A infecção pelo vírus Zika induz alterações ultraestruturais, disfunção mitocondrial e estresse oxidativo em linhagem de células trofoblásticas humanas HTR-8/SVneo

Abstract

Zika fever gained importance in Brazil in 2015 due to the association between infection and the occurrence of congenital syndrome. Zika virus (ZIKV) crosses the placenta and infects the fetus; however, the pathogenesis of the disease remains incompletely understood. This study aimed to investigate the consequences of ZIKV infection in placental cells using the HTR-8/SVneo trophoblast cell line. Initially, an infection protocol was standardized by varying the multiplicity of infection (MOI) (0.1, 0.2, or 1) of the virus or Mock (control) for 24 or 48 hours. Cell viability was assessed through membrane integrity, and the number of infected cells was determined through immunofluorescence and flow cytometry. Infected and control cells were processed for transmission electron microscopy analysis. Mitochondrial membrane potential of infected cells was evaluated by flow cytometry, and the expression of genes involved in mitochondrial dynamics was analyzed by RT-qPCR. The antioxidant response was assessed by RT-qPCR and immunofluorescence targeting the genes and proteins: SOD, CAT, and NRF2, in addition to measuring the enzymatic activity of SOD and CAT. The presence of 8-OHdG was detected, and quantified reactive species such as malondialdehyde (MDA) and nitric oxide (NO) by immunofluorescence and/or colorimetric assays. Mitochondrial quality control was evaluated by detecting Parkin protein levels by immunofluorescence and pink1 gene expression by RT-qPCR. Immunofluorescence indicated that infection with ZIKV at an MOI of 1 for 24 hours resulted in the highest expression of the NS1 protein, establishing this as the optimal infection condition. This result was confirmed by flow cytometry, which showed the highest percentage of infected cells (62.53%). Moreover, cell viability was higher after 24 hours of infection (89%) compared to 48 hours (28.1%), confirming 24 hours as the optimal infection duration. Ultrastructural analysis of infected cells revealed mitochondrial damage, such as ruptured membranes and loss of cristae, as well as an abundant endoplasmic reticulum with dilated cisternae, clusters of viral particles, and the presence and secretion of vesicles. In contrast, control cells displayed preserved morphology. Infected cells also exhibited a loss of mitochondrial membrane potential and decreased transcript levels of genes involved in mitochondrial dynamics, indicating organelle impairment. Gene transcript analysis suggested reduced expression of sod, cat and nrf2 in infected cells. However, these cells showed increased immunolabeling of the corresponding proteins and elevated SOD activity, suggesting the activation of compensatory mechanisms. Conversely, CAT activity was reduced in infected cells, possibly due to enzyme exhaustion. Infected cells also presented significant increases in 8-OHdG, MDA, and NO levels, indicating oxidative stress-induced damage. Lastly, infected cells showed reduced Parkin protein levels and decreased pink1 expression, suggesting inhibition of mitophagy. These results indicate that ZIKV infection induces mitochondrial dysfunction and oxidative stress in HTR8-SV/neo trophoblastic cells, contributing to the understanding of the cellular mechanisms involved in the infection and elucidating its effects on placental cells.