O Zika Virus (ZIKV) e um flavivirus que causa comprometimento neurologico caracterizando a Sindrome Congenita do ZIKV (SCZ). Embora as celulas precursoras neuronais e os neurônios sejam as celulas mais afetadas, os astrocitos sao mais suscetiveis a infeccao pelo ZIKV, uma vez que toleram maiores cargas virais, sofrem menos apoptose e sao utilizados para replicação viral. Varios reguladores de processos biologicos estao envolvidos na suscetibilidade a SCZ, incluindo os microRNAs (miRNAs). Os miRNAs sao pequenos RNAs nao codificantes que interagem com sequencias alvo de mRNA complementares para repressao pos-transcricional. Nesse contexto, o objetivo principal e investigar as interacoes miRNAs-mRNA durante a transmissao vertical do ZIKV, com foco em astrocitos. Primeiramente, demonstramos que o ZIKV aumenta a expressao miRNAs no cerebro total de camundongos neonatos nascidos de maes infectadas, estimulando a autofagia. A regulacao positiva do miR-295 limita a expressão do gene regulador da autofagia Bcl2l11 in vivo e em astrocitos in vitro. Alem disso, comparando o perfil de miRNAs em astrocitos primarios de camundongos SJL, observamos o aumento da expressao dos miR-295 e miR-302d. Utilizando plataformas bioinformaticas, mimetizadores e inibidores dos miRNAs, e ensaio de luciferase, demonstramos que os genes Ahrr, Neurod4 e Neurod6 sao regulados negativamente pelos miR-295 e miR-302d. Curiosamente, os miR-295 e miR-302d sao transcritos juntamente com os genes Nlrp12 e Larp7, respectivamente. E para entender melhor o mecanismo usado pelo ZIKV para induzir estes miRNAs, avaliamos fatores que levam a transcricao dos genes Nlrp12 e Larp7. De modo interessante, o ZIKV aumentou a expressao dos fatores de transcricao CEBP/b e da subunidade p65 de NF-kB. Corroborando, o ZIKV induziu o recrutamento de CEBP/b para o promotor de Nlrp12 e Larp7 e p65 para o promotor de Larp7. Ainda, a inibicao de Rela e Cebpb por siRNAs, diminuiu Nlrp12, Larp7, premiR-295, premiR-302d, miR-295 e miR-302d, enquanto aumentou a expressao dos genes Ahrr, Neurod4 e Neurod6. Em seguida, demonstramos que a ativação dessa via descrita e um mecanismo de suscetibilidade a SCZ, uma vez que nao observamos aumento da expressao de Cebpb, Rela, Nlrp12, premiR-295, Larp7, premiR-302d, miR-295 e miR-302d, e dos seus genes alvos, Ahrr, Neurod4, Neurod6 em astrocitos de camundongos C57BL/6 WT resistentes a microcefalia causada pelo ZIKV. Dessa forma, demonstramos que o ZIKV ativa os fatores de transcricao C/EBPb e NF-kB para aumentar a transcricao de Nlrp12 e Larp7 e, consequentemente, dos miR-295 e miR-302d em astrocitos SJL. Estes miRNAs levam a diminuicao da expressao dos genes Ahrr, Neurod4 e Neurod6, contribuindo para o desenvolvimento de SCZ em camundongos SJL suscetiveis, mas nao em camundongos C57BL/6 WT resistentes. Em conjunto, sugerimos um mecanismo de suscetibilidade ao desenvolvimento da SCZ.

The Zika Virus (ZIKV) is a flavivirus that leads to neurological impairment characterizing the Congenital ZIKV Syndrome (CZS). Although neuronal precursor cells and neurons are the most affected cells, astrocytes are more susceptible to ZIKV infection, once tolerate higher viral loads, suffer less apoptosis and are used for viral replication. Curiously, it is known that several regulators of biological processes are involved in the susceptibility to CZS, including microRNAs (miRNAs). MiRNAs are small non-coding RNAs, which work downstream of transcription factors by interacting with complementary mRNA target sequences for further post-transcriptional repression. In this context, the principal aim is investigated miRNAsmRNA interactions during vertical transmission of ZIKV, focusing on astrocytes. First, we demonstrated that ZIKV increases miRNAs expression in the brain of mice neonates from infected mothers, stimulating autophagy. Upregulation of miR-295 limits the expression of Bcl2l11, an autophagy regulator gene, in vivo and in astrocytes in vitro. Furthermore, comparing miRNAs profile in SJL astrocytes, we observed an increased expression of miR-295 and miR-302d. Using bioinformatics platforms, miRNAs mimics and inhibitors, and luciferase assay, we demonstrated that Ahrr, Neurod4 and Neurod6 genes are downregulated by miR- 295 and miR-302d. Interestingly, miR-295 and miR-302d are transcribed along with Nlrp12 and Larp7 genes, respectively. To better understand the mechanism used by ZIKV to induce miRNAs, we evaluated transcription factors for Nlrp12 and Larp7 transcription. Interestingly, ZIKV increased the expression of CEBP/b and NF-kB p65 subunit. Corroborating, ZIKV induced the recruitment of CEBP/b to Nlrp12 and Larp7 promote, p65 to Larp7 promoter. Moreover, Rela and Cebpb inhibition by siRNAs decreased Nlrp12, Larp7, premiR-295, premiR-302d, miR- 295 and miR-302d, while increased Ahrr, Neurod4 and Neurod6 gene expression. Following, we demonstrate that the activation of this described pathway is a susceptibility mechanism to CZS, since we did not observe increased expression of Cebpb, Rela, Nlrp12, premiR-295, Larp7, premiR-302d, miR-295 and miR-302d, and their target genes, Ahrr, Neurod4, Neurod6 in astrocytes from C57BL/6 WT resistant mice to microcephaly caused by ZIKV. Thus, we demonstrate that ZIKV activates transcription factors C/EBPb and NF-kB to increase transcription of Nlrp12 and Larp7, and consequently, miR-295 and miR-302d in SJL astrocytes. These miRNAs lead to decreased expression of Ahrr, Neurod4 and Neurod6 genes, contributing to CZS development in susceptible SJL mice, but not in resistant C57BL/6 WT mice. Together, we suggest a mechanism of susceptibility to CZS development.