Infecção congênita é aquela transmitida da mãe ao feto antes do nascimento. A transmissão vertical pode ocorrer por via transplacentária ou por contato direto com o patógeno durante o parto. A fonte de infecção é o microrganismo presente no sangue da gestante durante a infecção primária ou crônica. Estima-se que as infecções perinatais representam 2% a 3% de todas anomalias congênitas e as mais comuns são representadas pela sigla TORCH, que inclui Toxoplasmose, Outras (como sífilis e varicela-zoster), Rubeóla, Citomegalovírus e Herpes. A maioria das infecções TORCH causa morbidade materna leve, assintomática, mas tem consequências fetais graves e, em geral, o tratamento da infecção materna não tem impacto sobre o resultado fetal. Por isso, o reconhecimento do contágio materno e o monitoramento fetal são de extrema importância na prevenção de doenças congênitas. O presente projeto visa o desenvolvimento de um produto multiparamétrico utilizando a tecnologia xMAP®, criada pela companhia norte-americana Luminex Corporation, para detecção simultânea de anticorpos da classe IgG anti-Toxoplasmose, anti-Rubéola e anti- Citomegalovírus em amostras de sangue coletado em papel de filtro. O produto, inédito no mercado nacional, tem o objetivo de atender à demanda específica da triagem pré-natal no país. Como objetivos específicos pode-se citar: a melhoria da eficiência dos programas de triagem pré-natal, graças à economia de tempo, amostras e reagentes; a contribuição financeira para o Brasil, uma vez que será produzido nacionalmente, gerando emprego e renda; a possibilidade de ampliar o mercado a partir do desenvolvimento futuro de novos produtos baseados na mesma metodologia. Para tanto, utilizaram-se antígenos específicos acoplados à microesferas de poliestireno e anticorpos de detecção conjugados à estreptavidina-ficoeritrina. Foram analisadas 1499 amostras de gestantes, coletadas em papel de filtro, cedidas e previamente triadas pela APAE-Goiânia, com o objetivo de comparar os resultados obtidos através da análise com o protótipo desenvolvido com aqueles já confirmados pelo laboratório utilizando a tradicional metodologia de ELISA. Os resultados de Concordância e Sensibilidade foram superiores a 78% para todos os parâmetros. Por outro lado, os valores de Especificidade foram mais baixos, principalmente para os parâmetros Rubéola e Citomegalovírus. Importante ressaltar o pequeno número de amostras com resultado negativo para Citomegalovírus e Rubéola disponível, o que refletiu diretamente no cálculo da Especificidade do produto / Congenital infections are transmitted from the mother to the fetus before birth. Vertical transmission can occur via placenta or by direct contact with the pathogen during childbirth. The source of infection is the microorganism present in the pregnant woman\'s blood during primary or chronic infection. It is estimated that perinatal infections are responsible for 2% to 3% of all congenital abnormalities and the most common are represented by the acronym TORCH, including Toxoplasmosis, Others (such as syphilis and varicella-zoster), Rubella, Cytomegalovirus (CMV) and Herpes. Most infections TORCH causes mild maternal morbidity, asymptomatic, but has serious fetal consequences fetal and, generally, maternal infection treatment has no impact on fetal outcome. Therefore, the recognition of maternal infection and fetal monitoring are extremely important in preventing birth defects. This project aims to develop a product using the multiplex xMAP® technology, created by US company Luminex Corporation, for simultaneous detection of IgG antibodies anti-toxoplasmosis, antirubella and anti-cytomegalovirus in blood samples collected in filter paper. The product, unprecedented in brazilian market, aims to meet the specific demand of prenatal screening in Brazil. The specific objectives are: improving the efficiency of prenatal screening programs, thanks to savings in time, samples and reagents; the financial contribution for Brazil, as it will be produced nationally, generating jobs and income; the possibility of expanding the market from the future development of new products based on the same methodology. For this purpose, were used specific antigens coupled to polystyrene beads and antibodies conjugated to streptavidin-phycoerythrin. Were analyzed 1499 samples of pregnant women, collected on filter paper, pre-screened by APAE-Goiânia, in order to compare the results obtained from the analysis with the prototype developed with those already confirmed by the laboratory using traditional ELISA methodology. The results of Concordance and Sensitivity were higher than 78% for all parameters. In contrast, the Specificity values were lower, especially for Rubella and Cytomegalovirus parameters. Importantly the small number of negative samples negative for Cytomegalovirus and Rubella provided by APAE-Goiânia, which is directly reflected in the product specificity value.

