Made available in DSpace on 2015-12-06T23:47:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2008 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O Torque Teno Vírus (TTV) foi primeiramente identificado em 1997, no Japão, em urn paciente com hepatite aguda pós-transfusão, de etiologia desconhecida, sendo após, caracterizado como urn pequeno vírus DNA circular de fita simples e não envelopado. Recentemente, o Torque Teno Vírus foi classificado em urn novo gênero chamado Anellovírus, que compreende também, o Torque Teno Mini Vírus (TTMV), Torque Teno Midi Vírus (TTMDV) e pequenos anelovírus (SAV). 0 TTV tern sido detectado em uma ampla gama de primatas nao humanos, bem como em animais domésticos. Este trabalho teve por objetivo pesquisar o TTV no soro e sangue total de primatas nao humanos e em plasma de frangos domésticos de corte (Gallus g. domesticus), pela aplicação da Nested-PCR da região não codificadora (UTR) e pel a Semi­Nested-PCR da região codificadora N22, seguido de seqüenciamento genômico e de análise filogenética. Através da Nested-PCR da região não codificadora (UTR) o DNA do TTV foi detectado no soro de 4 (5,3%) de 75 Cebus apella, 2 (40%) de 5 Alouata fusca, 1 (20%) de 5 Alouata caraya, 1 (5.2%) de 19 Callithrix penicilata, 1 (4%) de 25 Callithrix jacchus, 1 (20%) de 5 Saimiri sciureus e 1 (25%) de 4 Leontopithecus chrysomelas. Nao se obteve amplificação por PCR-UTR em nenhuma amostra de sangue total. A análise filogenética revelou que as seqüências detectadas em 8 amostras apresentaram maior identidade com as seqüências de TTV isoladas de macacos japoneses do novo mundo: So-TTV2 (Saüíinus oedipus) e At-TTV3 (Aotes Trivirgatus). Trt!!s seqüências (uma de Callithrix penicilata, uma de Leontopithecus crysomelas e uma de Cebus apella) mostraram similaridade com uma seqüência de Torque Teno Mini Vírus (TTMV) de humanos. Não se obteve amplificação por PCR-UTR em nenhuma amostra de plasma de frangos domésticos de corte (Gallus g. domesticus). Três amostras foram positivas pela amplificação da região ORF2. 0 DNA do TTV da região ORF2 foi detectado em uma amostra de soro de Cebus apella e em uma amostra de sangue total de Callithrix jacchus, e em uma de plasma de frango doméstico de corte (Gallus g. domesticus). As seqüências amplificadas pela região ORF2 nao mostraram diferenças entre as de humano, primatas nao humanos e de frango doméstico Pela Semi-Nested-PCR da região codificadora (N22), o DNA do TTV foi detectado no sangue total de 3 (4%) de 75 Cebus apella e de 1 (25%) de 4 Leontopithecus chrysomelas. Não se obteve amplificação por PCR em nenhuma amostra de soro. A análise filogenética revelou que uma amostra de Cebus apella agrupou-se com seqüências de macacos japoneses do novo mundo: Saguinus oedipus (So-TTV2) e Aotes trivirgatus (At-TTV3); duas amostras de Cebus apella mostraram similaridade com uma seqüência de Torque Teno Mini Vírus (TTMV) de humanos e uma (Cebus apella) mostrando similaridade com uma seqüência de chimpanzé (Pt-TTV6) e com uma seqüência de TTV de humano, cepa protótipo denominada TA278. Não se obteve amplificação por PCR-N22 em nenhuma amostra de plasma de frangos domésticos de corte (Gallus g. domesticus). Os resultados apresentados mostraram que este é o primeiro relato da ocorrência de Torque Teno Vírus e Torque Teno Mini Vírus em primatas não humanos do novo mundo e em frangos de corte (Gallus g. domesticus) no Brasil. / Torque Teno Virus (TTV) was first identified in the serum of a patient with acute post-transfusion hepatitis of unknown etiology in 1997 and was characterized as a small nonenveloped virus with a circular, single-stranded DNA. Recently, Torque Teno Virus has been classified to the newly genus called Anellovirus, which also comprises Torque Teno Mini Virus (TTMV), Torque Teno Midi Virus (TTMDV) and small Anellovirus. TTV has been detected in a range of non-human primates as well as domestic animals. The purpose of this study was to search TTV in the serum and