L. hoogstraali. The organization of these regions is similar to those of other eukaryotes. The intergenic spacer region was approximately 4 kb in L. tarentolae and 5.5 kb in L. hoogstraali. The size difference was due to a greater number of 63-bp repetitive elements in the latter species. This region also contained another element, repeated twice, that had an inverted octanucleotide with the potential to form a stem-loop structure that could be involved in transcription termination or processing events. The ribosomal RNA gene localization showed a distinct pattern with one chromosomal band (2.2 Mb) for L. tarentolae and two (1.5 and 1.3 Mb) for L. hoogstraali. The study also showed sequence differences in the external transcribed region that could be used to distinguish lizard Leishmania from the mammalian Leishmania. The intergenic spacer region structure features found among Leishmania species indicated that lizard and mammalian Leishmania are closely related and support the inclusion of lizard-infecting species into the subgenus Sauroleishmania proposed by Saf'janova in 1982.

Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.

In situ hybridization of hepatitis C virus RNA in liver cells of an experimentally infected rhesus macaque

The liver tissue of a rhesus macaque inoculated with hepatitis C virus (HCV) has been analyzed for the presence of HCV RNA using the technique of in situ hybridization, both at light and electron microscopy levels. The animal was inoculated by the intrasplenic route using a HCV infected autogenic hepatocyte transplant. The serum sample used to infect the hepatocyte cells was characterized by polymerase chain reaction technique and shown to be positive for HCV RNA, genotype 3 with 10(7) RNA copies/ml. In situ hybridization was performed using a complementary negative strand probe made with the specific primer. We were able to detect and localize viral RNA in altered membranes of the rough endoplasmic reticulum of infected liver cells, showing evidence of virus replication in vivo.

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4%), followed by genotype 3 (21.4%), and genotype 2 (7.2%). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4%), mild (57.2%), and moderate (21.4%). Viral RNA was detected in liver cells from nine patients (64.3%). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.

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.

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.