Estabelecimento de modelo experimental para síndrome congênita por vírus Zika utilizando embriões de galinha

Após a epidemia de casos de infecção por Zika vírus (ZIKV) e o subsequente aumento abrupto no número de crianças nascendo com microcefalia, a associação entre a exposição à infecção durante a gestação e o desenvolvimento dessas anomalias congênitas foi estabelecida. Assim, em 2016, o ZIKV foi definido como novo teratógeno humano, sendo o causador da Síndrome Congênita por ZIKV (SCZ). Apesar de muitos estudos terem sido realizados visando elucidar os mecanismos de ação do ZIKV, ainda há muitas perguntas sem resposta. Portanto, existe a necessidade do estabelecimento de modelos experimentais que sirvam como ferramenta para a maior compreensão dos mecanismos teratogênicos do vírus. Neste contexto, os embriões de galinha são uma excelente alternativa como como modelo experimental principalmente por apresentarem fácil manipulação e baixo custo. Dessa forma, o objetivo desse trabalho foi estabelecer um modelo experimental para a SCZ utilizando embriões de galinha. Para tanto, primeiramente foi realizada uma avaliação do efeito de diferentes doses virais de ZIKV para a seleção daquela ideal, ou seja, capaz de gerar malformações, porém sem causar altas taxas de mortalidade. A avaliação completa dos fenótipos relacionados ao desenvolvimento encefálico também foi realizada. Em seguida foram conduzidos experimentos de acompanhamento fenotípico dos embriões durante cinco dias após a infecção. A infecção viral foi realizada através de aplicação do vírus sobre o embrião nos estágios iniciais de desenvolvimento do sistema nervoso central (SNC). Embriões que foram expostos a infecção por ZIKV apresentaram maior taxa mortalidade, tamanho encefálico reduzido e maior taxa de malformações de SNC, seguindo um padrão dose-dependente. As malformações se tornaram mais evidentes no quarto dia após a infecção, mesmo tempo em que se observou um pico na carga viral. O estabelecimento desse novo modelo experimental utilizando embriões de galinha possibilita a realização de novos estudos visando compreender os mecanismos moleculares envolvidos na ação teratogênica do ZIKV. Além disso, esse modelo experimental terá uma contribuição importante para o desenvolvimento de estratégias de prevenção e resgate dos fenótipos causados pela exposição ao vírus, sendo possível testar o potencial preventivo de substâncias farmacológicas conhecidas ou em desenvolvimento.After the epidemy of Zika virus (ZIKV) and the following abrupt increase in the number of children born with microcephaly, the association between the exposure to the infection during pregnancy and the development of these malformations was established. Consequently, in 2016, ZIKV was identified as new human teratogen, being the causative agent of the Congenital ZIKV Syndrome (CZS). Despite there are many studies trying to better understand the mechanism related to CZS, there are still unanswered questions. Therefore, there is a need of establishing experimental models to be used as tools to a better understanding of the ZIKV teratogenic mechanisms. In this context, chicken embryos are an excellent alternative as experimental model, since they are easy to manipulate and cheap. Hence, the aim of this work was to establish an experimental model for CZS using chicken embryos. For that, firstly an evaluation of different ZIKV viral doses was performed to select the best dose, capable to induce to malformations without generating high rates of embryo mortality. A complete evaluation of the phenotypes after the exposure to ZIKV was also conducted. Then, phenotype follow-up was performed during five days post-infection. The viral infection was performed by applying the virus over the embryo in the initial stages of central nervous system (CNS) development. Embryos exposed to ZIKV infection presented high mortality rate, reduced brain size and a higher rate of CNS malformations, following a dose-dependent pattern. The malformations became more evident by day the fourth postinfection, at the same day when the ZIKV reached the peak of replication. By the establishment of this new experimental model for CZS using chicken embryos, other studies aiming to understand the molecular mechanisms involved in the ZIKV teratogenic action can be performed. Furthermore, this experimental model will contribute to the development of prevention and rescue approaches to the malformations caused by the exposure to ZIKV. Therefore, it will be possible to test the preventive potential of pharmacological substances already known or in development

Avaliação de polimorfismos nos genes TP53 e MDM2 ao longo de quatro gerações e sua influência na longevidade