total blood of nonhuman primates and in plasma of domestic chickens of cut (Gallus g. domesticus) by application the nested-PCR of the non-coding region (UTR) and by semi-nested-PCR of the coding region (N22), followed by a genomic sequence and phylogenetic analysis. By nested-PCR of non-coding region (UTR), the TTV DNA was detected in sera from 4 (5.3%) of 75 Cebus apella, 2 (40%) of 5 Alouata fusca, 1 (20%) of 5 Alouata caraya, 1 (5.2%) of 19 Callithrix penicilata, 1 (4%) of 25 Callithrix jacchus, 1 (20%) of 5 Saimiri sciureus e 1 (25%) of 4 Leontopithecus chrysomelas. No PCR-UTR amplification in any total blood sample was obtained. Phylogenetic analysis revealed that sequences detected in 8 samples had presented greater identity with TTV sequences isolated of Japanese new world non-human primates (So-TTV2 - Sagüínus oedipus and At-TTV3 - Aotes Trivirgatus). Three sequences (1 of Callithrix penicilata, 1 of Leontopithecus crysomelas and 1 of Cebus apella) showed similarity with a human Torque Teno Mini Virus (TTMV) sequence. No PCRUTR amplification in any domestic chicken plasma sample was obtained. Three additional samples were positive by the amplification of the ORF-2 region. TTV ORF2 DNA was detected in one sera sample of Cebus apella and one whole blood sample of Callithrix jacchus and in one plasma sample of domestic chicken of cut (Gallus g. domesticus). The sequences amplified by the ORF2 region showed no differences between human, non-human primates and domestic chicken. By semi-nested-PCR of coding region (N22), the TTV DNA was detected in total blood from 3 (4%) out of 75 Cebus apella and from 1 (25%) out of 4 Leontopithecus chrysomelas. No PCR amplification in any serum sample was obtained. Phylogenetic analysis revealed that one sample of Cebus apella clustered with sequences isolated of Japanese new world non-human primates (Saguinus oedipus - So-TTV2 and Aotes trivirgatus - At-TTV3); two samples of Cebus apella showed similarity with a human Torque Teno Mini Virus (TLMV) sequence. The other sample of Cebus apella showed similarity with one sequence of the chimpanzee (Pt-TTV6) and with the human TTV strain called TA278. No PCR amplification any domestic chicken plasma sample was obtained. The presented results showed that this is the first occurrence of Torque Teno Virus and Torque Teno Mini Virus in new world non-human primates and domestic chicken of cut (Gallus g. domesticus) in Brazil. / BV UNIFESP: Teses e dissertações

Torque teno virus (TTV) is a single stranded circular DNA virus and, despite its widespread nature in the human population, its pathogenesis is still unknown. Factors complicating TTV research include its huge genetic diversity, difficulties identifying an uninfected control population, the lack of a small animal model and lack of a good cell culture system for viral propagation. Recently we have identified a TTV homologue (RoTTV) in wild rodents. RoTTV was frequently observed in wood mice and field voles. RoTTV infections were also found in bank voles but not in Mus musculus populations. Analysis of complete genome sequencing shows that several genetic variants are found in wild rodent population with two distinct species containing several diverse genotypes. Furthermore, multiple variants were present in single individuals, consistent with infection patterns seen in humans. RoTTV transcripts in infected wild wood mice have also been detected and fully sequenced. Predicted protein coding regions from these transcripts have been expressed in cell culture and show the different expression patterns. Using cloned genomic DNA it has also been possible to observe the transcription from the virus in vitro and it was shown RoTTV viral titer in the supernatant of culture fluid increased. In addition, RoTTV propagation was observed by using the supernatant of culture fluid. Using cloned genomic DNA and the culture supernatant, an in vivo model system in naïve laboratory wood mice was developed.

