<div><p>Zika virus (ZIKV) is a mosquito-borne pathogen that stayed unnoticed for over half a century. Only after the 2015-16 Brazilian outbreak did the severity of the infectious outcome, particularly the Congenital Zika Syndrome, become apparent. ZIKV is associated with severe neurodevelopmental impairments in human fetuses, including microencephaly, ventriculomegaly, retinopathy, and sensorineural hearing loss. Though the pandemic is now under control in the Latin American countries, several tropical countries could still be at risk of widespread infection. This warrants a better understanding of the congenital Zika syndrome; this project attempts to contribute towards this goal.</p><p><br></p><p>Previous reports examining neural progenitor tropism of ZIKV in organoid and animal models did not address whether the virus infects all neural progenitors uniformly. To explore this, ZIKV was injected into the neural tube of 2-day-old chicken embryos, resulting in non-uniform periventricular infection 3 days later. Recurrent foci of intense infection were present at specific signaling centers that influence neuroepithelial patterning at a distance through secretion of morphogens. ZIKV infection reduced transcript levels for 3 morphogens, SHH, BMP7, and FGF8, expressed at the midbrain basal plate, hypothalamic floor plate, and isthmus, respectively. Levels of Patched1, a SHH-pathway downstream gene, were also reduced and a SHH-dependent cell population in the ventral midbrain was shifted in position. Thus, the diminishment of signaling centers through ZIKV-mediated apoptosis may yield broader, non-cell autonomous changes in brain patterning.</p><p><br></p></div><p>Sensorineural hearing loss is a relatively understudied consequence of congenital Zika syndrome, and balance disorders are essentially unreported to date. ZIKV pathogenesis was explored in the developing inner ear using the accessible chicken embryo model system. One goal was to assess the spatiotemporal susceptibility of otic epithelial-derived structures to ZIKV infectivity. Direct injections of the inner ear or the inner ear primordium were performed <i>in ovo</i>with subsequent harvests at 2 to 8 days-post-infection. The degree of infection in sensory/prosensory organs was evaluated histologically to determine the susceptibility of one auditory and five vestibular organs. ZIKV infection of the sensory as well as non-sensory epithelia was observed at most stages of analysis, with no apparent preference for one over the other. The lagena, the ventral most tip of the chicken inner ear, and the endolymphatic sac/duct were least frequently infected. In this report, two novel findings in sequela of ZIKV infection are presented: the vestibular labyrinth can present with stalled canal morphogenesis, and the auditory ganglion can be severely shrunken, perhaps due to an increased cell death upon early ZIKV infection of the inner ear.</p><p><br></p><p>Additional methods of peripheral infection in the chicken embryos were tested to examine ZIKV transmission to the central nervous system: E3 blood vessel, E4 limb bud, and E10 chorioallantoic membrane infections. Although none of these methods resulted in a histologically significant infection of the developing brain 3 to 6 days-post-infection, evidence of ZIKV genome replication and viremia was detected in several tissue types.<br></p>

and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...

Arbovirus infections are widespread, and their disease burden has increased in the past decade. In Africa, arbovirus infections and fever with unknown etiology are common. Due to the lack of well-established epidemiologic surveillance systems and accurate differential diagnosis in most African countries, little is known about the prevalence of human arbovirus infections in Africa. The aim of this review is to summarize the available epidemiological data and diagnostic laboratory tools of infections with dengue, yellow fever, Zika, and chikungunya viruses, all transmitted by Aedes mosquitoes. Studies indicate that these arboviral infections are endemic in most of Africa. Surveillance of the incidence and prevalence of the infections would enable medical doctors to improve the diagnostic accuracy in patients with typical symptoms. If possible, arboviral diagnostic tests should be added to the routine healthcare systems. Healthcare providers should be informed about the prevalent arboviral diseases to identify possible cases.

