**4. Overview of microglial activation in encephalomyelitis: amplifier of virus-induced neuropathology**

In the context of viral encephalitis that is characterized by an inflammatory response with meningeal, perivascular, and parenchymal infiltrates of peripheral leukocytes, studies have revealed that microglial activation acts as a double-edged sword [16]. On the one hand they promote multiple antiviral functions; microglia sense the ATP released by virus-infected neurons through the purinergic receptor P2Y12 and quickly migrate towards the infected neurons to exert their phagocytic activity [54]. They directly exert their antiviral effect by producing type 1 interferon (IFN-1), inducing IFN-stimulated gene (ISG) to activate corresponding signaling pathways [55]. Additionally, microglia induce autophagy and secrete cytokines to clear the virus from the tissue [56–58]. On the other hand, their persistent activation leads to tissue damage due to autophagy and apoptotic pathway activation, presynaptic membrane damage in the hippocampus mediated by the complement system activation, which further results in long-term memory impairment and cognitive dysfunction in patients with viral encephalitis [59–62].

The most common and important example of virus-induced chronic brain infection is the HIV [63]. HIV-induced encephalitis is typified by the accumulation of activated microglia in nodules-like phenotype throughout the parenchyma [64, 65]. HIV enters the CNS via the myelomonocytic cells such as monocytes, perivascular cells, and microglia [66]. HIV particularly targets and disables microglia in the CNS and T cells in the periphery, the key players in neuroinflammation [64, 67]. In fact, the persistence of HIV in microglia indicates that the virus uses the cells as the reservoir [68]. Even though reports suggest that microglia may perform protective functions early on during HIV infections, their functions are considerably compromised. Most studies suggest that active infection of microglia results in their secretion of a variety of neurotoxins, increasing neural apoptosis and neuronal autophagy [16]. Astrogliosis is another characteristic pathology following microglial activation that, together with microgliosis, ultimately leads to myelin paleness and neuronal loss. Patients with advanced AIDS are likely to develop severe encephalitis upon human cytomegalovirus (HCMV) infection [69, 70]. HCMV infection is characterized by microglial nodular encephalitis consisting of microglia, astrocytes, and giant cells, and ventriculoencephalitis and is the main cause of dementia in AIDS patients [71].

Microglia are susceptible to congenital Zika virus (ZIKV) infection [61]. Histopathological analysis showed that ZIKV infects and activates microglia in the perivascular regions causing localized neuroinflammation [61]. Further, the virus is disseminated throughout the parenchyma, which is later associated with neuron damage, especially in the cortical regions [72]. Pronounced neuronal injury results in microcephaly noted in many cases of congenital ZIKV infection [72].

E3 ubiquitin ligase pellino (pelia) expressed by microglia promotes the replication of West nile virus (WNV) in microglia and neurons [73]. It also induces NF-κB and/ or p38-MAPK signaling in the microglia that causes an upregulation of inflammatory cytokines and chemokines, leading to peripheral leukocytes infiltration [73, 74]. The robust neuroinflammation may lead to lethal WNV encephalitis.

It is interesting to note that many viruses from diverse families of viruses have been studied in microglia depletion models. Results from most studies showed increased viral replication upon microglial depletion [16, 75–77]. Additionally, microglial depletion was also associated with overt neurological symptoms and/or death along with high viral burden, which indicated their importance in survival in encephalitis [77]. Though confirmatory results on how these protective functions are exerted by microglia are lacking, some studies show a dependence of T cell responses on microglia activation.

Studies on several mouse models of viral encephalitis have shown that viral clearance depends on efficient T cell responses, including WNV, MHV, and TMEV [78–81]. TMEV model shows strain-specific differences in disease phenotype and viral clearance, which was associated with underlying differences in CD8<sup>+</sup> T cell responses subject to Treg suppression [82, 83]. Further investigation revealed that microglia depletion did not impact CD8<sup>+</sup> T cell recruitment but resulted in increased infiltration of Tregs, which caused clinical severity in C57BL/6 mice which is normally not susceptible to TMEV induced disease [84]. In the MHV-induced neuroinflammation model, both CD8+ and CD4+ T cells are implicated in viral clearance, but CD4+ T cells have also been reported to contribute to pathogenesis [80, 85, 86]. A study showed that microglial depletion in mice infected with JHMV strain of MHV, rJ2.2 significantly reduced the infiltration of CD4+ T cells and Tregs in the CNS along with a significant reduction in IFN-γ expression by CD4<sup>+</sup> T cells, but there was no impact on the CD8<sup>+</sup> T cell population [77]. Thus, showing the importance of microglia in especially orchestrating virus-specific CD4+ T cell response.