Contexte : Le virus Torque Teno (TTV) est un petit virus à ADN fortement prévalent dont la réplication est étroitement liée à l'état immunitaire. Un nombre croissant de publications soulignent le potentiel de la charge virale du TTV en tant qu'indicateur d'immunosuppression chez les patients transplantés. Des tests cliniques sont nécessaires pour caractériser davantage son potentiel en tant que marqueur d'immunosuppression.Objectifs : Présentation de la faisabilité/optimisation et démonstration des performances analytiques et cliniques du premier test standardisé utilisé pour détecter et quantifier l'ADN du TTV humain dans le sang total et le plasma de diverses populations. La charge virale du TTV a été analysée sur des échantillons de sang total prélevés chez 31 volontaires sains, chez 53 donneurs de reins décédés et cours du suivi de 42 receveurs d’une greffe de rein.Résultats : La limite de détection de la trousse développée était de 2,2 log10 copies/ml dans le sang total et le plasma, la linéarité et la précision ont été démontrées entre 1,61 et 10,61 log10 copies/ml dans le sang total. La prévalence de l'ADN du TTV dans le sang variait significativement entre les groupes : 45% chez les volontaires sains, 74% chez les donneurs et 83% chez les receveurs de rein. Chez les receveurs de rein, les cinétiques TTV précoces étaient comparables à celles précédemment présentées dans la littérature (dans d'autres contextes de transplantation) : la charge virale est passée d'une moyenne de 4,3 log10 à 7,9 log10 copies/mL dans les 75 premiers jours post transplantation. L’analyse des séquences de TTV par séquençage haut débit nous a permis de décrire la prévalence des génotypes de TTV chez 20 donneur et receveurs appariés.Conclusion : Ce test TTV a montré une sensibilité analytique, une spécificité, une linéarité et une précision élevée. Avec ce test, la cinétique précoce chez les receveurs de rein est proche de celle précédemment décrite chez les receveurs de greffe de poumon. C'est un outil standardisé utile pour évaluer davantage la charge de TTV en tant que biomarqueur de l'état immunitaire qui pourrait améliorer la stratégie de traitement individuel. / Background: Torque teno viruses (TTV) are small DNA viruses with high prevalence whose replication is closely linked to immune status. A growing number of publications underlined the potential of TTV viral load as an indicator of immunosuppression. Clinical assays are necessary to further characterize their potential as immunosuppression markers.Objectives: To demonstrate the performance of the first standardized Research Use Only assay to detect and quantify human TTV DNA in whole blood and plasma.Study design: We established analytical and clinical performances for TTV load measurement in various populations. The TTV kinetics were followed in kidney recipients. TTV viral load was analyzed on whole blood samples from 31 healthy volunteers, 53 kidney deceased donors and 42 kidney recipients follow-up.Results: Limit of detection was 2.2 log copies/mL in whole blood and plasma, linearity and precision were demonstrated over the range 1.61 to 10.61 log10 copies/mL in whole blood. Prevalence of TTV DNA in blood differed significantly among groups: 45% in healthy volunteers, 74% in donors and 86% in kidney recipients. In kidney recipients, early TTV kinetics were comparable to those previously observed with in-house assays in other transplant settings: viral load increased from an average of 4.3 log10 to 7.9 log10 copies/mL within the first 75 days post transplantation. Sequence analysis of TTV by high throughput sequencing allowed us to describe the prevalence of TTV genotypes in 20 matched donors and recipients.Conclusion: This TTV assay showed high analytical sensitivity, specificity, linearity and precision. With this assay, early kinetics in kidney recipients are close to that previously described in lung transplant recipients. It is a useful standardized tool to further evaluate TTV load as a biomarker of immune status that could improve individual treatment strategy.

To protect public health, drinking water systems are monitored for indicator organisms that correlate with fecal contamination and suggest the presence of human pathogens. Total coliforms, fecal coliforms, and E. coli are the most commonly used indicator organisms. These bacteria generally colocate with fecal pollution, but some limitations exist. In particular, the ability of indicator bacteria to predict the presence of enteric viruses is questionable because of distinct transport and survival characteristics of bacteria and viruses. Although viral indicators of enteric viruses have been proposed, none have been implemented into the current regulatory framework. In this thesis, the correlation of bacteria and viruses in drinking water sources and treatment systems is reviewed, and the potential of Torque Teno virus (TTV) to qualify as an indicator virus is discussed. TTV is unique among enteric viruses as it infects approximately 80% of healthy individuals worldwide, is transmitted by the fecal-oral route, causes no observable illness, and lacks seasonal fluctuations.