As principais matérias primas necessárias para a preparação de testes diagnósticos são as proteínas dos patógenos que necessariamente apresentem as estruturas nativas. O objetivo do presente estudo foi a obtenção das proteínas não estruturais 1 (NS1) dos vírus da dengue (DENV) e da zika (ZIKV) a partir dos corpúsculos de inclusão (CI) produzidos em bactérias Escherichia coli. Mostramos que a combinação de alta pressão hidrostática (APH) e pH alcalino é eficiente para a solubilização de NS1-CI. A incubação em 2,4 kbar das suspensões de NS1-CI em pH alcalino mostrou-se eficiente para a solubilização da NS1. A presença de Arg promove a dissociação de oligômeros. A aplicação de 2,4 kbar às suspensões de NS1-CI em pH de 10,5 (DENV) e de 11,5 (ZIKV) na presença de Arg e um par redox, seguida de diálise em tampão em pH 8,5, foram as condições escolhidas para o reenovelamento de NS1. Obtivemos ambas NS1 com rendimentos entre 75% e 90% em relação às quantidades totais das proteínas presente nos correspondentes CI de NS1. As NS1 reenoveladas apresentaram reatividade comparável às proteínas obtidas utilizando um protocolo convencional estabelecido, com rendimentos mais de 25 vezes superiores. Foi obtido um processo altamente eficiente para o reenovelamento de NS1 apresentando características biológicas preservadas em relação a reatividade com anticorpos específicos de antígeno, incluindo soro de paciente infectado com zikv e que, portanto, podem ser usados como antígeno para o desenvolvimento de vacinas ou testes de diagnóstico. Além disso, este estudo descreve a criação de um processo inovador, que é a utilização concomitante de APH e pH alcalino, para solubilização e posterior reenovelamento de NS1-CI que podem ser utilizados para outras proteínas relevantes. / The main products for the preparation of diagnostic tests are as proteins of the pathogens that necessarily present as the native structures. The objective of the present study was to obtain non-structural proteins 1 (NS1) from dengue virus (DENV) and zika virus (ZIKV) from the inclusion bodies (IBs) produced in Escherichia coli bacteria. We show that it is a combination of high hydrostatic pressure (HHP) and alkaline pH is efficient for a solubilization of NS1-IB. A 2.4 kbar incubation of NS1-IB suspensions at alkaline pH proved to be efficient for NS1 solubilization. The presence of Arg promotes the dissociation of oligomers. The application of 2.4 kbar to the suspensions of NS1-IB at pH 10.5 (DENV) and 11.5 (ZIKV) in the presence of Arg and a redox pair, dialysis in pH 8.5 buffer were as conditions chosen for the refolding of NS1. We obtained both NS1 at yields between 75% and 90% relative to the total amounts of the proteins present in the corresponding NS1 IB. Refolded NS1 showed similar to proteins obtained using an established standard protocol, with yields more than 25 times higher. A highly efficient process for the refolding of NS1 was obtained with preserved biological features regarding reactivity with antigen-specific antibodies, including sera of zikv-infected patients and that can be used as antigen for the development of vaccines or diagnostic tests. In addition, this study describes the creation of an innovative process, which is a concomitant use of HHP and alkaline pH, for solubilization and subsequent refolding of NS1-IB that can be used for other relevant proteins.

As principais matérias primas necessárias para a preparação de testes diagnósticos são as proteínas dos patógenos que necessariamente apresentem as estruturas nativas. O objetivo do presente estudo foi a obtenção das proteínas não estruturais 1 (NS1) dos vírus da dengue (DENV) e da zika (ZIKV) a partir dos corpúsculos de inclusão (CI) produzidos em bactérias Escherichia coli. Mostramos que a combinação de alta pressão hidrostática (APH) e pH alcalino é eficiente para a solubilização de NS1-CI. A incubação em 2,4 kbar das suspensões de NS1-CI em pH alcalino mostrou-se eficiente para a solubilização da NS1. A presença de Arg promove a dissociação de oligômeros. A aplicação de 2,4 kbar às suspensões de NS1-CI em pH de 10,5 (DENV) e de 11,5 (ZIKV) na presença de Arg e um par redox, seguida de diálise em tampão em pH 8,5, foram as condições escolhidas para o reenovelamento de NS1. Obtivemos ambas NS1 com rendimentos entre 75% e 90% em relação às quantidades totais das proteínas presente nos correspondentes CI de NS1. As NS1 reenoveladas apresentaram reatividade comparável às proteínas obtidas utilizando um protocolo convencional estabelecido, com rendimentos mais de 25 vezes superiores. Foi obtido um processo altamente eficiente para o reenovelamento de NS1 apresentando características biológicas preservadas em relação a reatividade com anticorpos específicos de antígeno, incluindo soro de paciente infectado com zikv e que, portanto, podem ser usados como antígeno para o desenvolvimento de vacinas ou testes de diagnóstico. Além disso, este estudo descreve a criação de um processo inovador, que é a utilização concomitante de APH e pH alcalino, para solubilização e posterior reenovelamento de NS1-CI que podem ser utilizados para outras proteínas relevantes. / The main products for the preparation of diagnostic tests are as proteins of the pathogens that necessarily present as the native structures. The objective of the present study was to obtain non-structural proteins 1 (NS1) from dengue virus (DENV) and zika virus (ZIKV) from the inclusion bodies (IBs) produced in Escherichia coli bacteria. We show that it is a combination of high hydrostatic pressure (HHP) and alkaline pH is efficient for a solubilization of NS1-IB. A 2.4 kbar incubation of NS1-IB suspensions at alkaline pH proved to be efficient for NS1 solubilization. The presence of Arg promotes the dissociation of oligomers. The application of 2.4 kbar to the suspensions of NS1-IB at pH 10.5 (DENV) and 11.5 (ZIKV) in the presence of Arg and a redox pair, dialysis in pH 8.5 buffer were as conditions chosen for the refolding of NS1. We obtained both NS1 at yields between 75% and 90% relative to the total amounts of the proteins present in the corresponding NS1 IB. Refolded NS1 showed similar to proteins obtained using an established standard protocol, with yields more than 25 times higher. A highly efficient process for the refolding of NS1 was obtained with preserved biological features regarding reactivity with antigen-specific antibodies, including sera of zikv-infected patients and that can be used as antigen for the development of vaccines or diagnostic tests. In addition, this study describes the creation of an innovative process, which is a concomitant use of HHP and alkaline pH, for solubilization and subsequent refolding of NS1-IB that can be used for other relevant proteins.