In addition to these studies using the JHMV strain, MHV-A59 or its isogenic recombinant strain RSA59 have also elucidated a critical communication between microglia and CD4+ T cell response. Using a CD4−/− mice model very recent study demonstrated for the first time that the mice are highly susceptible to RSA59 induced chronic demyelination with axonal loss [80]. Though the overall inflammation was not affected during the early time-points (day 5–6) i.e., the acute neuroinflammation phase, the CD11b + microglial activation was significantly impaired. The entire inflammatory response was skewed towards an M2 type which was also reflected in the persistence of characteristic amoeboid shaped phagocytic microglia in the CNS of the mice during the chronic phase (day 30 p.i.). Encephalitis, which usually resolves after the acute phase in RSA59 infection, persisted for as long as day 30 p.i. The brain stems of CD4−/− mice were populated with CD11b + microglia surrounding bulbar vacuolated pathology, which signified axonal death and damage [80]. Additionally,

## *Neurotropic Virus-Induced Meningoencephalomyelitis DOI: http://dx.doi.org/10.5772/intechopen.102674*

CD4+ T cell deficiency resulted in severe grey matter inflammation in the form of poliomyelitis in the spinal cords as well as the dorsal root ganglion (**Figure 3**). Together these results showed a critical interdependence of microglia and CD4+ T cells in RSA59 infection. Typically, M2 microglial activation fails to resolve during the chronic infection, rendering mice more susceptible to demyelination and axonal bulbar vacuolation [80].

A very recent study on neurotropic coronavirus MHV-RSA59 infection in Ifit2−/− mice revealed that Ifit2 protects mice from uncontrolled replication and spread throughout the brain parenchyma as well as the spinal cord. Ifit2 deficiency showed pronounced morbidity and mortality in RSA59 infected mice. Furthermore, microglial activation in the CNS was impaired in infected Ifit2−/− mice compared to WT infected mice, and as a consequence, peripheral lymphocyte specifically NK1.1 T

#### **Figure 3.**

*CD4 deficiency causes poliomyelitis and the dorsal root ganglionic inflammation at acute phase and abnormal bulbar vacuolation at chronic phase of RSA59 infection. Serial sections of spinal cord, dorsal root ganglion and brain from CD4+/+ and CD4−/− mouse were immunostained with anti-CD45, LFB and/or H&E. CD45 immunostining showed heightened poliomyelitis/inflammation of gray matter and inflammation of the dorsal root ganglia in the CD4−/− mice compared to CD4+/+ mice at the acute phase of infection. Spinal cord sections form these mice when further analyzed at chronic stage for demyelination by LFB showed increased myelin loss in CD4−/− mice compared to CD4+/+ mice, CD45 + inflammatory cells were observed in demyelinating lesions of both wildtype and CD4 deficient mice but were elevated in case of CD4−/− mice. Sagittal sections of the brain at chronic stage, stained with H&E showed large number of vacuoles in the brain stem region denoting abnormal bulbar vacuolation which was populated with and Cd11b + micgrogila/monocyte macrophages. Adapted from Chakravarty et al [41].*

cells and CD4<sup>+</sup> T cells migration to the CNS was restricted in the Ifit2−/− mice possibly contributing to the lack of viral clearance. Impaired microglial activation and reduced migration of inflammatory cells in the CNS may be associated with less encephalitis and devoid of mounting host immunity. These deficiencies were associated with a lower level of microglial expression of CX3CR1, the cognate receptor of the CX3CL1 (fractalkine) chemokine, which plays a critical role in both microglial activation and leukocyte recruitment. These findings highlighted a pivotal role of interferon stimulating genes and its tetratricopeptide protein as host cell factors in the induction of encephalitis and uncovered a new potential role of an interferon-induced protein in immune protection (**Figure 4**) [30].

Taking the above-mentioned experimental evidence of the role of CD4<sup>+</sup> T cells and monocyte/macrophage activation in viral-induced neuroinflammation, further studies were geared to explore the interactome between the CD4+ T cell expressed CD40 Ligand and CD40 expressed on microglia. CD40-CD40L dyad is an important immune dyad that controls both CD4+ T cell and microglia functions [87, 88]. Our studies in CD40L−/− mice showed that the absence of CD40L renders mice highly susceptible to RSA59 infection due to reduced microglia/macrophage activation during the acute phase of infection required to eliminate the virus (**Figure 5**) [89]. Effector CD4<sup>+</sup> T recruitment to the CNS is significantly dampened, and due to the

