Expression of microRNAs in trophoblasts infected \in vitro\ by Zika and Chikungunya viruses and experimental model of vertical transmission of Chikungunya virus in pregnancy and breastfeeding

Author(s)

Juliano de Paula Souza

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2021-09-29

Examining board members

Eurico de Arruda Neto; Jose Simon Camelo Junior; Marcelo Alves Pinto

• Advisor: Eurico de Arruda Neto

Abstract

In the present study, we investigated two unexplored aspects of the Zika virus (ZIKV) and Chikungunya virus (CHIKV) vertical transmission mechanisms during pregnancy and lactation. ZIKV is known to be teratogenic and able to infect several cells in the human placenta, including trophoblasts, which are cells intrinsically resistant to viruses by several mechanisms. The expression of microRNAs (miRNAs) that regulate antiviral responses is very important to sustain this resistance. The susceptibility of these cells to ZIKV and CHIKV and the impact of changes in the expression of host miRNAs was studied by new generation sequencing (NGS) in this work. Using the repertoire of miRNAs in infected cells, we carried out computational prediction of negatively and positively regulated pathways during viral infections. We identified the CREB3 regulatory factor (CREBRF) as a potential target of several up-regulated miRNAs, what has an impact on autophagy induction. The experimental validation was performed by silencing CREBRF with interference RNAs (RNAi). Interestingly, we identified distinct impacts of CREBRF silencing on ZIKV and CHIKV infections: the replication of ZIKV increased, while CHIKV suffered a significant reduction in its genome replication. These results indicate that these miRNAs can regulate pro or antiviral pathways, based on the distinct virus-cell interactions between them. There are several reports of vertical transmission of CHIKV, but little is known about routes and mechanisms of transmission. In the present study, an experimental model of CHIKV infection was developed, using female mice of the BALB/c strain infected during pregnancy and lactation, which enabled the confirmation of transplacental transmission in 8.4% of the analyzed fetuses, with induction of teratogenic effects. Taking advantage that most animals born and breastfed by CHIKV infected females survive, the profile of immune response of these animals throughout adulthood against this virus was evaluated. The results showed that mice breastfed in infected females develop active humoral and cellular immune responses against the virus in adulthood. More importantly, these animals are more resistant to experimental CHIKV infection 90 days after birth, indicating that the response induced by breastfeeding is effective and long lasting. We observed that the mice mammary glands are sites of replication for CHIKV, and the transmission of viral antigens to neonates via milk occurs. These results will be very important for future investigations about the basic biological mechanisms of vertical transmission of ZIKV and CHIKV, as well as for translational approaches in pregnant women and newborns infected by these agents. (AU)

• FAPESP's process: 16/06490-8 - The role of placental exosomes and microRNAs in the protection against the arboviruses Zika, Chikungunya and Oropouche
• Grantee: Juliano de Paula Souza
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)