Avec une prévalence mondiale de 0,7%, l’infection in utero à cytomégalovirus (CMV) représente la première cause de handicap neurologique congénital d’origine infectieuse. Les modalités de prise en charge de cette infection restent débattues notamment en raison de l’absence de marqueurs pronostics fiables et d’inconnus sur sa physiopathologie notamment celle de l’atteinte du cerveau fœtal. Le premier objectif de notre travail était de décrire et valider des marqueurs immuno-virologiques prédictifs de la transmission verticale et de séquelles néonatales. Le deuxième objectif était d’étudier les corrélats immuno-virologiques in situ de la sévérité de l’atteinte du cerveau fœtal. Nous avons pu établir à partir du bilan virologique maternel (avidité des IgG et ADN CMV sanguin) un score de risque de transmission verticale du virus en cas de primo-infection maternelle. Nous avons montré que le niveau du réservoir viral fœtal, reflété par la charge virale dans le liquide amniotique et dans le sang fœtal, était un marqueur prédictif des séquelles néonatales. Ainsi, la combinaison de la mesure du réservoir viral fœtal avec l’imagerie fœtale a permis d’établir des scores pronostics avec des valeurs prédictives positives et négatives de 80 à 100% respectivement. Nous avons mis en évidence par immuno-histochimie couplée à une analyse quantitative d’images que la multiplication virale ainsi que la réponse immunitaire innée (cellules NK) et adaptative (CD8+ et plasmocytes) étaient significativement plus élevées dans les cerveaux fœtaux les plus sévèrement atteints. Ce résultat paradoxal nous a incité à quantifier la présence du marqueur PD-1 et celle de son récepteur PD-L1. PD-1 était significativement plus exprimé dans les cerveaux sévèrement atteints. L’analyse par cytométrie de flux montrait que PD-1 était exprimé par 96% des CD8+ mais aussi par plus de 70% des lymphocytes B et des cellules NK. Ces résultats témoignent de l’existence dans les cerveaux fœtaux infectés d’un épuisement immunitaire touchant la réponse adaptative mais aussi innée. Enfin, l’analyse par cytométrie de flux montrait la présence d’une réplication virale dans les différents types de cellules neuronales (cellules souches, neurones, astrocytes). En conclusion, les résultats de notre travail ont permis d’améliorer les algorithmes de prise en charge de l’infection à CMV in utero grâce à la validation de marqueurs prédictifs immuno-virologiques. Par ailleurs, le fait qu’un épuisement immunitaire et une forte multiplication virale soient associés à la sévérité de l’atteinte cérébrale est important pour l’élaboration de stratégies thérapeutiques in utero. / CMV congenital infection has a worldwide incidence estimated at about 0.7% of all life births and represents the major cause of neurological handicap of infectious origin. The management of this infection remains highly debated. Several factors contribute to this and among them are the absence of recognized prognostic markers and gaps in the knowledge of its pathogenicity particularly that of the fetal brain. The first objective of this work was to describe and validate immune and virological predictive markers of vertical transmission and of neonatal sequelae. The second objective was to study in situ immune and virological correlates of the severity of fetal brain infection. We first validated a model of materno-fetal transmission based on maternal virological results (IgG avidity and blood CMV DNA). We then showed that the viral reservoir level, estimated by the viral load in the amniotic fluid and the fetal blood, was a predictive marker of neonatal sequelae. Prognosis models combining quantification of the viral reservoir to fetal imaging allow to reach positive and negative predictive values up to 80% and 100% respectively. We showed using immunohistochemistry and quantitative image analysis that viral multiplication as well as both innate immune responses (NK cells) and adaptive immune responses (CD8+ and plasma cells) were significantly higher in the most severely infected fetal brains. This paradox drove us to quantify PD-1 and its receptor PD-L1, PD-1expression was significantly higher in severely affected fetal brains. Cytometry flow analysis evidenced that PD-1 was expressed in 95% of CD8+ cells but also in at least 70% of NK cells and of B cells. These results demonstrate immune exhaustion of both adaptive and innate responses in fetal infected brains. Finally, viral replication was evidenced in stem cells, neurons and mature astrocytes after separation by flow cytometry of these neuronal cell types. In conclusion, the validation of immune-virological markers obtained within this work has usefully improved the algorithms for the clinical management of in utero CMV infection. Moreover, the demonstration that immune exhaustion and high viral multiplication are responsible of severe fetal brain affection is important to elaborate in utero treatment strategies.

