Epidemiological profile of Zika, Dengue and Chikungunya virus infections identified by medical and molecular evaluations in Rondonia, Brazil

Several arboviruses have emerged and/or re-emerged in North, Central and SouthAmerican countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list

Genomic and phylogenetic evidence of VIPER retrotransposon domestication in Trypanosomatids

Submitted by Luciane Willcox ([email protected]) on 2017-04-17T16:08:38Z No. of bitstreams: 1 Genomic and phylogenetic evidence of VIPER retrotransposon domestication in trypanosomatids.pdf: 931672 bytes, checksum: 2c8fdb1dd8f87a451c995991f61d7c04 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2017-07-28T14:46:35Z (GMT) No. of bitstreams: 1 Genomic and phylogenetic evidence of VIPER retrotransposon domestication in trypanosomatids.pdf: 931672 bytes, checksum: 2c8fdb1dd8f87a451c995991f61d7c04 (MD5)Made available in DSpace on 2017-07-28T14:46:35Z (GMT). No. of bitstreams: 1 Genomic and phylogenetic evidence of VIPER retrotransposon domestication in trypanosomatids.pdf: 931672 bytes, checksum: 2c8fdb1dd8f87a451c995991f61d7c04 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná, Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná, Curitiba, PR, Brasil.Transposable elements are important residents of eukaryotic genomes and eventually the host can domesticate them to serve cellular functions. We reported here a possible domestication event of the vestigial interposed retroelement (VIPER) in trypanosomatids. We found a large gene in a syntenic location in Leishmania braziliensis, L. panamensis, Leptomanas pyrrhocoris, and Crithidia fasciculata whose products share similarity in the C-terminal portion with the third protein of VIPER. No remnants of other VIPER regions surrounding the gene sequence were found. We hypothesise that the domestication event occurred more than 50 mya and the conservation of this gene suggests it might perform some function in the host species

Preliminary multiplex microarray IgG immunoassay for the diagnosis of toxoplasmosis and rubella

Submitted by Manoel Barata ([email protected]) on 2017-08-22T15:31:15Z No. of bitstreams: 1 BaschirottoPrelim.pdf: 448080 bytes, checksum: 7d854782ba7b2fd9f9720dbe64d3d676 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2017-08-28T13:40:36Z (GMT) No. of bitstreams: 1 BaschirottoPrelim.pdf: 448080 bytes, checksum: 7d854782ba7b2fd9f9720dbe64d3d676 (MD5)Made available in DSpace on 2017-08-28T13:40:36Z (GMT). No. of bitstreams: 1 BaschirottoPrelim.pdf: 448080 bytes, checksum: 7d854782ba7b2fd9f9720dbe64d3d676 (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.During pregnancy, toxoplasmosis and rubella can cause serious damage to the mother and the foetus through vertical transmission. Early diagnosis enables implementation of health measures aimed at preventing vertical transmission and minimising damage caused by these diseases. Here, we report the development of a multiplex assay for simultaneous detection of IgG antibodies produced during toxoplasmosis and rubella infection. This assay is based on xMap technology. Initially, by singleplex assays, we evaluated the following antigens: one Toxoplasma gondii lysate; two antigenic extracts of T. gondii (TOX8131 and TOX8122); fragments of T. gondii antigens [SAG-1 (amino acids 45-198), GRA-7 (24-100), GRA-1 (57-149), ROP-4, and MIC-3 (234-306)]; two chimeric antigens composed of fragments of SAG-1, GRA-7, and P35 (CTOX and CTOXH); and fragments of Rubella virus antigens [E-1 (157-176, 213-239, 374-390), E-2 (31-105), and C (1-123)]. A multiplex assay to simultaneously diagnose toxoplasmosis and rubella was designed with the best-performing antigens in singleplex and multiplex assays, which included CTOXH, T. gondii lysate, TOX8131, E-1, and E-2. The multiplex assay showed 100% sensitivity and specificity for anti-T. gondii IgG detection and 95.6% sensitivity and 100% specificity for anti-R. virus IgG detection. We found that, despite the difficulties related to developing multiplex systems, different types of antigens (extracts and recombinant proteins) can be used to develop high-performance diagnostic tests. The assay developed is suitable to screen for prior T. gondii and R. virus infections, because it is a rapid, high-throughput, low-cost alternative to the current standard diagnostic tools, which require multiple individual tests

Isolation of an aptamer that binds specifically to E. Coli.

