During 2010 and 2011, the Loreto region of Peru experienced a dengue outbreak of unprecedented magnitude and severity for the region. This outbreak coincided with the reappearance of dengue virus-2 (DENV-2) in Loreto after almost 8 years. Whole-genome sequence indicated that DENV-2 from the outbreak belonged to lineage II of the southeast Asian/American genotype and was most closely related to viruses circulating in Brazil during 2007 and 2008, whereas DENV-2 previously circulating in Loreto grouped with lineage I (DENV-2 strains circulating in South America since 1990). One amino acid substitution (NS5 A811V) in the 2010 and 2011 isolates resulted from positive selection. However, the 2010 and 2011 DENV-2 did not replicate to higher titers in monocyte-derived dendritic cells and did not infect or disseminate in a higher proportion of Aedes aegypti than DENV-2 isolates previously circulating in Loreto. These results suggest that factors other than enhanced viral replication played a role in the severity of this outbreak.

Os Flavivirus são transmitidos por mosquitos (Diptera: Culicidae) que se refugiam em remanesctentes de Mata Atlântica. Essas áreas verdes correspondem às Unidades de Conservação e parques urbanos, que estão espalhados pela região metropolitana de São Paulo. Este estudo foi realizado com o intuito de conhecer as espécies de culicídeos que circulam na Área de Proteção Ambiental (APA) Capivari-Monos, na zona Sul do município de São Paulo, e no Parque Estadual da Cantareira (PEC), na zona Norte do mesmo município, e de investigar infecção natural por Flavivirus na fauna de culicídeos amostrada. Também foi proposto relacionar a variedade, a quantidade e identidade dos Flavivirus detectados com os padrões de riqueza, abundância e diversidade das assembleias de mosquitos. Foram realizadas 14 coletas, mensalmente, em quatro pontos de coleta na APA e três no PEC, todos com diferentes níveis de intervenção antrópica, no período de março de 2016 a abril de 2017. Armadilhas automáticas luminosastipo CDC (com atração de CO2 e ácido lático) foram instaladas na copa das árvores e no nível do solo. O esforço amostral foi equivalente para os todos os pontos, sendo que foram instaladas duas armadilhas em cada ponto (uma na copa e outra no solo), com 18 horas de coleta, permitindo amostragem de culicídeos de hábitos diurnos, crepusculares e noturnos. Os espécimes foram transportados com vida para o Laboratório de Saúde Pública da Faculdade de Saúde Pública da Universidade de São Paulo, criopreservados a temperatura -70ºC, identificados morfologicamente e agrupados em pools (com até 10 indivíduos). Os pools foram submetidos à técnica de isolamento viral em cultura de células (C6/36), seguida do teste de imunofluorescência indireta. Os pools positivos foram submetidos à reação de RT-qPCR e, posteriormente, sequenciados. Duas árvores de similaridade foram construídas para confirmação dos Flavivirus. No total, 1216 exemplares de culicídeos foram amostrados (13 gêneros), cuja riqueza foi de 42 táxons. A APA registrou a maior abundância (878 espécimes) e maior riqueza (37 táxons). A Cachoeira foi o ponto de coleta na APA que amostrou a maior riqueza e abundância, contudo, com a mais baixa diversidade. Entretanto, a Borracharia obteve alta riqueza, baixa abundância e a maior diversidade. O PEC amostrou 338 indivíduos e a riqueza foi de 23 táxons. Dentre os pontos do PEC, a Trilha do Pinheirinho amostrou a maior riqueza e abundância. An. (Ker.) cruzii, Cx. (Cux.) sp, Cx. (Mel.) vaxus, Li. durhami, Wy. (Prl.) confusa e Wy. (Pho.) theobaldi foram detectadas com infecção natural por Flavivirus. O sequenciamento revelou infecção por ZIKV em An. (Ker.) cruzii, Li. durhami e Wy. (Prl.) confusa, e infecção por DENV-2 em Cx. (Cux.) sp e Cx. (Mel.) vaxus. Concluiu-se que a riqueza, abundância e diversidade estão relacionadas entre si e, juntas, influenciaram na detecção de espécies de culicídeos naturalmente infectadas por Flavivirus, sendo que estes foram detectados em espécies provenientes de pontos de coleta cuja riqueza e abundância foram altas, e a diversidade baixa. A quantidade e a variedade dos Flavivirus também foram influenciadas por esses três fatores, para ocorrer na natureza. Não foi possível correlacionar a identidade dos Flavivirus com os três fatores uma vez que as espécies detectadas com infecção natural por esses vírus não são apontadas como potenciais vetoras. Além