Infecção congênita é aquela transmitida da mãe ao feto antes do nascimento. A transmissão vertical pode ocorrer por via transplacentária ou por contato direto com o patógeno durante o parto. A fonte de infecção é o microrganismo presente no sangue da gestante durante a infecção primária ou crônica. Estima-se que as infecções perinatais representam 2% a 3% de todas anomalias congênitas e as mais comuns são representadas pela sigla TORCH, que inclui Toxoplasmose, Outras (como sífilis e varicela-zoster), Rubeóla, Citomegalovírus e Herpes. A maioria das infecções TORCH causa morbidade materna leve, assintomática, mas tem consequências fetais graves e, em geral, o tratamento da infecção materna não tem impacto sobre o resultado fetal. Por isso, o reconhecimento do contágio materno e o monitoramento fetal são de extrema importância na prevenção de doenças congênitas. O presente projeto visa o desenvolvimento de um produto multiparamétrico utilizando a tecnologia xMAP®, criada pela companhia norte-americana Luminex Corporation, para detecção simultânea de anticorpos da classe IgG anti-Toxoplasmose, anti-Rubéola e anti- Citomegalovírus em amostras de sangue coletado em papel de filtro. O produto, inédito no mercado nacional, tem o objetivo de atender à demanda específica da triagem pré-natal no país. Como objetivos específicos pode-se citar: a melhoria da eficiência dos programas de triagem pré-natal, graças à economia de tempo, amostras e reagentes; a contribuição financeira para o Brasil, uma vez que será produzido nacionalmente, gerando emprego e renda; a possibilidade de ampliar o mercado a partir do desenvolvimento futuro de novos produtos baseados na mesma metodologia. Para tanto, utilizaram-se antígenos específicos acoplados à microesferas de poliestireno e anticorpos de detecção conjugados à estreptavidina-ficoeritrina. Foram analisadas 1499 amostras de gestantes, coletadas em papel de filtro, cedidas e previamente triadas pela APAE-Goiânia, com o objetivo de comparar os resultados obtidos através da análise com o protótipo desenvolvido com aqueles já confirmados pelo laboratório utilizando a tradicional metodologia de ELISA. Os resultados de Concordância e Sensibilidade foram superiores a 78% para todos os parâmetros. Por outro lado, os valores de Especificidade foram mais baixos, principalmente para os parâmetros Rubéola e Citomegalovírus. Importante ressaltar o pequeno número de amostras com resultado negativo para Citomegalovírus e Rubéola disponível, o que refletiu diretamente no cálculo da Especificidade do produto / Congenital infections are transmitted from the mother to the fetus before birth. Vertical transmission can occur via placenta or by direct contact with the pathogen during childbirth. The source of infection is the microorganism present in the pregnant woman\'s blood during primary or chronic infection. It is estimated that perinatal infections are responsible for 2% to 3% of all congenital abnormalities and the most common are represented by the acronym TORCH, including Toxoplasmosis, Others (such as syphilis and varicella-zoster), Rubella, Cytomegalovirus (CMV) and Herpes. Most infections TORCH causes mild maternal morbidity, asymptomatic, but has serious fetal consequences fetal and, generally, maternal infection treatment has no impact on fetal outcome. Therefore, the recognition of maternal infection and fetal monitoring are extremely important in preventing birth defects. This project aims to develop a product using the multiplex xMAP® technology, created by US company Luminex Corporation, for simultaneous detection of IgG antibodies anti-toxoplasmosis, antirubella and anti-cytomegalovirus in blood samples collected in filter paper. The product, unprecedented in brazilian market, aims to meet the specific demand of prenatal screening in Brazil. The specific objectives are: improving the efficiency of prenatal screening programs, thanks to savings in time, samples and reagents; the financial contribution for Brazil, as it will be produced nationally, generating jobs and income; the possibility of expanding the market from the future development of new products based on the same methodology. For this purpose, were used specific antigens coupled to polystyrene beads and antibodies conjugated to streptavidin-phycoerythrin. Were analyzed 1499 samples of pregnant women, collected on filter paper, pre-screened by APAE-Goiânia, in order to compare the results obtained from the analysis with the prototype developed with those already confirmed by the laboratory using traditional ELISA methodology. The results of Concordance and Sensitivity were higher than 78% for all parameters. In contrast, the Specificity values were lower, especially for Rubella and Cytomegalovirus parameters. Importantly the small number of negative samples negative for Cytomegalovirus and Rubella provided by APAE-Goiânia, which is directly reflected in the product specificity value.