Les maladies virales à transmission vectorielle émergentes et ré-émergentes comme la dengue, le chikungunya ou le zika sont responsables de nombreuses épidémies sévères à travers le monde. Récemment, la propagation rapide et très étendue du virus zika (ZIKV) ainsi que les complications neurologiques graves liées à l’infection par ZIKV ont incité l’Organisation Mondiale de la Santé (OMS) à déclarer le ZIKV comme une urgence de santé publique mondiale. Aujourd’hui, les mesures préventives ou curatives de l’infection par ZIKV sont quasiment inexistantes. D’autre part, la flore endémique de La Réunion est connue comme une source riche, renouvelable et prometteuse en produits naturels anti-infectieux. L’inscription à la pharmacopée française de 19 plantes médicinales réunionnaises souligne le potentiel thérapeutique des substances naturelles issues de la biodiversité locale. Les travaux ont été consacrés à l’identification de substances naturelles anti-ZIKV issues d’une sélection de sept plantes médicinales réunionnaises inscrites à la pharmacopée française. Dans une première étape, une extraction sans solvant assistée par micro-ondes a été appliquée sur les sept plantes médicinales sélectionnées afin d’obtenir des éco-extraits. Dans une deuxième étape, le criblage de l’activité antivirale, en utilisant un clone moléculaire du ZIKV avec un gène rapporteur, a permis d’identifier deux éco-extraits actifs provenant de Aphloia theiformis et de Psiloxylon mauritianum. Après avoir vérifié l’absence de cytotoxicité et de génotoxicité des extraits actifs, l’activité antivirale a été aussi démontrée sur d’autres types d’extraits réalisés via des méthodes d’extractions traditionnelles (infusion, décoction et macération). L’activité antivirale a été validée sur différentes souches de ZIKV (historique et épidémique) ainsi que sur les 4 sérotypes du virus de la dengue. Enfin, le mode d’action des deux extraits actifs a été étudié. Il a été montré que les éco-extraits d’A. theiformis et de P. mauritianum ciblent les phases précoces du cycle viral en inhibant l’attachement du virus à la cellule hôte. À l’aide de la microscopie électronique, il a été montré que l’éco-extrait d’A. theiformis déforme la particule virale empêchant cette dernière de s’attacher à la membrane de la cellule hôte. Ces résultats démontrent l’importance des plantes médicinales réunionnaises comme source de substances naturelles anti-infectieuses. / Emerging and re-emerging vector-borne viral diseases such as dengue, chikungunya or zika are responsible for many severe epidemics worldwide. Recently, the rapid spread of zika virus (ZIKV) worldwide and the serious neurological complications associated with ZIKV infection have prompted the World Health Organization (WHO) to declare ZIKV a public health emergency. Today, preventive or curative measures against ZIKV infection are almost non-existing. On the other hand, the endemic flora of Reunion Island is known as a rich, renewable and promising source of natural anti-infective products. The registration of 19 medicinal plants from Reunion Island in the French pharmacopoeia highlights the therapeutic potential of natural substances derived from local biodiversity. The work was dedicated to the identification of natural anti-ZIKV substances from a selection of seven medicinal plants from La Reunion registered in the French pharmacopoeia. In a first step, in collaboration with a local start-up (Bourbon Extracts), a solvent-free microwave-assisted extraction was applied to the seven selected plants in order to obtain eco-extracts. In a second step, the screening for antiviral activity, using a ZIKV molecular clone with a reporter gene, allowed the identification of two candidate plants: Aphloia theiformis and Psiloxylon mauritianum. After verifying the absence of cytotoxicity and genotoxicity of the active extracts, the antiviral activity was also demonstrated on other types of extracts using traditional extraction methods (infusion, decoction and maceration). The antiviral activity has been validated on different strains of ZIKV (historical and epidemic) as well as on the four serotypes of the dengue virus. Finally, the mode of action of the two active extracts has been studied. It has been shown that the eco-extracts from A. theiformis and P. mauritianum target the early steps of the viral cycle by inhibiting the attachment of the virus to the host cell. Using electron microscopy, it has been shown that the eco-extract of A. theiformis deforms the viral particle preventing its attachment to the membrane of the host cell. These results demonstrate the importance of medicinal plants from Reunion Island as a source of natural anti-infectious substances.

