Human microglial models to study host-virus interactions.

Microglia, the resident macrophage of the central nervous system, are increasingly recognized as contributing to diverse aspects of human development, health, and disease. In recent years, numerous studies in both mouse and human models have identified microglia as a "double edged sword" in the progression of neurotropic viral infections: protecting against viral replication and cell death in some contexts, while acting as viral reservoirs and promoting excess cellular stress and cytotoxicity in others. It is imperative to understand the diversity of human microglial responses in order to therapeutically modulate them; however, modeling human microglia has been historically challenging due to significant interspecies differences in innate immunity and rapid transformation upon in vitro culture. In this review, we discuss the contribution of microglia to the neuropathogenesis of key neurotropic viral infections: human immunodeficiency virus 1 (HIV-1), Zika virus (ZIKV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Herpes simplex virus (HSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We pay special attention to recent work with human stem cell-derived microglia and propose strategies to leverage these powerful models to further uncover species- and disease-specific microglial responses and novel therapeutic interventions for neurotropic viral infections.

Human Brain Organoids as Models for Central Nervous System Viral Infection.

Pathogenesis of viral infections of the central nervous system (CNS) is poorly understood, and this is partly due to the limitations of currently used preclinical models. Brain organoid models can overcome some of these limitations, as they are generated from human derived stem cells, differentiated in three dimensions (3D), and can mimic human neurodevelopmental characteristics. Therefore, brain organoids have been increasingly used as brain models in research on various viruses, such as Zika virus, severe acute respiratory syndrome coronavirus 2, human cytomegalovirus, and herpes simplex virus. Brain organoids allow for the study of viral tropism, the effect of infection on organoid function, size, and cytoarchitecture, as well as innate immune response; therefore, they provide valuable insight into the pathogenesis of neurotropic viral infections and testing of antivirals in a physiological model. In this review, we summarize the results of studies on viral CNS infection in brain organoids, and we demonstrate the broad application and benefits of using a human 3D model in virology research. At the same time, we describe the limitations of the studies in brain organoids, such as the heterogeneity in organoid generation protocols and age at infection, which result in differences in results between studies, as well as the lack of microglia and a blood brain barrier.

Imaging of Virus-Infected Cells with Soft X-ray Tomography.

Viruses are obligate parasites that depend on a host cell for replication and survival. Consequently, to fully understand the viral processes involved in infection and replication, it is fundamental to study them in the cellular context. Often, viral infections induce significant changes in the subcellular organization of the host cell due to the formation of viral factories, alteration of cell cytoskeleton and/or budding of newly formed particles. Accurate 3D mapping of organelle reorganization in infected cells can thus provide valuable information for both basic virus research and antiviral drug development. Among the available techniques for 3D cell imaging, cryo-soft X-ray tomography stands out for its large depth of view (allowing for 10 µm thick biological samples to be imaged without further thinning), its resolution (about 50 nm for tomographies, sufficient to detect viral particles), the minimal requirements for sample manipulation (can be used on frozen, unfixed and unstained whole cells) and the potential to be combined with other techniques (i.e., correlative fluorescence microscopy). In this review we describe the fundamentals of cryo-soft X-ray tomography, its sample requirements, its advantages and its limitations. To highlight the potential of this technique, examples of virus research performed at BL09-MISTRAL beamline in ALBA synchrotron are also presented.

In Silico Analysis of Possible Interaction between Host Genomic Transcription Factors (TFs) and Zika Virus (ZikaSPH2015) Strain with Combinatorial Gene Regulation; Virus Versus Host-The Game Reloaded.

In silico analysis is a promising approach for understanding biological events in complex diseases. Herein we report on the innovative computational workflow allowed to highlight new direct interactions between human transcription factors (TFs) and an entire genome of virus ZikaSPH2015 strain in order to identify the occurrence of specific motifs on a genomic Zika Virus sequence that is able to bind and, therefore, sequester host's TFs. The analysis pipeline was performed using different bioinformatics tools available online (free of charge). According to obtained results of this in silico analysis, it is possible to hypothesize that these TFs binding motifs might be able to explain the complex and heterogeneous phenotype presentation in Zika-virus-affected fetuses/newborns, as well as the less severe condition in adults. Moreover, the proposed in silico protocol identified thirty-three different TFs identical to the distribution of TFBSs (Transcription Factor Binding Sites) on ZikaSPH2015 strain, potentially able to influence genes and pathways with biological functions confirming that this approach could find potential answers on disease pathogenesis.