disso, a abundância e a diversidade pareceram ter uma relação inversa entre si. / Flaviviruses are transmitted by mosquitoes (Diptera: Culicidae) that take refuge in remnants of the Atlantic Forest. These green areas correspond to Conservation Units and urban parks which are spread throughout the metropolitan region of São Paulo. This study was carried out in order to identify the Culicidae fauna that circulate in Capivari-Monos Environmental Protection Area (APA), located in the South area of the city of São Paulo, and in Cantareira State Park (PEC), North area of the same municipality and to investigate natural Flaviviruses infection in this sampled Culicidae fauna. It was also proposed to relate the variety, quantity and identity of the Flaviviruses detected with patterns of richness, abundance and diversity of mosquito assemblages. Fourteen collections were carried out monthly at four collection sites in the APA and three in the PEC, all sites with different levels of anthropogenic intervention, during March 2016 to April 2017. CDC automatic traps (with attraction of CO2 and lactic acid) were installed in the canopy and on ground. The sampling effort was equivalent for all the points, and two traps were installed at each point (one in the canopy and the other on ground), with 18 hours of sampling, allowing sampling culicidae of daytime, morning and evening twilight, and nightlyl habits. The specimens were carried alive to the Public Health Laboratory of the School of Public Health of the University of São Paulo, were cryopreserved at a -70ºC temperature, identified morphologically and grouped in pools (with up to 10 individuals). The pools were submitted to the virus isolation technique in cell culture tissue (C6 / 36), followed by the indirect immunofluorescence test. The positive pools were submitted to the RT-qPCR reaction and, subsequently, sequenced. Two similarity trees were made only to confirm Flaviviruses infection. In total, 1216 specimens of culicidae were sampled (13 genera), and the richness was 42 taxa. In addition to APA recorded the highest abundance (878 specimens) and also highest richness (37 taxa). Cachoeira was the collection site in APA that showed the greatest richness and abundance as well, however, with the lowest diversity. In addition, Borracharia obtained high richness, low abundance and highest diversity. PEC sampled 338 specimens and the richness was 23 taxa. Among the collection sites of the PEC, Pinheirinho Trail showed the highest richness and also abundance. An. (Ker.) cruzii, Cx. (Cux.) sp, Cx. (Mel.) vaxus, Li. durhami, Wy. (Prl.) confusa and Wy. (Pho.) theobaldi were detected with natural Flaviviruses infection. The sequencing analyzes revealed ZIKV infection in An. (Ker.) cruzii, Li. durhami and Wy. (Prl.) confusa, and DENV-2 infection in Cx. (Cux.) sp and Cx. (Mel.) vaxus. It has concluded that the richness, abundance and also diversity are related to each other and, together, influenced the detection of species of culicidae naturally infected by Flaviviruses, which were detected in species from collection sites whose richness and abundance were high. About quantity and variety of Flaviviruses, these were also influenced by the three factors on nature. It was not possible to correlate the identity of the Flaviviruses with the three factors since the species detected with natural infection by these viruses are not indicated as potential vectors. Moreover, abundance and diversity appeared to have an inverse relation.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-25T23:51:26Z No. of bitstreams: 1 RenatoFerreiraDeAlmeidaJunior_DISSERT.pdf: 1495100 bytes, checksum: f007a8b773603ddf1cf491e66172e839 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-28T20:17:55Z (GMT) No. of bitstreams: 1 RenatoFerreiraDeAlmeidaJunior_DISSERT.pdf: 1495100 bytes, checksum: f007a8b773603ddf1cf491e66172e839 (MD5) / Made available in DSpace on 2016-04-28T20:17:55Z (GMT). No. of bitstreams: 1 RenatoFerreiraDeAlmeidaJunior_DISSERT.pdf: 1495100 bytes, checksum: f007a8b773603ddf1cf491e66172e839 (MD5) Previous issue date: 2015-03-16 / Os v?rus dengue pertence ? fam?lia Flaviviridae e ao g?nero Flavivirus, sendo composto por 4 sorotipos antigenicamente distintos, s?o considerados os arbov?rus mais importantes no mundo por causar altas taxas de morbidade e mortalidade em regi?es tropicais e subtropicais do planeta, colocando em risco at? 3,6 bilh?es