The spatial and temporal scales of local dengue virus transmission in natural settings:a retrospective analysis

Background Dengue is a vector-borne disease caused by the dengue virus (DENV). Despite the crucial role of Aedes mosquitoes in DENV transmission, pure vector indices poorly correlate with human infections. Therefore there is great need for a better understanding of the spatial and temporal scales of DENV transmission between mosquitoes and humans. Here, we have systematically monitored the circulation of DENV in individual Aedes spp. mosquitoes and human patients from Caratinga, a dengue endemic city in the state of Minas Gerais, in Southeast Brazil. From these data, we have developed a novel stochastic point process pattern algorithm to identify the spatial and temporal association between DENV infected mosquitoes and human patients. Methods The algorithm comprises of: (i) parameterization of the variogram for the incidence of each DENV serotype in mosquitoes; (ii) identification of the spatial and temporal ranges and variances of DENV incidence in mosquitoes in the proximity of humans infected with dengue; and (iii) analysis of the association between a set of environmental variables and DENV incidence in mosquitoes in the proximity of humans infected with dengue using a spatio-temporal additive, geostatistical linear model. Results DENV serotypes 1 and 3 were the most common virus serotypes detected in both mosquitoes and humans. Using the data on each virus serotype separately, our spatio-temporal analyses indicated that infected humans were located in areas with the highest DENV incidence in mosquitoes, when incidence is calculated within 2.5–3 km and 50 days (credible interval 30–70 days) before onset of symptoms in humans. These measurements are in agreement with expected distances covered by mosquitoes and humans and the time for virus incubation. Finally, DENV incidence in mosquitoes found in the vicinity of infected humans correlated well with the low wind speed, higher air temperature and northerly winds that were more likely to favor vector survival and dispersal in Caratinga. Conclusions We have proposed a new way of modeling bivariate point pattern on the transmission of arthropod-borne pathogens between vector and host when the location of infection in the latter is known. This strategy avoids some of the strong and unrealistic assumptions made by other point-process models. Regarding virus transmission in Caratinga, our model showed a strong and significant association between high DENV incidence in mosquitoes and the onset of symptoms in humans at specific spatial and temporal windows. Together, our results indicate that vector surveillance must be a priority for dengue control. Nevertheless, localized vector control at distances lower than 2.5 km around premises with infected vectors in densely populated areas are not likely to be effective

Análise global do transcriptoma de pequenos RNAs e RNAs mensageiros durante a Interação Macrófago -Trypanosoma cruzi