Cytomegalovirus (CMV) is a widespread infection affecting 50-90% of the human population. A typical silent primary infection is followed by life-long persistence in the host under control by virus-specific CD8 (“killer”) and CD4 (“helper”) T cells. Although harmless in most people, CMV may cause disease and sequelae in patients with deficient cellular immunity, such as AIDS patients, recipients of organ transplants and children who have acquired the virus before birth. In this thesis we have characterized the cellular immunity to CMV in immunocompetent subjects, in patients receiving transplants and in infants. In healthy individuals with latent CMV, the frequencies of CMV-specific CD8 T cells varied considerably between the donors. Within the same individual, the changes over time were usually small. In patients with primary, symptomatic CMV infection, the frequencies of CMV-specific CD8 T cells peaked within the first month after the appearance of symptoms. The frequencies then declined to levels similar to those in latently infected CMV carriers. The CD4 T-cell function followed the same pattern, but with lower peak values. Immunosuppressed renal transplant patients with latent CMV had CMV-specific CD4 cell function similar to healthy controls. The frequencies of CMV-specific CD8 T cells were also comparable, but their function was impaired. When renal transplant recipients were investigated longitudinally, we found that their CMV-specific T cells decreased rapidly after transplantation. Whereas the frequencies and function of CD8 T cells rebounded within 3 months, CD4 T-cell recovery was impaired during the entire first year after transplantation. Finally, the frequencies and function of CMV-specific T-cells were investigated in children with congenital and postnatal CMV. CMV-specific CD8 T cells could be detected in even the youngest children, suggesting that these cells can develop early in life. In contrast, CMV specific CD4 T cells were low or absent in the youngest children but increased slowly with age.

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No. of bitstreams: 2 Dissertacão - Taynara Cristina Gomes - 2016.pdf: 2697158 bytes, checksum: efd1f7ed4fcc4ac2659d5cde95cc18fd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-07-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The congenital transmission is considered one of the main pathways of Trypanosoma cruzi infection. Retrospective studies of pregnant women population with Chagas disease (CD) allow the study of the epidemiological profile of this population which contributes to the knowledge of the congenital CD prevalence in Brazil. This study evaluated the triage of T. cruzi congenital infection in new-borns through the newborn screening aiming the precocious detection of congenital transmission as well as its retrospective analysis of the seroepidemiological profile of the infected pregnant women dwelling in Goias State in a period of three years (2013-2015). The retrospective study used the data base of the Association of Parents and Friends of Special Needs Individuals (APAE) of Goiania. The analysis showed that the prevalence of CD infected pregnant women from 0.24% to 0.17%, with a decrease throughout the years. Most of these pregnant women had ≥31 years old, which is in accordance with the success of Triatoma infestans vectorial transmission control and transfusional control programmes, improvement in dwellings and better efficacy in diagnostics and therapeutics. The prospective study used the IFI and ELISA techniques for serology and filter paper detection. A total of 967 samples of dry blood in filter paper were collected in three public health units in Goiania and Aparecida de Goiania. From these samples 19 (1.96%) which were collected in filter paper from newborns were from mothers who declared to have CD. Results showed that 8 (42%) from the 19 samples were reagent to IgG anti-T. cruzi in both technique used. Peripheral blood was collected in seven mothers and their respective children. The serum samples were analyzed and the results