de pessoas em mais de 100 pa?ses. Por ser uma doen?a com amplo espectro cl?nico e por n?o possuir vacina ou tratamento eficaz, o estudo de poss?veis antivirais que visam diminuir a viremia do paciente ? de suma import?ncia, j? que este ? um dos fatores que pode levar a febre hemorr?gica da dengue e a s?ndrome do choque da dengue que s?o as formas grave da doen?a. No presente estudo foi avaliado o potencial antiviral do extrato da folhada planta Caesalpinia echinata contra o v?rus dengue-2 (DENV-2) em cultura de c?lulas C6/36 e Vero e a a??o antiviral da Rifampicina em c?lulas Vero. A escolha da Caesalpinia echinata se deve ao fato de que j? foi observada sua a??o antiinflamat?ria e antimal?rica, al?m de n?o ter sido encontrado nenhum trabalho que tenha avaliado seu potencial de a??o frente a v?rus. A Rifampicina foi escolhida por demonstrado a??o antiviral, principalmente contra os poxvirus, por?m poucos s?os os relatos da utiliza??o deste f?rmaco contra v?rus de RNA. O resultado foi obtido atrav?s da quantifica??o da carga viral pela t?cnica da qRT-PCR em Tempo Real. As c?lulas infectadas por DENV-2 foram submetidas ao tratamento pelo per?odo de 7 dias em diferentes concentra??es do extrato da planta Caesalpinia echinataque variou entre 0,68 a 0,0068mg/mL. N?o foi poss?vel observar neste estudo, evid?ncias de inibi??o significativa da replica??o do v?rus DENV-2 em ambas as culturas celulares. ARifampicina foi utilizada em diferentes condi??es de tratamento, no qual foi avaliado ao longo de 72 horas a carga viral produzida nas c?lulas Vero.Nas condi??es de tratamento p?s-infec??o e no ensaio virucida o f?rmaco apresentou atividade antiviral, reduzindo a taxa de replica??o em 100 vezes em rela??o ao controle. De acordo com os nossos resultados conclui-se que a Rifampicina mostrou-se eficaz no combate a infec??o do DENV-2 em cultura de c?lulas Vero. / The dengue virus belongs to the Flaviviridae family and the Flavivirus genus, consisting of four serotypes antigenically distinct, are considered the most important arbovirus in the world to cause high rates of morbidity and mortality in tropical and subtropical regions of the world, threatening to 3, 6 billion people in over 100 countries. Because it is a disease with a wide clinical spectrum and has no vaccine or effective treatment, the study of possible antiviral drugs aimed at reducing viremia of patients is of paramount importance, since this is one of the factors which can lead to dengue hemorrhagic fever and dengue shock syndrome that are severe forms of the disease. In the present study we evaluated the antiviral potential of the leaf extract of the plant Caesalpinia echinata against dengue-2 virus (DENV-2) in cultured C6/36 and Vero cells and the antiviral action of Rifampicin on Vero cells. The choice of Caesalpinia echinata is due to the fact that has been observed its action anti-inflammatory and antimalarial , and not have been found no study evaluated its antiviral effect. Rifampin was chosen was chosen for demonstrated antiviral action, especially against poxviruses, but few sane reports usage of this drug against RNA viruses. The result was obtained by quantifying the viral load bythe technique of Real-time qRT-PCR. Cells infected with DENV-2 were subjected to treatment for 7 days in different concentrations of plant extract Caesalpinia echinata (0.68 - 0,0068mg / ml). As a result, we could not observe a inhibition significant of virus replication in both cell cultures. The Rifampicin was used for different treatment condition,which were evaluated over 72 hours the amount of viral load produced in Vero cells, In the conditions treatment and the test virucidal theantiviral activity, which is capable of reducing the rate of 100X replication as compared to control. According to our results we can conclude that Rifampicin was effective in action against to infection DENV-2 in Vero cell culture.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-25T23:51:26Z No. of bitstreams: 1 TabataLoiseCunhaLima_DISSERT.pdf: 1403767 bytes, checksum: 7424c70f286f04743b45ac7531e6829b (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-28T20:23:59Z (GMT) No. of bitstreams: 1 TabataLoiseCunhaLima_DISSERT.pdf: 1403767 bytes, checksum: 7424c70f286f04743b45ac7531e6829b (MD5) / Made available in DSpace on 2016-04-28T20:23:59Z (GMT). No. of bitstreams: 1 TabataLoiseCunhaLima_DISSERT.pdf: 1403767 bytes, checksum: 7424c70f286f04743b45ac7531e6829b (MD5) Previous issue date: 2015-03-13 / A dengue ? uma doen?a de notifica??o compuls?ria e cerca de 50 a 100 milh?es de casos s?o registrados anualmente. Possui amplo espectro cl?nico e ? transmitida ao homem atrav?s da picada dos mosquitos do g?nero Aedes, tendo como principal vetor a esp?cie Aedes aegypti. O agente etiol?gico da doen?a ? o v?rus dengue (DENV) pertencente ao g?nero Flavivirus, fam?lia Flaviviridae e s?o conhecidos quatro sorotipos antigenicamente distintos (DENV-1, DENV-2, DENV-3 e DENV-4). Atualmente o tratamento da dengue ? apenas de suporte, feito atrav?s de intensa hidrata??o. Ainda n?o existe uma vacina comprovadamente eficaz ou tratamento espec?fico, o estudo de poss?veis antivirais que possam diminuir a viremia no paciente ? de alt?ssima relev?ncia, uma vez que a carga viral ? um dos fatores associado ao aparecimento das formas graves da doen?a (febre hemorr?gica da dengue e s?ndrome do choque da dengue). No presente estudo n?s avaliamos o potencial antiviral de extratos brutos obtidos a partir das folhas das plantas do Nordeste brasileiro Annona muricata (graviola) e Spondias mombin (caj?) contra o DENV-2 em cultura de c?lulas C6/36 e Vero. A avalia??o da a??o dos extratos brutos foi feita por meio da quantifica??o da carga viral atrav?s da PCR em Tempo Real (qRT-PCR) e pela t?cnica de contagem de unidades formadoras de placa (PFU). As concentra??es dos extratos de ambas as plantas utilizadas foram: 0,01, 0,1 e 1mg/mL. As culturas de c?lulas infectadas foram submetidas ao tratamento com os extratos durante os per?odos de 24-168h horas (7 dias). C?lulas Vero tratadas com o extrato da S. mombin n?o apresentaram redu??o na carga viral. Em contrapartida, quando estas c?lulas foram tratadas com o extrato da A. muricata, uma hora ap?s infec??o, observou-se uma redu??o significativa na carga viral nas primeiras horas (24h), quando comparadas com as c?lulas n?o tratadas utilizadas como controle positivo. Ao serem tratadas em intervalos de 24 horas apresentaram uma redu??o na carga viral nos dias subsequentes (at? o s?timo dia). N?o foi observada redu??o na carga viral em c?lulas C6/36 tratadas com ambos os extratos. De acordo com os nossos resultados, o extrato da planta A. muricata possui potencial antiviral promissor contra a infec??o pelo DENV-2 em cultura de c?lulas Vero. / Dengue is a reportable disease and about 50 to 100 million cases are reported annually. It has a wide clinical spectrum and is transmitted to humans through the bite of Aedes mosquitos, the main vector the Aedes aegypti species. The causative agent of disease is dengue virus (DENV) belonging to the genus Flavivirus, family Flaviviridae and are known four antigenically distinct serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). Currently the treatment of dengue is supportive, made by intense hydration. Although there is no proven effective vaccine or specific treatment, the study of potential antiviral drugs that can reduce viremia in patients is very high importance, since the viral load is one of the factors associated with the development of severe forms of the disease (hemorrhagic fever dengue and dengue shock syndrome). In the present study we evaluated the antiviral potential of crude extracts obtained from the leaves of plants in Northeastern Brazil Annona muricata (soursop) and Spondias mombin (caja) against DENV-2 in cultured C6/36 and Vero. The evaluation of the activity of the crude extracts was performed by the quantification of viral load by RT-PCR (qRT-PCR) and counting technique of plaque forming units (PFU). The concentrations of extracts of both plants used were 0.01, 0.1 and 1 mg/mL. The infected cell cultures were subjected to treatment with the extracts during periods of 24-168h hours (7 days). Vero cells treated with the S. mombin extract showed no reduction in viral load. In contrast, when these cells were treated with the extract of A. muricata, one hour after infection, significant reductions in viral load in the first hour was observed (24 h) when compared to untreated cells used as positive control. When they are treated at 24 hour intervals showed a reduction in viral load in subsequent days (until day). There was no reduction in viral load in C6/36 cells treated with both extracts. According to our results, the plant extract has antiviral A. muricata promising potential against infection by DENV-2 in Vero cell culture.