Some viral metagenomic serum samples contain a huge amount of Anellovirus, which is a genetically diverse family with a few conserved regions making it hard to efficiently characterize. Multiple sequence alignment of the Anelloviruses found in the sample must be constructed to get a clear picture of Anellovirus diversity and to identify stable regions. Using available multiple sequence alignment software directly on these fragments results in an MSA of a very poor quality due to their diversity, misaligned regions and low-quality regions present in the sequence. An efficient MSA must be constructed in order to characterize these Anellovirus present in the samples. Pairwise alignment is used to align one fragment to the database sequences at a time. The fragments are then aligned to the database sequences using the start and end position from the pairwise alignment results. The algorithm will also exclude non-aligned portions of the fragments, as these are very hard to handle properly and are often products of misassembly or chimeric sequenced fragments. Other tools to aid further analysis were developed, such as finding a non-overlapping window that contains the most fragments, find consensus of the alignment and extract any regions from the MSA for further analysis. An MSA was constructed with a high percent of correctly aligned bases compared to an MSA constructed using MSA softwares. The minimal number of genomes found in the sampled sequence was found as well as a distribution of the fragments along the database sequence. Moreover, highly conserved region and the window containing most fragments were extracted from the MSA and phylogenetic trees were constructed for these regions.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-10-23T13:30:15Z No. of bitstreams: 1 carlos_a_p_nascimento_ioc_bcm_0045_2011.pdf: 1006058 bytes, checksum: e670c764b773d1d5bd9fa107d877a76b (MD5) / Made available in DSpace on 2012-10-23T13:30:15Z (GMT). No. of bitstreams: 1 carlos_a_p_nascimento_ioc_bcm_0045_2011.pdf: 1006058 bytes, checksum: e670c764b773d1d5bd9fa107d877a76b (MD5) Previous issue date: 2011 / CNPq FAPERJ / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O Torque teno virus (TTV) é um vírus de DNA do gênero Alphatorquevirus da família Anelloviridae. O TTV é altamente prevalente em populações de todo o mundo. Isolados foram classificados em cinco grupos filogenéticos (1-5) com grande distância genética entre eles. A presença do TTV já foi detectada nas fezes, porém, não se sabe se todos os cinco genogrupos do TTV são excretados nas fezes, e qual a distribuição do TTV entre os genogrupos. A fim de avaliar a presença, a diversidade genômica e a carga viral do TTV em fezes, 135 amostras de pacientes com gastroenterite foram analisadas. O DNA do TTV foi extraído de suspensão fecal e três diferentes métodos de reação em cadeia da polimerase (PCR), dois qualitativos e um quantitativo, foram avaliados. Nas amostras de fezes estudadas, 123 (91,1%) foram positivas em pelo menos um dos três métodos. O DNA do TTV pertencente aos genogrupos de 1 a 5 foi detectado em 37 (27,4%), 27 (20,0%), 57 (42,2%), 29 (21,5%) e 33 (24,4%) amostras, respectivamente. Co-infecções com dois, três, quatro e cinco genogrupos do TTV foram encontradas em 23 (17,0%), 15 (11,1%), 7 (5,2%) e 7 (5,2%) amostras fecais, respectivamente. Assim, 52 (38,5%) amostras continham mais de um genogrupo de TTV. A carga viral variou de 2,6 a 6,5 log de genoma equivalentes por grama de fezes. No entanto, variações de carga viral foram observadas em função do genogrupo detectado e do número de genogrupos presentes simultaneamente. Os resultados encontrados são os primeiros a mostrar a alta prevalência e a diversidade de TTV nas fezes humanas. / Torque teno virus (TTV) is a DNA virus of the genus Alphatorquevirus of Anelloviridae family. The TTV is highly prevalent in populations from around the world. Isolates have been classified into at least five main phylogenetic groups (1-5) showing a large genetic distance between them. The presence of TTV has been detected in feces. However, are presently unknown whether all five TTV genogroups are excreted in feces and the genogroup distribution. To evaluate the presence and the genomic distribution of TTV DNA in feces, 135 samples of patients with gastroenteritis were analyzed. The DNA was extracted of fecal suspension and three different PCR methods, two qualitative and one quantitative, were used. One hundred and twenty three (91.1%) samples were positive with at least one method. The TTV DNA belonging to the genogroups 1 to 5 was detected in 37 (27.4%), 27 (20.0%), 57 (42.2%), 29 (21.5%) and 33 (24.4%) fecal samples, respectively. Coinfections with two, three, four and five TTV genogroups were found in 23 (17.0%), 15 (11.1%), 7 (5.2%) and 7 (5.2%) fecal samples, respectively. Thus, 52 (38.5%) samples contained more than one TTV genogroup. Viral loads ranged from 2.6 to 6.5 log genome equivalents per gram of feces. However, variations of viral load were noted depending on genogroup and number of coinfecting TTV genogroups. These results are the first to show high prevalence and the diversity of TTV isolates in human feces.