O Zika virus (ZIKV) é um flavívirus, tal qual o virus da dengue e, assim como esse, também é transmitido pelos mosquitos do gênero Aedes, especialmente o Aedes aegipty Restrito ao continente africano até 2007, o ZIKV chegou as Américas em 2013, com os primeiros registros brasileiros da infecção datando de 2014 - 2015. Em 2016, a OMS classificou o surto de ZIKV como Emergência de Saúde Mundial. O ZIKV pode acarretar na Sindrome Congênita do ZIka Vírus (SCZ) em bebês que sofreram infecção gestacional. Além da manifestação mais temida e marcante, a microcefalia, e de demais danos neurológicos, a SCZ tem sido principalmente associada com anomalias visuais e oftalmológicas. Entretanto, poucas informações se encontram disponíveis na literatura sobre o impacto da infecção por ZIKV nas funções visuais e desenvolvimento visual em crianças sem microcefalia. Pouco se conhece também do impacto da infecção em diferentes populações brasileiras, de localidades distintas. Dessa forma, o presente trabalho se propôs a preencher essa lacuna, avaliando a acuidade visual (AV) e desenvolvimento da acuidade visual em crianças expostas gestacionalmente ao ZIKV, utilizando-se o Teste dos Cartões de Acuidade de Teller (CAT). As crianças avaliadas vieram de duas populações distintas da região Sudeste do Brasil, a cidade de Jundiaí (SP) e a cidade do Rio de Janeiro (RJ). A Coorte Zika Jundiaí foi composta de 118 crianças divididas entre grupo controle saudável (ZC) com 63 crianças, grupo experimental, dividido em dois subgrupos, baseado em exame de RT- qPCR, sorologia ou avaliação clínica, o subgrupo Exposto (ZE) com 23 crianças, em que apenas as mães tiveram infecção confirmada por ZIKV e o subgrupo Infectado (ZI), com 24 crianças no qual mãe e criança tiveram infecção confirmada. Oito crianças formaram o grupo Controle Postivo (M), com crianças com microcefalia em decorrência de outras causas. A Coorte IFF Fiocruz Rio de Janeiro foi composta de 32 crianças, todas com infecção confirmada pelo ZIKV (ZI), por exame de RT-qPCR, sorologia ou avaliação clínica. Os resultados do presente trabalho mostram que a exposição gestacional ao ZIKV, na ausência de infecção, parece não ter efeito sobre a AV ou seu desenvolvivemento. No entanto, quando ocorre infecção gestacional por ZIKV, pode haver prejuízos à AV mesmo quando a criança não apresenta anomalias neurológicas e/ou oftalmológicas aparentes. Esse trabalho, entretanto, concorda com estudos anteriores que mostram uma maior chance de haver perdas de AV concomitantementes a um comprometimento cognitivo e/ou oftalmologico. As análises do subgrupo ZI de Jundiaí dão suporte a noção de que crianças gestacionalmente infectadas por ZIKV tendem a apresentar um desenvolvimento anormal (mais lento) da AV. Esse achado, se reproduzido, possuí repercussões mais gerais, dado que quaisquer danos à função visual podem indicar deficiências no sistema nervoso central. Assim sendo, tais achados tem importantes repercussões na saúde pública. Além disso, as taxas diferentes de danos à AV, bem como de incidência de microcefalia e comprometimento oftalmológico entre as populações estudadas neste trabalho, apoiam a hipótese da existência de demarcadas diferenças regionais entre as linhagens de ZIKV circulantes no país, assim como de diferenças de suscetibilidade de hospedeiros entre as populações estudadas / The Zika virus (ZIKV) is a flavivirus, such as the dengue virus, and like the dengue virus is also transmitted by the Aedes osquitoes, especially the Aedes aegipty Restricted to the African Country until 2007, the ZIKV arrived in the Americas in 2013, with the first brazillian cases of the infection dating to 2014 - 2015. In 2016, WHO classified the ZIKV spread as a World Health Emergency. The ZIKV, in gestationally exposed children, can lead to Congenital Zika Syndrome (CZS). Beyond its most marked and feared manifestation, microcephaly, and its other neurological damages, CZS is mostly associated with visual and ophthalmologic anomalies. However, little information is available on the literature about the impact of the ZIKV infection in the visual function and on the visual development in infected children without microcephaly. Little is also known about the impact of the infection in different brazillian populations from distinct areas. With that in mind, the present work intends to fill this gap in literature, evaluating the visual acuity (VA) and visual acuity development in children gestationally exposed to the ZIKV, using the Teller Acuity