Submitted by Luciane Willcox ([email protected]) on 2017-04-17T16:53:35Z No. of bitstreams: 1 Isolation of an Aptamer that Binds.pdf: 6317653 bytes, checksum: db250b5635644e003a8867e2cefefa81 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2017-07-24T18:50:41Z (GMT) No. of bitstreams: 1 Isolation of an Aptamer that Binds.pdf: 6317653 bytes, checksum: db250b5635644e003a8867e2cefefa81 (MD5)Made available in DSpace on 2017-07-24T18:50:41Z (GMT). No. of bitstreams: 1 Isolation of an Aptamer that Binds.pdf: 6317653 bytes, checksum: db250b5635644e003a8867e2cefefa81 (MD5) Previous issue date: 2016-04-22Instituto de Biologia Molecular do Paraná. Department of Research and Development. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Department of Research and Development. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Department of Research and Development. Curitiba, PR, Brasil./ Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, BrasilInstituto de Biologia Molecular do Paraná. Department of Research and Development. Curitiba, PR, Brasil.Escherichia coli is a bacterial species found ubiquitously in the intestinal flora of animals, although pathogenic variants cause major public health problems. Aptamers are short oligonucleotides that bind to targets with high affinity and specificity, and have great potential for use in diagnostics and therapy. We used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers–called P12-31—is highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis associated E. coli (MNEC) clinical isolates, and is the first aptamer described with potential for use in the diagnosis of MNEC-borne pathologies

In silico analysis of amino acid variation in human respiratory Syncytial virus: insights into immunodiagnostics

Submitted by Manoel Barata ([email protected]) on 2018-02-09T15:58:00Z No. of bitstreams: 1 ZanchinInSil.pdf: 4932859 bytes, checksum: d66e391fe69434a3a2b08d848d6f169c (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2018-02-26T19:12:12Z (GMT) No. of bitstreams: 1 ZanchinInSil.pdf: 4932859 bytes, checksum: d66e391fe69434a3a2b08d848d6f169c (MD5)Made available in DSpace on 2018-02-26T19:12:12Z (GMT). No. of bitstreams: 1 ZanchinInSil.pdf: 4932859 bytes, checksum: d66e391fe69434a3a2b08d848d6f169c (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil / Universidade Federal do Paraná. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil / Universidade Federal do Paraná. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.The highly contagious nature of human respiratory syncytial virus (HRSV) and the gravity of its infection in newborns and vulnerable adults pose a serious public health problem. Thus, a rapid and sensitive diagnostic test for viral detection that can be implemented upon the first appearance of symptoms is needed. The genetic variation of the virus must be considered for immunodiagnostic purposes.This paper has the objectiv of to analyse HRSV genetic variation and discuss the possible consequences for capture immunoassay development. We performed a wide analysis of N, F and G protein variation based on the HRSV sequences currently available in the GenBank database. We also evaluated their similarity with homologous proteins from other viruses. It found that the mean amino acid divergences for the N, F, and G proteins between HRSV-A and HRSV-B were determined to be approximately 4%, 10% and 47%, respectively. Due to their high conservation, assays based on the full-length N and F proteins may not distinguish HRSV from human metapneumovirus and other Mononegavirales viruses, and the full-length G protein would most likely produce false negative results due to its high divergence. How main conclusions, we have identified specific regions in each of these three proteins that have higher potential to produce specific results, and their combined utilisation should be considered for immunoassay development

Trypanosoma cruzi response to Sterol Biosynthesis Inhibitors: morphophysiological alterations leading to cell death

Submitted by Luciane Willcox ([email protected]) on 2016-10-03T13:10:06Z No. of bitstreams: 1 Trypanosoma cruzi Response to Sterol Biosynthesis Inhibitors.PDF: 5416806 bytes, checksum: 129fb4ef9e35aed5b882214a68eb086b (MD5)Approved for entry into archive by Luciane Willcox ([email protected]) on 2016-10-03T13:16:47Z (GMT) No. of bitstreams: 1 Trypanosoma cruzi Response to Sterol Biosynthesis Inhibitors.PDF: 5416806 bytes, checksum: 129fb4ef9e35aed5b882214a68eb086b (MD5)Made available in DSpace on 2016-10-03T13:16:47Z (GMT). No. of bitstreams: 1 Trypanosoma cruzi Response to Sterol Biosynthesis Inhibitors.PDF: 5416806 bytes, checksum: 129fb4ef9e35aed5b882214a68eb086b (MD5) Previous issue date: 2013-01-31Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.The protozoan parasite Trypanosoma cruzi displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of T. cruzi cells following treatment with the sterol biosynthesis inhibitors (SBIs) ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC50/72 h) or killing all cells within 24 hours (EC100/24 h). Incubation with inhibitors at the EC50/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC50/72 h. By contrast, treatment with SBIs at the EC100/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP), culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the “point of no return” in the cell death cascade, and propose a model for the necrotic cell death of T. cruzi. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in T. cruzi. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite