Beneficial and detrimental functions of microglia during viral encephalitis

During homeostasis, microglia are the most abundant immune cell type within the central nervous system. Different microglia subsets in different brain areas maintain brain function and integrity.During virus-induced encephalitis, microglia are essential for antiviral defence and protection of the brain.Microglia activation and antiviral defence are regulated by crosstalk between neurons, astrocytes, and other cell types in the brain.Microglia activation may have detrimental effects by causing direct or indirect loss of neurons and disturbance of tissue integrity, eventually causing long-term sequelae.Innovative methodologies are being used to study microglial function in experimental and clinical settings to refine concepts about the roles of microglia in viral encephalitis.Improved understanding of microglial function in the virus-infected brain is essential for the development of novel treatments for viral encephalitis. Microglia are resident immune cells of the central nervous system (CNS) with multiple functions in health and disease. Their response during encephalitis depends on whether inflammation is triggered in a sterile or infectious manner, and in the latter case on the type of the infecting pathogen. Even though recent technological innovations advanced the understanding of the broad spectrum of microglia responses during viral encephalitis (VE), it is not entirely clear which microglia gene expression profiles are associated with antiviral and detrimental activities. Here, we review novel approaches to study microglia and the latest concepts of their function in VE. Improved understanding of microglial functions will be essential for the development of new therapeutic interventions for VE. Microglia are resident immune cells of the central nervous system (CNS) with multiple functions in health and disease. Their response during encephalitis depends on whether inflammation is triggered in a sterile or infectious manner, and in the latter case on the type of the infecting pathogen. Even though recent technological innovations advanced the understanding of the broad spectrum of microglia responses during viral encephalitis (VE), it is not entirely clear which microglia gene expression profiles are associated with antiviral and detrimental activities. Here, we review novel approaches to study microglia and the latest concepts of their function in VE. Improved understanding of microglial functions will be essential for the development of new therapeutic interventions for VE. Microglia are resident myeloid cells of the CNS. They are part of the innate immune system and have miscellaneous functions. Microglia are considered glial cells and mononuclear phagocytes. They are involved in pathogen recognition as well as in the initiation and maintenance of local immune responses. Microglia have been shown to be crucially involved in orchestrating responses to viral infections of the CNS, which are mostly associated with brain inflammation and accordingly are often referred to as 'viral encephalitis' (VE) [1.Rock R.B. et al.Role of microglia in central nervous system infections.Clin. Microbiol. Rev. 2004; 17: 942-964Crossref PubMed Scopus (504) Google Scholar,2.Chhatbar C. Prinz M. The roles of microglia in viral encephalitis: from sensome to therapeutic targeting.Cell. Mol. Immunol. 2021; 18: 250-258Crossref PubMed Scopus (7) Google Scholar]. Under homeostatic conditions, microglia contribute to the maintenance of brain plasticity and function by supporting synaptic wiring and spatial patterning of the developing and adult CNS [3.Schafer D.P. Stevens B. Microglia function in central nervous system development and plasticity.Cold Spring Harb. Perspect. Biol. 2015; 7a020545Crossref PubMed Scopus (167) Google Scholar, 4.Ransohoff R.M. Cardona A.E. The myeloid cells of the central nervous system parenchyma.Nature. 2010; 468: 253-262Crossref PubMed Scopus (576) Google Scholar, 5.Ransohoff R.M. El Khoury J. Microglia in health and disease.Cold Spring Harb. Perspect. Biol. 2015; 8a020560PubMed Google Scholar, 6.Prinz M. Priller J. The role of peripheral immune cells in the CNS in steady state and disease.Nat. Neurosci. 2017; 20: 136-144Crossref PubMed Scopus (274) Google Scholar]. In some situations, myeloid cells other than microglia, such as peripheral macrophages and dendritic cells, are recruited to the CNS. Although microglia share several characteristics with infiltrating myeloid cells in the CNS, microglia are unique in terms of their ontogeny. Fate-mapping studies and developmental analyses revealed that microglia are yolk sac derived. Specifically, yolk sac‐derived myeloid precursors (i.e., mesodermal progenitors) differentiate to yolk sac‐derived macrophages that, during early embryonic development, travel to the brain, where they differentiate to immature microglia [7.Butovsky O. et al.Identification of a unique TGF-β dependent molecular and functional signature in microglia.Nat. Neurosci. 2014; 17: 131-143Crossref PubMed Scopus (627) Google Scholar, 8.Bruttger J. et al.Genetic cell ablation reveals clusters of local self-renewing microglia in the mammalian central nervous system.Immunity. 2015; 43: 92-106Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 9.Ginhoux F. et al.Origin and differentiation of microglia.Front. Cell. Neurosci. 2013; 7: 45Crossref PubMed Scopus (430) Google Scholar, 10.Li Q. Barres B.A. Microglia and macrophages in brain homeostasis and disease.Nat. Rev. Immunol. 2018; 18: 225-242Crossref PubMed Scopus (556) Google Scholar]. Microglia also share functions and phenotypes with other macrophages located within the CNS, including perivascular, meningeal, and choroid plexus macrophages, which are also referred to as 'border-associated macrophages' and 'CNS-associated macrophages' (BAMs and CAMs, respectively) [11.Jordão M.J.C. et al.Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation.Science. 2019; 363eaat7554Crossref PubMed Scopus (246) Google Scholar,12.Van Hove H. et al.A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment.Nat. Neurosci. 2019; 22: 1021-1035Crossref PubMed Scopus (220) Google Scholar]. Therefore, particularly under inflammatory conditions, it is difficult to unambiguously classify different myeloid cell subsets within the CNS. Recent transcriptional analyses revealed genes that are specifically expressed by microglia and that allow classification of myeloid cells. Core signature genes of homeostatic BAMs/CAMs comprise Mrc1, Pf4, Ms4a7, Stab1, Cbr2, and CD163, whereas homeostatic microglia express Hexb, Cx3cr1, Csf1r, P2ry12, Tmem119, Gpr34, Tgfbr1, Fcrls, Siglech, Slc2a5, and Sall1 (see following text) [11.Jordão M.J.C. et al.Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation.Science. 2019; 363eaat7554Crossref PubMed Scopus (246) Google Scholar, 12.Van Hove H. et al.A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment.Nat. Neurosci. 2019; 22: 1021-1035Crossref PubMed Scopus (220) Google Scholar, 13.Schwabenland M. et al.Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T-cell interactions.Immunity. 2021; 54: 1594-1610.e11Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. Since microglia play multiple roles during health and disease, there is tremendous interest in better understanding their diverse functions, particularly during VE. Here, we review concepts of microglial functions during VE, including insights from recent studies gained through novel methodologies in in vivo as well as ex vivo settings. Microglial biology and functions have been studied for many decades, which brought up essential understanding of this unique brain-resident immune cell type (reviewed in [14.Prinz M. et al.Microglia biology: one century of evolving concepts.Cell. 2019; 179: 292-311Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar]). Notable methodologies in the study of microglia include immunohistochemical visualisation and flow cytometric analysis, as well as imaging of microglia both in vitro and in vivo. Recent progress in microglia classification using the aforementioned methods allowed an improved understanding of microglial morphology (e.g., by quantitative analysis of ramification and cell shape), activation status (e.g., by analysis of cell surface marker expression profiles and cell motility), and functions (e.g., by cell–cell interaction analysis by in vivo imaging). However, differentiating microglia from other myeloid cell subsets within the CNS continues to be challenging, underscoring the need for further methodology refinement. Substantial efforts have been undertaken to generate transgenic mouse lines showing microglia-selective Cre expression in order to target genes of interest selectively in this cell type. The most robust and frequently used models are conventional Cx3cr1-Cre and tamoxifen-inducible Cx3cr1-CreERT2 mice, both expressing Cre under the promoter of the Cx3 chemokine receptor 1 gene (Cx3cr1) [15.Jung S. et al.Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.Mol. Cell. Biol. 2000; 20: 4106-4114Crossref PubMed Scopus (1723) Google Scholar]. Even though microglia show more abundant CX3CR1 expression than other myeloid cells, the expression of this receptor is not strictly limited to microglia, highlighting an important limitation of these models [16.Prinz M. et al.Heterogeneity of CNS myeloid cells and their roles in neurodegeneration.Nat. Neurosci. 2011; 14: 1227-1235Crossref PubMed Scopus (478) Google Scholar, 17.Goldmann T. et al.A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation.Nat. Neurosci. 2013; 16: 1618-1626Crossref PubMed Scopus (399) Google Scholar, 18.Masuda T. et al.Novel Hexb-based tools for studying microglia in the CNS.Nat. Immunol. 2020; 21: 802-815Crossref PubMed Scopus (49) Google Scholar, 19.Goldmann T. et al.Origin, fate and dynamics of macrophages at central nervous system interfaces.Nat. Immunol. 2016; 17: 797-805Crossref PubMed Scopus (483) Google Scholar]. This limitation can be overcome by using Cx3cr1-CreERT2 mice 8 weeks after tamoxifen treatment, when bone marrow-derived myeloid cells have been renewed by bone marrow-derived precursors, whereas microglia did not change, because they are not bone marrow derived and renew locally at extremely slow rates (for the most part) [20.Yona S. et al.Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.Immunity. 2013; 38: 79-91Abstract Full Text Full Text PDF PubMed Scopus (1575) Google Scholar]. Another mouse line was generated based on inducible Cre expression under the control of the Sall1 promoter, which was described as being microglia specific [21.Buttgereit A. et al.Sall1 is a transcriptional regulator defining microglia identity and function.Nat. Immunol. 2016; 17: 1397-1406Crossref PubMed Google Scholar]. However, especially under varying conditions, the specificity and stability of Sall1 expression in microglia has not yet been fully validated. More recently, two additional transgenic mouse lines based on microglia-specific genes have been generated. In one of these lines, P2ry12-CreER mice, an inducible Cre was inserted into the gene of the purinergic receptor P2Y12 (P2ry12), which has been reported to be expressed during the entire lifespan of microglia [22.McKinsey G.L. et al.A new genetic strategy for targeting microglia in development and disease.eLife. 2020; 9e54590Crossref PubMed Scopus (30) Google Scholar]. The other transgenic line is based on the hexosaminidase subunit beta (Hexb) gene, which is specifically expressed by microglia. In brief, in these mice, the Hexb locus is modified in such a manner that (i) the open reading frame of Hexb is replaced by a cassette consisting of a T2A peptide that has self-cleaving activity and that is combined with the tdTomato reporter (T2A-tdTomato) (HexbtdTomato) or (ii) the T2A-CreERT2 cassette is introduced into the Hexb locus directly before the Hexb stop codon (HexbCreERT2) [18.Masuda T. et al.Novel Hexb-based tools for studying microglia in the CNS.Nat. Immunol. 2020; 21: 802-815Crossref PubMed Scopus (49) Google Scholar]. In the first case, the Cre-expressing allele is associated with a Hexb knockout, whereas in the second case, translation of the resulting polycistronic RNA results in a fusion protein that, due to the self-cleaving activity of the T2A peptide, gives rise to HEXB and the CreERT protein. Despite the potential of these two mouse lines to address microglia's roles in a wide range of scenarios, their utility in the context of infectious and noninfectious encephalitis models remains to be proven. Thus, depending on the scientific question at hand, each of the various aforementioned transgenic mouse lines can be useful to study certain aspects of microglia's phenotype and function during development, health, and disease. In recent years, single-cell RNA sequencing (scRNA-seq) provided deeper insight into the transcriptomic profiles of microglia [11.Jordão M.J.C. et al.Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation.Science. 2019; 363eaat7554Crossref PubMed Scopus (246) Google Scholar,12.Van Hove H. et al.A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment.Nat. Neurosci. 2019; 22: 1021-1035Crossref PubMed Scopus (220) Google Scholar,18.Masuda T. et al.Novel Hexb-based tools for studying microglia in the CNS.Nat. Immunol. 2020; 21: 802-815Crossref PubMed Scopus (49) Google Scholar,23.Hammond T.R. et al.Single-cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes.Immunity. 2019; 50: 253-271.e6Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar]. One of the main conclusions from these studies is that, under homeostatic conditions, microglia consist of at least two different subpopulations [23.Hammond T.R. et al.Single-cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes.Immunity. 2019; 50: 253-271.e6Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 24.Borst K. et al.Microglia: immune and non-immune functions.Immunity. 2021; 54: 2194-2208Abstract Full Text Full Text PDF PubMed Google Scholar, 25.Li Q. et al.Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing.Neuron. 2019; 101: 207-223.e10Abstract Full Text Full Text PDF PubMed Google Scholar, 26.Prinz M. et al.Microglia and central nervous system-associated macrophages – from origin to disease modulation.Annu. Rev. Immunol. 2021; 39: 251-277Crossref PubMed Scopus (0) Google Scholar, 27.Olah M. et al.Single cell RNA sequencing of human microglia uncovers a subset associated with Alzheimer's disease.Nat. Commun. 2020; 11: 6129Crossref PubMed Scopus (59) Google Scholar]. In contrast, during sterile and infectious neuroinflammation as well as during aging, microglia show a high extent of plasticity and heterogeneity. Depending on the context in which microglia are activated, they may show major changes in their transcriptomic profiles, and even their core signature genes, which until recently were considered to be stably expressed under all conditions, can be downmodulated. Earlier studies proposed that M1 and M2 microglia polarisation is of key relevance for the control of VE (Box 1). However, the concept of M1/M2 polarisation has been challenged [28.Ransohoff R.M. A polarizing question: do M1 and M2 microglia exist?.Nat. Neurosci. 2016; 19: 987-991Crossref PubMed Scopus (723) Google Scholar, 29.Colonna M. Butovsky O. Microglia function in the central nervous system during health and neurodegeneration.Annu. Rev. Immunol. 2017; 35: 441-468Crossref PubMed Scopus (621) Google Scholar, 30.Dumas A.A. et al.Current tools to interrogate microglial biology.Neuron. 2021; 109: 2805-2819Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar], and inspection of transcriptional profiles of microglia during viral infection generally does not support the concept of M1/M2 polarisation [13.Schwabenland M. et al.Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T-cell interactions.Immunity. 2021; 54: 1594-1610.e11Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar,31.Syage A.R. et al.Single-cell RNA sequencing reveals the diversity of the immunological landscape following central nervous system infection by a murine coronavirus.J. Virol. 2020; 94e01295-20Crossref PubMed Scopus (0) Google Scholar,32.Huang K.W. Sabatini B.L. Single-cell analysis of neuroinflammatory responses following intracranial injection of G-deleted rabies viruses.Front. Cell. Neurosci. 2020; 14: 65Crossref PubMed Scopus (2) Google Scholar]. Clarifying how precisely microglia respond to viral CNS infection, including in terms of transcriptional profiles, will require further investigations.Box 1M1 and M2 polarisation of microglia during VE?Early studies proposed the polarisation of microglia into proinflammatory M1 microglia, which are CD14-, CD16-, CD32-, CD40-, CD86-, MHC-II-, translocator protein-, and inducible nitric oxide synthase-positive, and anti-inflammatory M2 microglia expressing CD163, CD206, Arg1, and other markers [108.Jurga A.M. et al.Overview of general and discriminating markers of differential microglia phenotypes.Front. Cell. Neurosci. 2020; 14: 198Crossref PubMed Scopus (57) Google Scholar]. In the context of VE, it has been shown, for instance, that upon WNV infection of the CNS, microglia are activated and display upregulation of certain M1-like markers, such as Iba-1, as well as chemokines such as CCL2, CCL3, CCL5, and CCL7 [109.Quick E.D. et al.Activation of intrinsic immune responses and microglial phagocytosis in an ex vivo spinal cord slice culture model of West Nile virus infection.J. Virol. 2014; 88: 13005-13014Crossref PubMed Scopus (37) Google Scholar]. In contrast, treatment with the anti-inflammatory drug minocycline reduced the expression of M1-like markers and increased the expression of M2-like markers. Such M2-like microglia were associated with neuroprotective effects in WNV infection of the CNS [110.Quick E.D. et al.Minocycline has anti-inflammatory effects and reduces cytotoxicity in an ex vivo spinal cord slice culture model of West Nile virus infection.J. Virol. 2017; 91e00569-17Crossref PubMed Scopus (19) Google Scholar], suggesting that M1-like microglia are needed for the initial control of WNV infection, whereas M2-like microglia prevent excessive tissue damage during CNS inflammation. However, in recent years, the relevance of the M1/M2 microglial polarisation paradigm has been challenged [28.Ransohoff R.M. A polarizing question: do M1 and M2 microglia exist?.Nat. Neurosci. 2016; 19: 987-991Crossref PubMed Scopus (723) Google Scholar], and in particular, transcriptomic analyses of microglia did not indicate a clear separation into M1- and M2-like microglia (see also main text). Early studies proposed the polarisation of microglia into proinflammatory M1 microglia, which are CD14-, CD16-, CD32-, CD40-, CD86-, MHC-II-, translocator protein-, and inducible nitric oxide synthase-positive, and anti-inflammatory M2 microglia expressing CD163, CD206, Arg1, and other markers [108.Jurga A.M. et al.Overview of general and discriminating markers of differential microglia phenotypes.Front. Cell. Neurosci. 2020; 14: 198Crossref PubMed Scopus (57) Google Scholar]. In the context of VE, it has been shown, for instance, that upon WNV infection of the CNS, microglia are activated and display upregulation of certain M1-like markers, such as Iba-1, as well as chemokines such as CCL2, CCL3, CCL5, and CCL7 [109.Quick E.D. et al.Activation of intrinsic immune responses and microglial phagocytosis in an ex vivo spinal cord slice culture model of West Nile virus infection.J. Virol. 2014; 88: 13005-13014Crossref PubMed Scopus (37) Google Scholar]. In contrast, treatment with the anti-inflammatory drug minocycline reduced the expression of M1-like markers and increased the expression of M2-like markers. Such M2-like microglia were associated with neuroprotective effects in WNV infection of the CNS [110.Quick E.D. et al.Minocycline has anti-inflammatory effects and reduces cytotoxicity in an ex vivo spinal cord slice culture model of West Nile virus infection.J. Virol. 2017; 91e00569-17Crossref PubMed Scopus (19) Google Scholar], suggesting that M1-like microglia are needed for the initial control of WNV infection, whereas M2-like microglia prevent excessive tissue damage during CNS inflammation. However, in recent years, the relevance of the M1/M2 microglial polarisation paradigm has been challenged [28.Ransohoff R.M. A polarizing question: do M1 and M2 microglia exist?.Nat. Neurosci. 