confirmed IgG positive serology anti-T. cruzi with rates and titulation similar between mother and their respective newborn. Also there was accordance between the serology and the filter paper results which validates this strategy in the newborn screening triage from children from chronically infected mother. This study highlights the importance of the prenatal and neonatal care that precociously identifies the T. cruzi infection and also of programmes of health education about CD to the pregnant women population. / A transmissão congênita é considerada uma das principais vias de infecção pelo Trypanosoma cruzi. Estudos retrospectivos da população de gestantes com doença de Chagas (DC) viabilizam o estudo do perfil epidemiológico desta população, contribuindo para conhecimento da prevalência da DC congênita no Brasil. Este estudo avaliou a triagem de infecção por T. cruzi em recém-nascidos através do teste do pezinho, visando o diagnóstico precoce da transmissão congênita, como também a análise restrospectiva do perfil soroepidemiológico de gestantes infectadas residentes no estado de Goiás em um período de três anos (2013-2015). Para o estudo retrospectivo foi utilizado a base de dados da Associação de Pais e Amigos dos Excepcionais (APAE) de Goiânia. As análises mostraram que a prevalência de gestantes com DC variou de 0,24% a 0,17%, apresentando um declínio com o decorrer dos anos. Foi também possível evidenciar que a maioria destas gestantes possuía idade ≥31 anos, dados que corroboram com o sucesso de programas de controle da transmissão vetorial pelo Triatoma infestans e transfusional, melhoria habitacional e melhor eficiência do diagnóstico e da terapêutica. Para o estudo prospectivo foram utilizados os métodos de IFI e ELISA para a sorologia tanto em papel filtro (PF) e soro. Um total de 967 amostras de sangue seco em papel filtro (PF) foi coletado em três unidades de saúde pública em Goiânia e Aparecida de Goiânia, destes, 19 (1,96%), amostras de sangue em PF de RN foram de mães que se autodeclararam com a doença de Chagas. Os resultados mostraram que oito (42%) destas 19 amostras foram reagentes para IgG anti-T. cruzi em ambas as técnicas. Foi coletado sangue periférico em sete mães com seus respectivos filhos. As amostras de soro foram analisadas e os resultados confirmaram sorologia IgG anti-T. cruzi com índices e titulações semelhantes entre mães e seus respectivos RN, havendo concordância entre os resultados obtidos pela sorologia e o PF, validando esta estratégia na triagem dos RN de mães cronicamente infectadas. Este estudo salienta a importância de um acompanhamento pré-natal e neonatal que identifique precocemente a infecção por T. cruzi e também programas de conscientização sobre a DC na população de gestantes.

This work is part of a project focused on the study of the variability of human cytomegalovirus (HCMV) among clinical isolates with the aim to map the geographical distribution of HCMV genotypes, reveal the relationships between genotypes and the severity of HCMV-associated diseases, and identify regions in the HCMV genome with a potential for use as diagnostic and therapeutic targets. Attention was paid to the development of the methodology for the preparation of the material for next-generation sequencing (NGS) from HCMV clinical isolates and evaluation of the obtained sequencing data. Blood and urine samples collected from hematopoietic stem cell transplantat recipients and congenitally infected children were analyzed. Samples suitable for NGS were sequenced by the Illumina platform and sequences were created by de novo assembly followed by mapping assembly. Urine samples in comparison to blood samples had higher yield of material for NGS. Of the samples positive for HCMV DNA (7 of 50) after amplification in the cell cultures, only one sample had high purity of the viral DNA (98%) while six samples had purity of less than 7%. The sample containing 98% of the viral DNA was fully sequenced and the sequence was compared to the sequences of other clinical isolates from Belgium in 11 polymorphic...