Exportado OPUSMade available in DSpace on 2019-08-10T12:39:09Z (GMT). No. of bitstreams: 1 teseandrenicolau.pdf: 9942519 bytes, checksum: 101e237260281139a2d77959b0a56046 (MD5) Previous issue date: 28A doença de Chagas é causada pelo parasito intracelular Trypanosoma cruzi, que é capaz de infectar vários tipos celulares. A interação inicial entre parasito e hospedeiro envolve a ativação de vias responsáveis pela internalização do parasito, resposta imune inata do hospedeiro e o combate aos parasitos intracelulares. Diversas mudanças na expressão gênica acontecem na célula hospedeira em resposta ao patógeno e vice-versa. As vias de RNA de interferência (RNAi) estão relacionadas à regulação pós-transcricional da expressão gênica mediada por pequenos RNAs não codificantes como os microRNAs (miRNAs), que permitem um ajuste fino de várias respostas biológicas e são alteradas em infecções parasitárias. Macrófagos infectados por Leishmania sp. ou Toxoplasma sp. tem a expressão alterada de miRNAs que regulam a resposta à estes parasitos. Até o momento, não há descrição do papel de miRNAs durante a infeção do Trypanosoma cruzi em macrófagos murinos. Neste trabalho, realizamos o sequenciamento de pequenos RNAs de macrófagos murinos infectados com o parasito Trypanosoma cruzi nos tempos de 2, 4, 8 e 48 horas após infeção, para avaliar a expressão geral do transcriptoma de pequenos RNAs, especialmente os miRNAs do hospedeiro. A expressão da citocina pro-inflamatória TNF- foi avaliada como indicador da resposta imune inata durante a infeção, bem como o crescimento dos parasitos intracelulares. Não houve mudanças qualitativas no perfil de pequenos RNAs derivados do genoma do parasito ao longo da infeção. Em contraste, entre os pequenos RNAs do hospedeiro, observamos a expressão diferencial de vários miRNAs entre os tempos de 2 e 48 horas. Dos 18 miRNAs que apresentaram expressão diferencial em algum tempo da infeção, somente 1 foi expresso positivamente em pelo menos dois tempos analisados, o miR-34c-5p. Identificamos pela ferramenta TargetScan e pela base de dados StarBase, alvos preditos e validados para os miRNAs diferencialmente expressos e utilizamos os alvos encontrados em ambos para o estudo das funções dos miRNAs. A análise de enriquecimento dos mRNAs alvos para vias biológicas sugerem que os miRNAs estão relacionados com vias como, sinalização de TGF-, atividade transcricional das Smads, cascata de sinalização de PI3K, sinalização de morte celular via JNK bem como a ativação de NFKB via TRAF6. O miR-34c-5p mostrou regulação positiva significativa ao longo da infeção e validamos a expressão por RT-qPCR. A análise dos mRNAs alvos do miR-34c-5p mostrou enriquecimento para processos biológicos como resposta de apoptose e sinalização por Notch. Como os miRNAs agem através da regulação da estabilidade de seus mRNAs alvos, o transcriptoma dos macrófagos após 4 e 8 horas de infeção foi sequenciado e analisado. Ao todo, 274 mRNAs foram regulados: 33 foram regulados negativamente e 241 foram regulados positivamente em algum dos tempos analisados. Entretanto, não houve nenhuma correlação direta, positiva ou negativa, entre os alvos dos miRNAs diferencialmente expressos durante a infeção e sua expressão no transcriptoma. Apesar disto, nós observamos a intersecção de vias biológicas enriquecidas para os mRNAs alvos dos miRNAs diferencialmente expressos e vias biológicas enriquecidas para os mRNAs diferencialmente expressos no transcriptoma. As vias biológicas enriquecidas foram, sinalização de Rho GTPases, transporte de membrana, controle do ciclo celular, sinalização por Notch e transporte e modificação pelo complexo de Golgi. Assim, os miRNAs induzidos pela infeção do T. cruzi em macrófagos estão, provavelmente, envolvidos no ajuste fino de atividades das vias biológicas importantes na resposta à infeção, podendo atuar como reguladores indiretos dos componentes dessas vias. Como perspectiva futura, os miRNAs diferencialmente expressos serão bloqueados e avaliados o seu impacto na reposta à infeção pelo T. cruzi em macrófagos murinos.Chagas disease is caused by the intracellular parasite Trypanosoma cruzi, which is capable of infecting several cell types. The initial interaction between parasite and host involves the activation of pathways responsible for the internalization of the parasite, the innate immune response of the host and the fight against intracellular parasites. Several changes in gene expression occur in the host cell in response to the pathogen and vice versa. Interfering RNA pathways (RNAi) are related to posttranscriptional regulation of gene expression mediated by small non-coding RNAs such as microRNAs (miRNAs), which allow fine-tuning of various biological responses and are altered in parasitic infections. Macrophages infected with Leishmania sp. or Toxoplasma sp. has the altered expression of miRNAs that regulate the response to these parasites. To date, there is no description of the role of miRNAs during Trypanosoma cruzi infection in murine macrophages. In this work, we performed the sequencing of small RNAs from murine macrophages infected with the parasite Trypanosoma cruzi at 2, 4, 8 and 48 hours after infection to evaluate the general expression of the transcriptome of small RNAs, especially the host miRNAs. Expression of the proinflammatory cytokine TNF- was evaluated as an indicator of the innate immune response during infection, as well as the growth of intracellular parasites. There were no qualitative changes in the profile of small RNAs derived from the genome of the parasite throughout the infection. In contrast, among the small host RNAs, we observed the differential expression of several miRNAs between the times of 2 and 48 hours. Of the 18 miRNAs that showed differential expression at some time of infection, only 1 was expressed positively in at least two analyzed times, miR-34c-5p. We identified the predicted and validated targets for the miRNAs differentially expressed by the TargetScan tool and the StarBase database, and used the targets found in both for the study of miRNAs functions. The enrichment analysis of target mRNAs for biological pathways suggests that miRNAs are related to pathways such as TGF- signaling, Smads transcriptional activity, PI3K signaling cascade, signaling of cell death via JNK as well as the activation of NFKB via TRAF6. The miR-34c-5p showed significant positive regulation throughout the infection and validated the expression by RT-qPCR. Analysis of miR-34c-5p target mRNAs showed enrichment for biological processes as apoptosis response and Notch signaling. As the miRNAs act by regulating the stability of their target mRNAs, the macrophage transcriptome after 4 and 8 hours of infection was sequenced and analyzed. In all, 274 mRNAs were regulated: 33 were down-regulated and 241 were down-regulated at any of the times analyzed. However, there was no direct correlation, either positive or negative, between the miRNA targets differentially expressed during infection and their expression in the transcriptome. In spite of this, we observed the intersection of enriched biological pathways for the mRNAs targets of the differentially expressed miRNAs and biological pathways enriched for the mRNAs differentially expressed in the transcriptome. Enriched biological pathways were Rho GTPases signaling, membrane transport, cell cycle control, Notch signaling and transport and modification by the Golgi complex. Thus, the miRNAs induced by T. cruzi infection in macrophages are probably involved in the fine-tuning of activities of the biological pathways important in the response to infection and may act as indirect regulators of the components of these pathways. As a future perspective, differentially expressed miRNAs will be blocked and evaluated for their impact on T. cruzi infection response in murine macrophages

Table_2_miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.PDF

<p>Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.</p

Table_4_miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.PDF

<p>Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.</p

miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection

Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells

Table_5_miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.PDF

<p>Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.</p

Table_1_miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.PDF

<p>Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.</p

Table_6_miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.PDF

<p>Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.</p