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Made available in DSpace on 2016-12-08T16:24:07Z (GMT). No. of bitstreams: 1 PGCA09MA052.pdf: 726948 bytes, checksum: 2a141d4d92b1c5ac552655f7c9ad3c1a (MD5) Previous issue date: 2009-11-06 / Many infectious agents have been associated with reproductive failure in swine, representing significantly economic losses for production. Recently, Porcine Circovirus Type 2 (PCV2), etiologic agent of PCVAD or PCV2 associated diseases, was associated with reproductive failure in swine around the world. To confirm the pathogenic potential of PCV2 inducing reproductive failure in sows, it s necessary the viral isolation and antigen and nucleic acid demonstration in fetuses. Other viral agent, Torque Teno Vírus (TTV), also have been recently associated with infections caused by PCV2. TTV alone has not showed pathogenic signals in swine, but, its role in co-infections with other pathogens has been investigated. The present study aimed the diagnostic of PCV2 in natural infections where there was reproductive failure, as well as to establish and apply the Polimerase Chain Reaction (PCR) technique for TTV from organs. Samples from field cases, as aborted fetuses, mummified, stillborn, fragile piglets and material from abattoir sows were collected and processed to diagnostic infection in order to detect PCV2 by PCR and immunohistochemistry (IHC). Samples were collected from 21 farms; and a total of 169 fetuses were necropsied. Moreover, reproductive samples from 83 abattoir sows were collected in 4 slaughterhouses of Santa Catarina State. In the present study was possible detect viral DNA by PCR in 29 (17,1%) of 169 analyzed fetuses, where heart and lymphoid tissues showed virus DNA more frequently, 41,4% and 37,8%, respectively. Viral presence was confirmed by IHC in tissues, which detected viral antigens in 17 PCV2 positives fetuses by PCR. Samples of reproductive tissues from sows also were tested by PCR and PCV2 was identified in 4 sows (4,8%). PCR technique aimed to detect TTV was established for viral DNA from organs. Samples of reproductive tissues from sows were tested, and were found both genogroups of TTV (TTV1 and TTV2), in 25 (30,1%) and 41 (49,3%) sows, respectively. Fetuses samples that resulted positive to PCV2 by PCR were also tested to TTV, and it was observed the occurrence of co-infection between these agents. The results obtained here suggest the involvement of PCV2 in reproductive failure in sows, besides show that TTV was present in analyzed samples, corroboring the association with PCV2 / Muitos agentes infecciosos têm sido associados às falhas reprodutivas na produção de suínos, representando significativas perdas econômicas para os suinocultores. Recentemente o Circovirus Suíno tipo 2 (PCV2), agente etiológico da circovirose suína, foi associado a falhas reprodutivas em suínos em diversas partes do mundo. Para confirmar o potencial patogênico do PCV2 causando falhas reprodutivas em porcas, é necessário o isolamento do vírus e demonstração de antígeno e ácido nucléico viral em fetos. Outro agente viral, o Torque Teno Vírus (TTV), também foi recentemente associado às infecções causadas pelo PCV2. O TTV sozinho ainda não tem se mostrado patogênico em suínos, porém, seu papel em co-infecções com outros patógenos vem sendo investigado. O presente trabalho teve por objetivos diagnosticar o PCV2 em infecções naturais onde existiam falhas reprodutivas, assim como padronizar e aplicar a técnica de Reação em Cadeia da Polimerase (PCR) para TTV a partir de órgãos. Amostras provenientes de casos clínicos de campo, como fetos abortados, mumificados, natimortos, leitões inviáveis e material de fêmeas descartadas foram coletadas e processadas para diagnóstico da infecção pelo PCV2 através de PCR e imunoistoquímica (IHQ). Foram colhidas amostras de 21 granjas produtoras de suínos, totalizando 169 fetos, que foram necropsiados para coleta de órgãos. Além disso, amostras de órgãos reprodutivos de 83 fêmeas descartadas foram colhidas em 4 abatedouros da região oeste catarinense. No presente estudo foi possível detectar DNA viral por PCR em 29 (17,1%) dos 169 fetos analisados, sendo coração e tecidos linfóides os órgãos onde o vírus foi identificado com maior freqüência, 41,4% e 37,8%, respectivamente. A presença do vírus foi confirmada por teste de IHQ dos tecidos, sendo encontrado antígeno viral em 17 fetos positivos para PCV2 por PCR. As amostras de tecido reprodutivo das fêmeas também foram testadas por PCR e o PCV2 foi identificado em 4 porcas (4,8%). Visando a detecção de TTV foram testadas por PCR amostras de órgãos reprodutivos de fêmeas suínas, sendo diagnosticados os dois genogrupos de TTV, TTV1 e TTV2 em 25 (30,1%) e 41 (49,3%) fêmeas, respectivamente. As amostras de fetos que resultaram positivas para PCV2 pela técnica de PCR também foram testadas para TTV, observando-se a ocorrência de coinfecção entre estes agentes. Os resultados obtidos evidenciam o provável envolvimento do PCV2 em falhas reprodutivas em fêmeas suínas, bem como mostram que o TTV está presente nas amostras analisadas, confirmando a associação com o PCV2