Cards Test (TAC). The evaluated children were from two different populations of the Southeast region of Brazil, the city of Jundiaí (SP) and the city of Rio de Janeiro (RJ). The Zika Jundiaí Cohort was composed of 118 children divided between the healthy control group (ZC) with 63 children and the experimental group, divided in two subgroups based on RT-qPCR, sorology or clinical evaluation, the Exposed subgroup (ZE) with 23 children, which only the mothers had ZIKV confirmed and the Infected subgroup (ZI), with 24 children, wich both mother and children had ZIKV confirmed infection. Eight children formed the Positive Control Group (M), composed of children with microcephaly due to other causes. The IFF Fiocruz Rio de Janeiro Cohort was composed of 32 children, all with confirmed ZIKV infection (ZI), either by RT-qPCR/sorology exam or by clinical evaluation. The results of the present work show that ZIKV exposition in absence of infection, does not seem to affect VA or its development. However when theres a gestational ZIKV infection, it may lead to VA damages even when the children does not show any apparent neurologic/ophtalmologic anomalies. This work, nonetheless, is in agreement with former studies that show a higher chance of VA losses when the children also have cognitive and/or ophtalmologic deficits. The Jundiaí ZI Subgroup analisies support the notion that ZIKV gestationally infected children tend to have an abnormal (slower) VA development. This finding, if replicated, has more general repercussions given that any visual function damages may indicate central nervous system deficiencies. Thus, these findings have important repercussions in the public health area. Furthermore, the different rates of VA damages as well as the different rates of microcephaly incidencies and ophthalmologic deficits between the populations studied in this work support the hypothesis of marked regional differences between the brazillian ZIKV strains, as well as host suscepitibilitiess differences between the studied populations

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-21T13:27:45Z No. of bitstreams: 2 Dissertacao_CristianeFracaoDiefenbach.pdf: 3471815 bytes, checksum: 5340dde1fa83817a1b28da306684977c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-21T13:27:45Z (GMT). No. of bitstreams: 2 Dissertacao_CristianeFracaoDiefenbach.pdf: 3471815 bytes, checksum: 5340dde1fa83817a1b28da306684977c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-12-21 / The Zika Virus (ZIKV) is a flavivirus, which is responsible for causing an emerging arbovirosis in Brazil what is primarily transmitted by the bite of infected mosquitoes of the genus Aedes. A much discussed transmission route lately is blood transfusion due to the large number of asymptomatic cases and the epidemic potential of ZIKV that causes epidemics of great proportions. As such, asymptomatic individuals may contaminate blood components and thereby participate in the transfusion transmission of this infection. The disease has a benign clinical picture but serious clinical consequences have been reported, such as Guillain-Barré syndrome (GBS) and fetal malformations such as microcephaly. Due to this fact, recently, the Health Ministry (HM) declared the infection by ZIKV a Public Health Emergency of National Importance (PHENI). Thus, the major challenge for blood banks is the transfusion prevention of ZIKV due to lack of licensed laboratory routine tests for blood donor screening. In the South region of Brazil there is no epidemiological study in this population. This study will allow a robust assessment of the current epidemiological situation in this region. The objective of the study is to estimate the seroprevalence and asymptomatic viremia of ZIKV in blood donors of a Hemotherapy Service in the southern region of Brazil. In this study, blood samples were collected from 500 donors in the period from December 2016 to January 2017. The biological materials were tested for the presence of ribonucleic acid (RNA) from ZIKV, by means of real-time reverse transcription polymerase chain reaction (RT-PCR) test by commercial kit (TaqPath™ Zika Virus Kit (ZIKV) and enzyme-linked immunosorbent assay (ELISA) serologically assessed for anti-ZIKV IgG using Anti-Zika Virus ELISA IgG kit (Euroimmun, Medizinische Labordiagnostika AG, Lübek, Germany). No samples showed positive results for ZIKV RNA, which shows the low risk of transfusion transmission in the region studied. However, the serologically assessed showed