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.

Zika virus (ZIKV) poses a serious threat to global public health due to its close relationship with neurological and male reproductive damage. However, deficiency of human testicular samples hinders the in-depth research on ZIKV-induced male reproductive system injury. Organoids are relatively simple in vitro models, which could mimic the pathological changes of corresponding organs. In this study, we constructed a 3D testicular organoid model using primary testicular cells from adult BALB/c mice. Similar to the testis, this organoid system has a blood-testis barrier (BTB)-like structure and could synthesize testosterone. ZIKV tropism of testicular cells and ZIKV-induced pathological changes in testicular organoid was also similar to that in mammalian testis. Therefore, our results provide a simple and reproducible in vitro testicular model for the investigations of ZIKV-induced testicular injury.

Autophagy is a conserved lysosomal degradation process, and abnormal autophagy has been associated with various pathological processes, e.g., neurodegeneration, cancer, and pathogen infection. Small chemical modulators of autophagy show the potential to treat autophagy-associated diseases. Diterpenoids, nature products found in various plants, exhibit a wide range of bioactivity, and we have recently isolated and characterized over 150 diterpenoids from Isodon species distributed in China. Here, we applied a high-content fluorescence imaging-based assay to assess these diterpenoids\' ability to affect autophagic flux in HeLa cells. We found that enanderinanin J, an ent-kauranoid dimer, is an autophagy inhibitor, manifested by its ability to increase lysosomal pH and inhibit the fusion between autophagosomes and lysosomes. Autophagy has been shown to be either positively or negatively involved in the life cycle of Zika virus (ZIKV), Japanese encephalitis virus (JEV), Dengue virus (DENV), and enterovirus-A71 (EV-A71). We found that enanderinanin J significantly inhibited the infection of ZIKV, DENV, JEV, or EV-A71. Interestingly, although ATG5 knockdown inhibited ZIKV or JEV infection, enanderinanin J further inhibited the infection of ZIKV or JEV in ATG5-knockdown cells. Taken together, our data indicate that enanderinanin J inhibits autophagosome-lysosome fusion and is a potential antiviral agent.

Mosquito-borne flaviviruses can cause disease in the nervous system, resulting in a significant burden of morbidity and mortality. Disease models are necessary to understand neuropathogenesis and identify potential therapeutics and vaccines. Non-human primates have been used extensively but present major challenges. Advances have also been made toward the development of humanized mouse models, but these models still do not fully represent human pathophysiology. Recent developments in stem cell technology and cell culture techniques have allowed the development of more physiologically relevant human cell-based models. In silico modeling has also allowed researchers to identify and predict transmission patterns and discover potential vaccine and therapeutic candidates. This review summarizes the research on in vitro and in silico models used to study three mosquito-borne flaviviruses that cause neurological disease in humans: West Nile, Dengue, and Zika. We also propose a roadmap for 21st century research on mosquito-borne flavivirus neuropathogenesis, prevention, and treatment.