2016; 19: 987-991Crossref PubMed Scopus (723) Google Scholar], and in particular, transcriptomic analyses of microglia did not indicate a clear separation into M1- and M2-like microglia (see also main text). Another strategy to perform microglia-specific gene profiling is the RiboTag approach. This strategy relies on Cre recombinase‐induced expression of a haemagglutinin tag in conjunction with the major ribosomal subunit (Rpl22 gene) [33.Sanz E. et al.Cell-type-specific isolation of ribosome-associated mRNA from complex tissues.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 13939-13944Crossref PubMed Scopus (438) Google Scholar]. Crossing this mouse line with a microglia-specific Cre line (e.g., Cx3cr1-CreERT) allows pull-down of microglia-derived ribosomes from whole-brain lysates. Following isolation of mRNA attached to tagged ribosomes, the microglia-associated translatome can be studied [34.Haimon Z. et al.Re-evaluating microglia expression profiles using RiboTag and cell isolation strategies.Nat. Immunol. 2018; 19: 636-644Crossref PubMed Scopus (81) Google Scholar]. By using this approach, cell isolation techniques that might affect gene expression profiles of the cells of interest can be avoided. Considering the aforementioned methodologies that enhanced microglia understanding under various conditions, this review focuses on new developments in elucidating the roles of microglia under homeostatic conditions as well as in sterile inflammation and VE, with a special emphasis on new methodologies (e.g., transcriptomics). Notably, only few studies using such methodologies to investigate microglia upon viral CNS infection are currently available. Microglia are the most abundant resident phagocytic cell type within the brain and represent approximately 10% of all cells in the CNS [4.Ransohoff R.M. Cardona A.E. The myeloid cells of the central nervous system parenchyma.Nature. 2010; 468: 253-262Crossref PubMed Scopus (576) Google Scholar,5.Ransohoff R.M. El Khoury J. Microglia in health and disease.Cold Spring Harb. Perspect. Biol. 2015; 8a020560PubMed Google Scholar]. During homeostatic conditions in the healthy CNS, microglia constantly scavenge the tissue and support neuronal functions, thus maintaining neuronal networks and connectivity [35.Paolicelli R.C. et al.Synaptic pruning by microglia is necessary for normal brain development.Science. 2011; 333: 1456-1458Crossref PubMed Scopus (1998) Google Scholar,36.Schafer D.P. et al.Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.Neuron. 2012; 74: 691-705Abstract Full Text Full Text PDF PubMed Scopus (1870) Google Scholar]. During normal brain function, the main roles of microglia are removal of cell debris, synaptic engulfment and pruning, myelin homeostasis, and tissue surveillance. Morphologically, homeostatic microglia are typically small, rod-shaped cells with numerous thin and highly ramified processes [37.Nimmerjahn A. et al.Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.Science. 2005; 308: 1314-1318Crossref PubMed Scopus (3515) Google Scholar]. Despite their homeostatic status, these microglial cells are not resting. On the contrary, they are continuously scanning and monitoring the proximal tissue to get alerted in case of detection of any irregularity. Notably, microglia express several core genes, which are specifically but not exclusively expressed in this cell type, such as Hexb, Cx3cr1, Csf1r, P2ry12, Tmem119, Gpr34, Tgfbr1, Fcrls, Siglech, Slc2a5, and Sall1 [11.Jordão M.J.C. et al.Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation.Science. 2019; 363eaat7554Crossref PubMed Scopus (246) Google Scholar,18.Masuda T. et al.Novel Hexb-based tools for studying microglia in the CNS.Nat. Immunol. 2020; 21: 802-815Crossref PubMed Scopus (49) Google Scholar]. scRNA-seq studies revealed that homeostatic microglia are rather heterogeneous and consist of several subsets. Initial studies proposed that homeostatic microglia comprise at least two subsets that are located in different brain areas and that presumably have overall similar functions [12.Van Hove H. et al.A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment.Nat. Neurosci. 2019; 22: 1021-1035Crossref PubMed Scopus (220) Google Scholar,18.Masuda T. et al.Novel Hexb-based tools for studying microglia in the CNS.Nat. Immunol. 