the detection of a properly positive sample for anti-ZIKV IgG (0.55%) and 4 samples with optical density (OD) values in the gray zone of the test. Due to the high rate of cross-reaction between the different flaviviruses all samples (including the positive) were assessed to confirmatory testing for anti-DENV IgG (Anti-Dengue Virus ELISA IgG, Euroimmun, Medizinische Labordiagnostika AG, Lübek, Germany). The positive sample for anti-ZIKV IgG was negative for anti-DENV IgG, confirming the positive result for anti-ZIKV IgG. However, 3 gray zone seroreactive samples showed very high title of anti-DENV IgG and one remained in the gray zone. Confirmatory analysis of this sample using in-house ELISA with anti-DENV IgG antibody adsorption revealed that it was negative for anti-DENV IgG. This indicates a 0.55% seroprevalence of ZIKV in the Santa Maria-RS region. Our results demonstrate that the transfusional risk of ZIKV in the South of the Country is relatively low, however, it cannot be underestimated and serological analyses of large numbers of samples are required to determine the exact seroprevalence of anti-ZIKV and anti- DENV IgG in blood donors in the region of Santa Maria. / O vírus Zika (ZIKV) é um flavivírus responsável por causar uma arbovirose emergente no Brasil que é transmitido principalmente por picada de mosquitos infectados do gênero Aedes. Uma via de transmissão muito discutida ultimamente é a transfusão de sangue devido ao grande número de casos assintomáticos e o potencial epidêmico do ZIKV que causa epidemias de grandes proporções. Nessas ocasiões, os indivíduos assintomáticos podem contaminar hemocomponentes e deste modo participar na transmissão transfusional dessa infecção. A doença tem quadro clínico benigno, porém consequências clínicas graves têm sido relatadas, tais como a síndrome de Guillain-Barré (SGB) e malformações fetais como microcefalia. Diante desse fato, recentemente, o Ministério da Saúde (MS) declarou a infecção por ZIKV como uma Emergência em Saúde Pública de Importância Nacional (ESPIN). Assim, o principal desafio para bancos de sangue é a prevenção transfusional do ZIKV devido a falta de teste de rotina laboratorial licenciado para triagem de doadores de sangue. Na região Sul do Brasil não há nenhum estudo epidemiológico nessa população. Este estudo permitirá de uma forma robusta avaliar a situação atual epidemiológica nessa região. O objetivo do estudo é estimar a soroprevalência e a viremia assintomática do ZIKV em doadores de sangue de um Serviço de Hemoterapia da região sul do Brasil. Neste estudo foram coletadas amostras sanguíneas de 500 doadores voluntários de sangue no período de dezembro de 2016 a janeiro de 2017. Os materiais biológicos foram testados pela presença do ácido ribonucleico (RNA) do ZIKV, por meio de um teste de reação em cadeia da polimerase por transcrição reversa (RT-PCR) em tempo real por kit comercial TaqPath™ Zika Virus Kit (ZIKV) e inquérito sorológico por teste de imunoabsorbância ligada a enzima (ELISA) Anti-Zika Virus ELISA IgG kit (Euroimmun, Medizinische Labordiagnostika AG, Lübek, Alemanha). Nenhuma amostra mostrou resultado positivo para o RNA do ZIKV, o que mostra o baixo risco de transmissão transfusional na região estudada. No entanto, o inquérito sorológico revelou a detecção de uma amostra devidamente positiva para anti-ZIKV IgG (0.55%) e 4 amostras com valores de densidade ótica (DO) na zona cinza do teste. Devido a alta taxa de reação cruzada entre os diferentes flavivírus todas as amostras (incluindo a positiva) foram submetidas a teste confirmatório para anti-DENV IgG (Anti-Dengue Virus ELISA IgG, Euroimmun, Medizinische Labordiagnostika AG, Lübek, Alemanha). A amostra positiva para anti-ZIKV IgG foi negativa para anti-DENV IgG, confirmando o resultado positivo para anti-ZIKV IgG. No entanto, três amostras sororeagentes na zona cinza mostraram título muito elevado de anti-DENV IgG e uma permaneceu na zona cinza. Análise confirmatória dessa amostra via utilização de ELISA in house com adsorção de anticorpo anti-DENV IgG revelou que a mesma é negativa para anti-DENV IgG. Isso demonstra uma soroprevalência de 0.55% de ZIKV na região de Santa Maria-RS. Nossos resultados demonstram que o risco transfusional do ZIKV no Sul do país é relativamente baixo, contudo, não pode ser subestimado e análises sorológicas de grande número de amostras são necessárias para determinação da soroprevalência exata do anti-ZIKV e anti-DENV IgG em doadores de sangue na região de Santa Maria.

