Human infection by different flaviviruses may cause severe neurologic syndromes, through pathogenic mechanisms that are still largely unknown. Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV), yellow fever virus (YFV), dengue virus (DENV), and tick-borne encephalitis virus (TBEV) are believed to reach the central nervous system by a hematogenous route, upon crossing the blood-brain barrier. Although the disruption of BBB during flavivirus infection has been largely evidenced in experimental models, the relevance of BBB breakdown for virus entering the brain was not completely elucidated. In vitro models of BBB had demonstrated that these viruses replicated in brain microvascular endothelial cells (BMECs), which induced downregulation of tight junction proteins and increased the permeability of the barrier. Other reports demonstrated that infection of BMECs allowed the basolateral release of infectious particles, without a remarkable cytopathic effect, what might be sufficient for virus invasion. Virus replication and activation of other cells associated to the BBB, mostly astrocytes and microglia, were also reported to affect the endothelial barrier permeability. This event might occur simultaneously or after BMECs infection, being a secondary effect leading to BBB disruption. Importantly, activation of BMECs, astrocytes, and microglia by flaviviruses was associated to the expression and secretion of inflammatory mediators, which are believed to recruit leukocytes to the CNS. The leukocyte infiltrate could further mediate viral invasion through a Trojan horse mechanism and might contribute to BBB breakdown and to neurological alterations. This review discussed the previous studies regarding in vitro and in vivo models of JEV, WNV, ZIKV, YFV, DENV, and TBEV infection and addressed the pathways for BBB overcome and invasion of the CNS described for each virus infection, aiming to increment the knowledge and stimulate further discussion about the role of BBB in the neuropathogenesis of flavivirus infection.

Zika virus (ZIKV) has been associated to central nervous system (CNS) harm, and virus was detected in the brain and cerebrospinal fluids of microcephaly and meningoencephalitis cases. However, the mechanism by which the virus reaches the CNS is unclear. Here, we addressed the effects of ZIKV replication in human brain microvascular endothelial cells (HBMECs), as an in vitro model of blood brain barrier (BBB), and evaluated virus extravasation and BBB integrity in an in vivo mouse experimental model. HBMECs were productively infected by African and Brazilian ZIKV strains (ZIKV_MR766 and ZIKV_PE243), which induce increased production of type I and type III IFN, inflammatory cytokines and chemokines. Infection with ZIKV_MR766 promoted earlier cellular death, in comparison to ZIKV_PE243, but infection with either strain did not result in enhanced endothelial permeability. Despite the maintenance of endothelial integrity, infectious virus particles crossed the monolayer by endocytosis/exocytosis-dependent replication pathway or by transcytosis. Remarkably, both viruses' strains infected IFNAR deficient mice, with high viral load being detected in the brains, without BBB disruption, which was only detected at later time points after infection. These data suggest that ZIKV infects and activates endothelial cells, and might reach the CNS through basolateral release, transcytosis or transinfection processes. These findings further improve the current knowledge regarding ZIKV dissemination pathways.

Protective adaptive immunity to Zika virus (ZIKV) has been mainly attributed to cytotoxic CD8⁺ T cells and neutralizing antibodies, while the participation of CD4⁺ T cells in resistance has remained largely uncharacterized. Here, we show a neutralizing antibody response, dependent on CD4⁺ T cells and IFNγ signaling, which we detected during the first week of infection and is associated with reduced viral load in the brain, prevention of rapid disease onset and survival. We demonstrate participation of these components in the resistance to ZIKV during primary infection and in murine adoptive transfer models of heterologous ZIKV infection in a background of IFNR deficiency. The protective effect of adoptively transferred CD4⁺ T cells requires IFNγ signaling, CD8⁺ T cells and B lymphocytes in recipient mice. Together, this indicates the importance of CD4⁺ T cell responses in future vaccine design for ZIKV.

In recent years, the Zika Virus (ZIKV) has caused pandemic outbreaks associated with a high rate of congenital ZIKV syndrome (CZS). Although all strains associated with worldwide outbreaks derive from the Asian lineage, the reasons for their enhanced spread and severity are not fully understood. In this study, we conducted a comparative analysis of miRNAs (miRNA-155/146a/124) and their cellular targets (SOCS1/3, SHP1, TRAF6, IRAK1), as well as pro- and anti-inflammatory and anti-viral cytokines (IL-6, TNF-α, IFN-γ, IL-10, and IFN-β) and peroxisome proliferator-activated receptor γ (PPAR-γ) expression in BV2 microglia cells infected with ZIKV strains derived from African and Asian lineages (ZIKV_MR766 and ZIKV_PE243). BV2 cells were susceptible to both ZIKV strains, and showed discrete levels of viral replication, with delayed release of viral particles without inducing significant cytopathogenic effects. However, the ZIKV_MR766 strain showed higher infectivity and replicative capacity, inducing a higher expression of microglial activation markers than the ZIKV_PE243 strain. Moreover, infection with the ZIKV_MR766 strain promoted both a higher inflammatory response and a lower expression of anti-viral factors compared to the ZIKV_PE243 strain. Remarkably, the ZIKK_PE243 strain induced significantly higher levels of the anti-inflammatory nuclear receptor—PPAR-γ. These findings improve our understanding of ZIKV-mediated modulation of inflammatory and anti-viral innate immune responses and open a new avenue to explore underlining mechanisms involved in the pathogenesis of ZIKV-associated diseases.

Endothelial dysfunction plays a central role in COVID-19 pathogenesis, by affecting vascular homeostasis and worsening thromboinflammation. This imbalance may contribute to blood–brain barrier (BBB) disruption, which has been reported in long COVID-19 patients with neurological sequelae. The kallikrein–kinin system (KKS) generates bradykinin (BK), a proinflammatory peptide that induces microvascular leakage via B2R. Under inflammatory conditions, BK is converted to Des-Arg-BK (DABK), which activates B1R, a receptor upregulated in inflamed tissues. DABK is degraded by ACE2, the main SARS-CoV-2 receptor; thus, viral binding and ACE2 downregulation may lead to DABK/B1R imbalance. Here, we investigated these interactions using human brain microvascular endothelial cells (HBMECs), as a model of the BBB. Since endothelial cell lines express low levels of ACE2, HBMECs were modified with an ACE2-carrying pseudovirus. SARS-CoV-2 replication was confirmed by RNA, protein expression, and infectious particles release. Infection upregulated cytokines and endothelial permeability, enhancing viral and leukocyte transmigration. Additionally, viral replication impaired ACE2 function in HBMECs, amplifying the response to DABK, increasing nitric oxide (NO) production, and further disrupting endothelial integrity. Our findings reveal a mechanism by which SARS-CoV-2 impacts the BBB and highlights the ACE2/KKS/B1R axis as a potential contributor to long COVID-19 neurological symptoms.