Durant la grossesse, le fœtus est séparé de la mère par le placenta qui constitue une barrière protectrice efficace. Cependant, cette barrière n’est pas totalement imperméable et permet de nombreux échanges (nutriments, hormones, déchets, ...) dans des zones bien spécifiques nommées interfaces materno-fœtales. Au niveau de ces zones, les cellules fœtales entrent en contact direct avec le sang et les tissus maternels. Parmi ces interfaces, on trouve notamment la decidua basalis (paroi de l’endomètre gestant) où les villosités choriales du placenta s’ancrent profondément et l’espace intervilleux où les villosités flottantes sont baignées par le sang maternel. L’accès au placenta au niveau de ces interfaces est un processus finement régulé par de nombreux mécanismes. Cependant, certains pathogènes qui infectent la mère peuvent détourner ces mécanismes, franchir la barrière placentaire et se disséminer au fœtus. La famille des pathogènes TORCH (Toxoplasmosis, Others, Rubella, Cytomegalovirus et Herpes) est la plus connue pour induire des infections congénitales. Cependant d’autres virus moins connus ou émergents sont aussi capables d’infecter les interfaces mère-fœtus et de causer des complications graves pouvant être fatales pour la mère et le fœtus. Parmi ces virus, on retrouve notamment le virus de l’Hépatite E (VHE) et le virus Zika (ZIKV). C’est dans ce contexte que s’insèrent mes travaux de thèse qui s’articulent autour de trois axes. Dans la première partie de ma thèse, nous nous sommes intéressés à la pathogenèse du VHE et du ZIKV à l’interface mère-fœtus en identifiant les cibles cellulaires des virus et en caractérisant les conséquences fonctionnelles de l’infection. Dans la seconde partie, nous avons étudié la fonction des cellules Natural Killer déciduales (dNK), qui représentent 30% des cellules de la decidua basalis. Ces cellules dNK ne sont pas cytotoxiques durant une grossesse physiologique mais elles sécrètent de nombreux facteurs solubles essentiels au bon déroulement de la grossesse. Nous avons démontré que les fonctions effectrices des cellules dNK sont directement régulées et dictées par le microenvironnement décidual. De plus, nous avons découvert que les cellules dNK sont capables de détecter et de limiter l’infection des cellules stromales déciduales par le ZIKV. Enfin, dans la dernière partie, nous nous sommes intéressés à la pathogenèse de l’infection par le VHE dans un autre groupe de patients à haut risque de formes graves, les personnes âgées. Nous avons alors mis en évidence que le développement de formes sévères est associé à l’émergence d’une population de lymphocytes T CD8 caractérisée par un fort état d’activation associé à des défauts fonctionnels. En conclusion, mes travaux de thèse ont permis de mieux comprendre la pathogenèse du VHE et du ZIKV durant la grossesse et au-delà. De plus, ils ont participé à prouver l’importance du microenvironnement local dans le contrôle de la plasticité des cellules immunitaires. / During pregnancy, the fetus is isolated from the mother by the placenta, which constitutes an efficient protective barrier. However, this barrier is not completely impermeable and allows various exchanges (nutrients, hormones, wastes …) in specific areas called maternal-fetal interfaces. In these areas, fetal cells are in direct contact with maternal blood and tissues. Among these interfaces, we can distinguish the decidua basalis (gestating endometrium wall) where the placental chorionic villi are deeply anchored, and the intervillous space where the floating villi bathe in the maternal blood. The access to the placenta is a process tightly regulated by different mechanisms. However, some pathogens that infect the mother can subvert these mechanisms, cross the placental barrier, and spread to the fetus. The family of TORCH pathogens (Toxoplasmosis, Others, Rubella, Cytomegalovirus and Herpes) is best known for inducing such congenital infections. Alternatively, other less known or emerging viruses like Hepatitis E virus (HEV) and Zika virus (ZIKV) are also able to infect the maternal-fetal interface and cause severe outcomes that can be lethal for both the mother and the fetus. It’s in this context that fit my thesis work, articulated around three research axes. In the first part of my work, we focused on the pathogenesis of HEV and ZIKV at the maternal-fetal interface by identifying the cellular targets of the viruses and deciphering the functional consequences of their infection. Then, we studied the role of the decidual Natural Killer (dNK) cells, which account for 30% of total cells within the decidua basalis. These dNK cells are devoid of cytotoxic function in healthy conditions but they rather secrete various soluble factors that are essential for the success of pregnancy. In the second part of my work, we demonstrated that the decidual microenvironment dictates and regulates the effector functions of dNK cells. Moreover, we found that dNK cells are able to detect and limit the infection of decidual stromal cells by ZIKV. Finally, in a last part, we investigated the pathogenesis of HEV infection in another group of patients at high risk of developing serious forms, the elderly people. Thus, we highlighted that the development of severe forms is associated with the emergence of a population of CD8 T cells characterized by a high activation status associated with functional defects. In conclusion, my thesis work has shed light on the pathogenesis of HEV and ZIKV during pregnancy and beyond. In addition, they helped to demonstrate the importance of the local microenvironment in controlling the plasticity of immune cells.