Congenital Zika syndrome can cause a number of changes in the child, including severe brain abnormalities. Injuries to the developing central nervous system result in sensorimotor disorders with repercussions on motor performance. Physical therapy allows the acquisition of motor skills based on the child's interaction with the environment and the task. The present study aimed to analyze the role of physiotherapy in the acquisition of the motor skills of children with congenital Zika syndrome. This is a clinical trial developed at the Physiotherapy Clinic of the University Hospital of Sergipe, with a sample of 46 children with congenital Zika syndrome. Data collection was from January 2016 to December 2017, at the physiotherapeutic admission of the child and at 18 months of age. The children were stratified according to the age group in the initial evaluation. In the first moment, a form was designed to record information on the identification and clinical characteristics of mothers and children, sociodemographic characteristics of families, neurological and musculoskeletal disorders, and type of neurological lesion. The severity of the neurological lesion was classified based on the data obtained in the medical record in the transfontanel ultrasonography report, based on the Noyola Classification System. Motor skills were assessed through the Alberta Infant Motor Scale (AIMS), stratified scores by age group and subgroup of neurological injury. The children were then submitted to a physiotherapy program, with a frequency of two weekly sessions and a duration of up to 50 minutes each. The program was based on the principles of neurodevelopment therapy (Bobath), muscle stretching and passive mobilizations. When children completed 18 months of age, motor skills reassessment was performed through the AIMS. The results showed that there was an increase in the total score comparing the initial evaluation and at 18 months of age in the children who started physical therapy with age from 0 to 6 months (p <0.0001). Despite this increase, the total scores of the study sample at 18 months were significantly lower than the normative values, with a significant difference (p <0.0001). In the initial evaluation, only children older than 4 months of age presented motor impairment when compared with normative values (p <0.0001). As for most neurological lesions, the degree was moderate to severe, with a predominance of multiple calcifications, mainly cortical-subcortical, severe cortical atrophy and agenesis / hypoplasia of the corpus callosum. The severity and type of neurological lesions in children with congenital Zika syndrome suggest impairment of neuronal migration and proliferation related to motor dysfunction. However, early physiotherapy promoted a difference in the behavior of the motor skills of these children. / A síndrome da Zika congênita pode causar um conjunto de alterações na criança, dentre elas graves anomalias cerebrais. As lesões do sistema nervoso central em desenvolvimento acarretam em desordens sensoriomotoras com repercussão no desempenho motor. A fisioterapia possibilita a aquisição de habilidades motoras a partir da interação da criança com o ambiente e com a tarefa. O presente estudo teve como objetivo analisar o papel da fisioterapia na aquisição das habilidades motoras de crianças com a síndrome da Zika congênita. Trata-se de um ensaio clínico, desenvolvido no ambulatório de fisioterapia do Hospital Universitário de Sergipe, com amostra constituída por 46 crianças com a síndrome da Zika congênita. A coleta de dados ocorreu de janeiro de 2016 a dezembro de 2017, na admissão fisioterapêutica da criança e ao completar 18 meses de idade. As crianças foram estratificadas de acordo com a faixa etária na avaliação inicial. No primeiro momento foi aplicado formulário construído para registro de informações referentes a identificação e características clinicas das mães e das crianças, características sociodemográficas das famílias, alterações neurológicas e musculoesqueléticas e tipo de lesão neurológica. Classificou-se a gravidade da lesão neurológica, a partir dos dados obtidos em prontuário no laudo da ultrassonografia transfontanela, baseada no Sistema de Classificação de Noyola. Foi realizada avaliação das habilidades motoras através da Alberta Infant Motor Scale (AIMS), estratificados escores por faixa etária e por subgrupo de lesão neurológica. Em seguida, as crianças foram submetidas a um programa de fisioterapia, com frequência de duas sessões semanais e duração de até 50 minutos cada. O programa baseou-se nos princípios da terapia do neurodesenvolvimento (Bobath), alongamentos musculares e mobilizações passivas. Quando as crianças completaram 18 meses de idade, foi realizada reavaliação das habilidades motoras por meio da AIMS. Os resultados apontaram que houve aumento do escore total comparando a avaliação inicial e