A conquista da longevidade é dependente de uma complexa interação entre fatores genéticos, epigenéticos e ambientais. Estudos prévios sugerem que variantes no gene TP53 (Tumor protein p53) possam estar relacionadas com a longevidade. Neste estudo, avalimos o papel de polimorfismos nos genes TP53 e MDM2 (Mouse double minute 2 p53 binding protein homolog) – seu principal regulador – na longevidade em indivíduos de diferentes famílias com quatro gerações vivas da população da cidade de Veranópolis, RS. Assim, foram avaliadas as frequências genotípicas e alélicas do polimorfismo P72R (rs1042522) de TP53 e do SNP309 (rs2279744) de MDM2 em indivíduos de doze famílias distribuídos em quatro gerações. Comparações entre gerações e outros subgrupos não apresentaram diferença entre as frequências genotípicas e alélicas dos dois polimorfismos avaliados. Porém, quando consideradas apenas mulheres divididas em dois grupos (gerações 1 e 2 vs. gerações 3 e 4) as frequências alélicas de G (34,1% e 43,0%, respectivamente) do SNP309 de MDM2 apresentaram diferença (p = 0,04). O aumento da frequência do alelo G nas gerações 3 e 4 pode sugerir que este esteja sendo selecionado através de gerações em mulheres. Este alelo está associado ao aumento da atividade de MDM2, que regula negativamente p53, diminuindo, assim, sua atividade apoptotica. Assim, propomos um modelo de estudo transversal e transgeracional que considera várias famílias e suas gerações na investigação de fatores genéticos e ambientais que possam ter papel na longevidade.The achievement of longevity is dependent on a complex interaction between genetic, epigenetic and environmental factors. Previous studies suggest that variants in TP53 gene (Tumor protein p53) might be related to longevity. We evaluated the role of polymorphisms in TP53 gene and MDM2 (Mouse double minute 2 p53 binding protein homolog) gene- its main regulator- in longevity in individuals of different families with four alive generations of Veranópolis, RS. Thereby, genotypic and allelic frequencies of P72R polymorphism of TP53 (rs1042522) and the SNP309 of MDM2 (rs2279744) were evaluated in individuals from twelve families over four generations. Comparisons between generations and other subgroups did not present difference between genotypic and allelic frequencies of both evaluated polymorphisms. However, when considering only womens divided in two groups (1 and 2 generations vs. 3 and 4 generations) a significant difference of allelic frequency of G (34,1% e 43,0%, respectively) of SNP309 polymorphism of MDM2 was found (p = 0,04). The increase of G allele frequency in 3 and 4 generations might suggest this allele is being selected in the transmission of generations in womens. This allele is associated with increase of MDM2 activity, that negatively regulates p53, decreasing its apoptotic activity. Herein we propose a transversal and transgenerational study model that considers different families and their generations in the research of genetic and environmental factors that might have a role in longevity

From the farm to the lab: how chicken embryos contribute to the field of teratology

Funding This study received no specific grant from any funding agency in the public, commercial or not-for402 profit sectors. Acknowlegment We would like to thank all previous researches that established chicken embryos as a really important and respected experimental model to the teratology field through the history. NV lab funded by Royal Society, Wellcome Trust, Sarcoma UK, NIH. LRF lab funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant number 424362/2018-0], Fundo de Incentivo a Pesquisa e Eventos (FIPE) of the Hospital de Clínicas de Porto Alegre (HCPA) [grant numbers 2019-0649 and 2017-0342] and PROPESQ/UFRGS through “Recently Hired Professors” [Call 001/2019]. The authors would like to Sophia Martins Simon de Matos for technical assistance on Figure 1 drawing.Peer reviewedPublisher PD

From the farm to the lab : how chicken embryos contribute to the field of teratology

Congenital anomalies and its causes, particularly, by external factors are the aim of the field called teratology. The external factors studied by teratology are known as teratogens and can be biological or environmental factors for example, chemicals, medications, recreational drugs, environmental pollutants, physical agents (e.g., X-rays and maternal hyperthermia) and maternal metabolic conditions. Proving the teratogenicity of a factor is a difficult task requiring epidemiology studies as well as experimental teratology evidence from the use of animal models, one of which is the chicken embryo. This model in particular has the advantage of being able to follow development live and in vivo, with rapid development hatching around 21 days, is cheap and easy to manipulate and to observe development. All this allows the chicken embryo to be used in drug screening studies, teratogenic evaluation and studies of mechanisms of teratogenicity. The chicken embryo shares morphological, biochemical and genetic similarities with humans as well as mammalian species, making them ideal to ascertain the actions of teratogens, as well as screen drugs to test for their safety. Pre-clinical trials for new drugs are carried out in rodents and rabbits, however, chicken embryos have been used to screen new compounds or analogs of thalidomide as well as to investigate how some drugs can lead to congenital malformations. Indeed, the chicken embryo has proved valuable in understanding how many congenital anomalies, seen in humans, arise following teratogen exposure. The aim of this review is to highlight the role of the chicken embryo as an experimental model for studies in teratology, exploring its use in drug screening studies, phenotypic evaluation and studies of teratogenic mechanisms of action. Here, we discuss many known teratogens, that have been evaluated using the chicken embryo model including some medicines, such as, thalidomide, valproic acid; recreational drugs including alcohol; environmental influences, such as viruses, specifically ZIKV, which is a newly discovered human teratogen. In addition, we discuss how the chicken embryo has provided insight on the mechanisms of teratogenesis of many compounds and also how this impact on drug safety

Molecular mechanisms of Zika virus teratogenesis from animal studies : a systematic review protocol

Background: Due to the diversity of studies in animal models reporting that molecular mechanisms are involved in the teratogenic effect of the Zika virus (ZIKV), the objective of the present study is to evaluate the methodological quality of these studies, as well as to demonstrate which genes and which molecular pathways are affected by ZIKV in different animal models. Methods: This search will be performed in four databases: PubMed/MEDLINE, EMBASE, Web of Science, and Scopus, as well as in the grey literature. The studies selection process will be reported through the PRISMA Statement diagram model. All studies describing the molecular mechanisms possibly involved in the development of malformations caused by embryonic/fetal ZIKV exposure in animal models with an appropriate control group and methodology will be included (including, for instance, randomized and non-randomized studies). All animals used as experimental models for ZIKV teratogenesis may be included as long as exposure to the virus occurred during the embryonic/fetal period. From the selected studies, data will be extracted using a previously prepared standard form. Bias risk evaluation will be conducted following the SYRCLE’s Risk of Bias tool. All data obtained will be tabulated and organized by outcomes (morphological and molecular). Discussion: With the proposed systematic review, we expect to present results about the methodological quality of the published studies with animal models that investigated the molecular mechanisms involved in the teratogenic effect of ZIKV, as well as to show the studies with greater reliability