2020; 21: 802-815Crossref PubMed Scopus (49) Google Scholar,19.Goldmann T. et al.Origin, fate and dynamics of macrophages at central nervous system interfaces.Nat. Immunol. 2016; 17: 797-805Crossref PubMed Scopus (483) Google Scholar,38.Tuan Y-L. et al.Microglial regional heterogeneity and its role in the brain.Mol. Psychiatry. 2020; 25: 351-367Crossref PubMed Scopus (84) Google Scholar]. Following activation, the morphology of microglia changes significantly (i.e., they show a swollen and larger cell body with thicker ramifications). Major effort has been invested in the characterisation of microglia by means of morphology, including analysis of process motility and ramifications under different conditions [29.Colonna M. Butovsky O. Microglia function in the central nervous system during health and neurodegeneration.Annu. Rev. Immunol. 2017; 35: 441-468Crossref PubMed Scopus (621) Google Scholar], which allows determining microglia status (i.e., homeostasis versus activation) [39.Davis E.J. et al.Cellular forms and functions of brain microglia.Brain Res. Bull. 1994; 34: 73-78Crossref PubMed Scopus (273) Google Scholar]. Similar to the morphological changes, also expression profiles of core signature genes change upon microglial activation, highlighting again the challenges associated with targeting and tracing these cells under experimental conditions [18.Masuda T. et al.Novel Hexb-based tools for studying microglia in the CNS.Nat. Immunol. 2020; 21: 802-815Crossref PubMed Scopus (49) Google Scholar]. Viral infections of the brain, which mostly are associated with the induction of inflammatory processes, are defined as VE and are the most common cause of encephalitis [40.Kennedy P.G. Viral encephalitis: causes, differential diagnosis, and management.J. Neurol. Neurosurg. Psychiatry. 2004; 75: i10-i15Crossref PubMed Google Scholar,41.Leahy C.B. et al.The clinical approach to managing herpes simplex virus encephalitis.Br. J. Hosp. Med. 2018; 79: 556-559Crossref Scopus (0) Google Scholar]. Many viruses that can enter the brain exhibit neurotropic characteristics, indicating that they predominantly infect neurons and other cells from the CNS [42.John C. et al.Global research priorities for infections that affect the nervous system.Nature. 2015; 527: S178-S186Crossref PubMed Scopus (66) Google Scholar]. Under certain conditions, also opportunistic viral infections can cause VE (e.g., in immunosuppressed patients). Mostly, VE is undiagnosed due to the unspecific, mild, flulike symptoms that patients develop (see also Box 2). Normally, VE is self-limiting [43.Klein C. et al.Neurological features of West Nile virus infection during the 2000 outbreak in a regional hospital in Israel.J. Neurol. Sci. 2002; 200: 63-66Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 44.Hanley D.F. et al.Yes, brain biopsy should be a prerequisite for herpes simplex encephalitis treatment.Arch. Neurol. 1987; 44: 1289-1290Crossref PubMed Google Scholar, 45.Eeg-Olofsson O. Virological and immunological aspects of seizure disorders.Brain Dev. 2003; 25: 9-13Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar], but still, severe and even fatal cases of encephalitis can occur after viral infection of the CNS. In some cases, patients affected by viral infection of the CNS can develop long-term neurological sequelae, such as epilepsy and cognitive dysfunctions, that often manifest years after the initial insult. In-depth understanding of the association between CNS infection and long-term sequelae is crucial for the development of new intervention strategies, and, accordingly, many ongoing preclinical studies are focussing on studying viral CNS infections and their effects on neural function.Box 2Clinical outcomes of VEAmong viruses that cause encephalitis, the most common known ones are herpes simplex virus (HSV) and other viruses of the Herpesviridae family, JEV, rabies virus, HIV, and measles virus [40.Kennedy P.G. Viral encephalitis: causes, differential diagnosis, and management.J. Neurol. Neurosurg. Psychiatry. 2004; 75: i10-i15Crossref PubM