aos 18 meses de idade, nas crianças que iniciaram a fisioterapia com idade de 0 a 6 meses (p<0,0001). Apesar deste aumento, os escores totais da amostra do estudo aos 18 meses são bastantes inferiores aos valores normativos, com diferença significativa (p<0,0001). Na avaliação inicial, somente as crianças com idade superior a 4 meses de idade apresentaram comprometimento motor ao comparar com os valores normativos (p<0,0001). Quanto as lesões neurológicas, em sua maioria, o grau foi de moderado a grave com predomínio de calcificações múltiplas, principalmente córtico-subcortical, atrofia cortical grave e agenesia/ hipoplasia de corpo caloso. A gravidade e o tipo das lesões neurológicas nas crianças com síndrome da Zika congênita, sugerem comprometimento da migração e proliferação neuronal, relacionados com a disfunção motora. Porém, a fisioterapia precoce promoveu uma diferença no comportamento das habilidades motoras dessas crianças. / Aracaju

Le virus Chikungunya (CHIKV), arbovirus en pleine ré-émergence, a envahi rapidement de nombreuses zones géographiques du monde. La propagation mondiale de ce virus constitue une menace pour la santé humaine car il n'y a pas de vaccin ou d'agents antiviraux appropriés pour contrôler l'infection virale. La transmission du virus s’effectue lors de la piqure d’un moustique infecté du genre Aedes, qui injecte sa salive contenant le virus dans la peau de l’hôte humain. Afin de contrôler la dissémination du virus, il est primordial de développer des recherches sur l’identification de molécules antivirales et de comprendre les mécanismes moléculaires impliqués dans les interactions hôte-virus et/ou vecteur-virus-hôte. En utilisant différentes stratégies moléculaires et cellulaires, nous avons étudié le potentiel antiviral de l'Imipramine, une molécule déjà commercialisée et qui a la capacité de perturber le transport du cholestérol intracellulaire. Nous avons démontré que cette molécule est capable d'inhiber la réplication du CHIKV dans les fibroblastes cutanés humains. Nous avons mis en évidence que l'Imipramine affectait à la fois les étapes de fusion et de réplication pendant le cycle de réplication du virus. En outre, la molécule a également fortement inhibé la réplication de plusieurs Flavivirus comme le virus Zika (ZIKV), le virus du Nil occidental et le virus de la Dengue. Nous avons également déterminé le profil protéomique global des fibroblastes humains infectés par le CHIKV ou le ZIKV. Cela nous a permis de mettre en évidence les modulations significatives de plusieurs protéines stimulées par l'interféron et de protéines impliquées dans à la défense anti-virale dans les cellules infectées. Plus important encore, nos résultats montrent pour la première fois le rôle de la protéine SAMHD1 dans l'infection des fibroblastes cutanés par les arbovirus. Enfin, compte tenu des fortes interactions entre l’hôte, le vecteur et le CHIKV, l'effet de la salive du moustique Ae. Aegypti sur l'infection virale a été étudié. À notre connaissance, cette étude est la première à montrer l’importance de la salive d’Ae. aegypti sur la facilitation de l’infection du CHIKV, dans des fibroblastes cutanés, à travers la régulation des gènes impliqués dans la réponse interféron de type I. / Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that has been spread worldwide. The dissemination of this virus is a threat to human health since there is no approved vaccine or appropriate antiviral agents to control viral infection. The global expansion of the virus is preceded by biting of infected Aedes mosquitos, which injects saliva containing the virus into the skin of the human host. Searching for effective antiviral compounds and understanding of the molecular mechanisms involved in host-virus or vector-virus-host interactions are crucial for controlling viral spread.Using different molecular and cellular strategies, we demonstrated that the FDA approved drug, imipramine, which has the capability to disturb intracellular cholesterol transport inhibits CHIKV replication in human skin fibroblasts. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. Moreover, it also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. We have also determined the global proteomic profile of Chikungunya and Zika virus infected human skin fibroblasts, and found that several interferon-stimulated proteins and antiviral response proteins are significantly up-regulated in the infected cells. More importantly, our results also provided for the first time a role of SAMHD1 in arbovirus infection of human skin fibroblasts. Finally, we demonstrated that Aedes aegypti saliva enhances CHIKV replication in human skin fibroblasts. To our knowledge, this is the first report showing the importance of Aedes aegypti saliva on promoting CHIKV infection via down regulation of the genes involving type I IFN secretion in the infected human cutaneous cells.