West Nile virus (WNV) is a member of the Flaviviridae family and belongs to the Japanese encephalitis serocomplex (Petersen et al, 2013). In nature, WNV is maintained between mosquitos and birds as vectors and amplifying hosts, respectively. Humans and horses are incidental hosts and are infected with WNV by mosquito bites. The clinical symptoms of WNV infection in humans include febrile illness, fetal meningoencephalitis, and acute flaccid paralysis (Davis et al, 2006). No WNV-specific therapy is available, and vaccines against WNV are only approved for use in horses (Ulbert, 2019). This indicates the need to develop an effective and safe treatment and vaccine based on an understanding of the pathogenesis of WNV.
WNV infects neuronal cells in the central nervous system (CNS) and induces neuronal cell death through caspase 3-dependent apoptosis (Clarke et al, 2014; Peng and Wang, 2019; Stonedahl et al, 2020). In addition, the capsid protein of WNV inhibits autophagy and induces protein aggregation in WNV-infected neuronal cells, leading to neuronal cell death and inflammation involving activated microglia (Kobayashi et al, 2020; Quick et al, 2014; Stonedahl et al, 2020).
Microglia are the resident immune cells of the CNS. The roles of microglia are involved in homeostasis and host defense against pathogens (Hickman et al, 2018). Under physiological conditions, microglia are morphologically characterized by small and circular soma with an intensive branching process; this is termed the ramified phenotype (Jurga et al, 2020). After recognition of pathogens, microglia become activated; their morphology transforms from the ramified to the amoeboid phenotype, which is characterized by a rounder cell body with short and thick pseudopodia (Chen et al, 2019; Jurga et al, 2020; Quick et al, 2014). Activated microglia are observed in several neurodegenerative diseases, including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (Kenkhuis et al, 2022; Takahashi, 2023). In mouse studies, activated microglia degraded Aβ plaque, which is the distinctive pathological hallmark of AD, and inhibited neuronal cell death in the early stages (Feng et al, 2020; Kadowaki et al, 2005). In another study, however, activated microglia chronically produced excess proinflammatory cytokines and suppressed the degradation of Aβ plaque, leading to neuronal injury and AD progression (Wendimu and Hooks, 2022). These reports indicate that activated microglia exhibit conflicting functions. Activated microglia were previously classified into proinflammatory (M1) and anti-inflammatory (M2) phenotypes, but this classification is insufficient for a complete understanding of these conflicting functions (Hashemiaghdam and Mroczek, 2020). The recent development of a novel technique showed that the presence of diverse phenotypes of microglia influences the pathogenesis of AD in the brain (Stratoulias et al, 2019). In addition, specific microglia phenotypes such as plaque-associated microglia or disease-associated microglia reportedly affect the pathogenesis of AD (Hashemiaghdam and Mroczek, 2020; Keren-Shaul et al, 2017; Stratoulias et al, 2019). Hence, analysis of the diversity of microglia phenotypes and identification of disease-associated microglia may promote a better understanding of how microglia are involved in the pathogenesis.
Activated microglia are detected in brain tissue infected with WNV (Kobayashi et al, 2012; Stonedahl et al, 2020). However, the roles of activated microglia remain controversial because they may exhibit either neuroprotective or neuroinjury-related roles (Chhatbar and Prinz, 2021; Ghoshal et al, 2007; Malmlov et al, 2019; Seitz et al, 2018). Therefore, analysis of the diversity of microglia phenotypes and identification of disease-associated microglia may help to understand the neuropathogenesis of WNV infection. In this study, the diversity of microglia phenotypes caused by WNV infection was examined by detection of various microglia markers. Furthermore, disease-associated microglia were investigated in WNV-infected mouse brain tissue.