#### **Figure 4.**

*Ifit2−/− mice upon a murine β-CoV RSA59 infection show increased viral spread and decreased microglia/ macrophage activation. About 4–5-week-old Ifit2−/− mice upon RSA59 infection showed a robust viral replication and antigen spread throughout the brain parenchyma compared to the wildtype mice infection. Infectious viral load was significantly higher in the Ifit2−/− mice when assessed by plaque assay (A). The cryosections form Ifit2−/− and wildtype mice brain sections showed similar overall distribution of RSA59 but the total EGFP expression (viral antigen) was more in Ifit2−/− mice (A). At day 5 p.i. H&E staining for the sagittal sections of the whole brain form WT (B) and Ifit2−/− mice (I) showed much milder meningitis (J) and encephalitis characterized by perivascular cuffing (K) and microglial nodule (L) formation in the Ifit2−/− compared to WT (C–E) mice. Similarly activated microglia/macrophage were much less in the Ifit2−/− mice (M–O) compared to the WT (F–H) mice as seen in Iba1 immunostaining. (P) Brain section of Ifit2−/− mice showed heightened viral infection as evident by the profuse viral N protein immunostaining. Ifit2−/− mice however showed a comparatively decreased Iba1 immunostaining indicative of impaired activation of microglia/macrophages compared to the wildtype mice. Adapted form Das Sarma et al [30].*

*Neurotropic Virus-Induced Meningoencephalomyelitis DOI: http://dx.doi.org/10.5772/intechopen.102674*

#### **Figure 5.**

*RSA59 infection in CD40L deficient mice showed impaired microglia/macrophage activation during acute phase of neuroinflammation but causes profuse chronic demyelination concurrent with diminished PLP staining. (a and b) CD40L−/− and WT mice at day 5 upon RSA59 infection showed acute encephalitis and myelitis.Iba1 immunostaining in these brain and spinal cord sections showed that the CD40L−/− mice showed reduced Iba1+ cells compared to WT mice. (c) At day 30 p.i., spinal cord of CD40L−/− mice showed more intense demyelination and reduced PLP staining compared to wildtype mice. Adapted from Saadi et al [89].*

impaired CD40-CD40L signaling in CD40L−/− mice, their priming is reduced substantially in the draining lymph nodes [89]. Effector CD4+ T cell population was reduced as well as the antiviral response was diminished, and phagocytic microglia persisted in the CNS at a substantial amount in the CD40L−/− mice. As a result, CD40L−/− mice exhibited greater demyelination, axonal loss, and persistent poliomyelitis at the chronic phase of infection [89]. Together, these studies highlight that migration of peripheral T cells and their interaction with microglia via CD40-CD40L is essential to eliminate the virus and provide long-term neuroprotection.

Independent of the effect on T cells, IFN produced by microglia acts on other cells that exert indirect antiviral effects [90]. For example, microglia induce antiviral functions in neurons via STING signaling and stimulate IFN-1 production in astrocytes by the TLR3 pathway [91]. Studies have shown that the Type 1 IFNAR signaling in astrocytes helps to protect the blood-brain barrier against virus infection and immunopathology [92]. Depletion of IFNAR signaling in astrocytes resulted in increased inflammatory cytokines and chemokines production, which caused blood-brain barrier inflammation during neurotropic viral infection [92].

Additionally, microglia also mediate viral clearance by autophagy [59]. Viral infections induce NFkB-dependent inflammatory effectors that produce antiviral molecules, including those promoting autophagy. ZIKV infection in Drosophila induces a stimulator of interferon genes (dSTING) in the brain, which promotes autophagy and helps protect the brain [93]. In mammals, autophagy has been shown to restrict HSV-1 infection [94]. Autophagy by microglia helps to clear the virus without causing cell death which protects mature neurons. Microglial phagocytosis is another mechanism that helps protect the neurons from severe damage. Microglia and neurons express C3aR that recognizes C3 cleavage products. In response to C3 and its cleavage products, microglia surround the neurons and phagocytose the presynaptic ends of the neurons [95]. This prevents the trans-synaptic spread of the virus and keeps neurons from firing abnormal signals that may result in cognitive impairment and physical disabilities.

Additionally, detrimental effects of microglia are also reported in many studies on viral encephalitis. Microglia are reported to remain persistently activated in several viral infections [80, 89]. Their activation is further associated with the production of TNF-α [96, 97], which can activate the astrocytic TNFR-1 pathway [98]. This signaling accentuates their crosstalk with neurons leading to modification of the excitatory synapses, which emerges in cognitive disabilities. TNF-α secreted by microglia can directly affect synaptic transmission and plasticity [62]. ZIKV infection has been associated with neurological damage among infants. Studies have found that ZIKV majorly infects the fetal microglia and activates them [61]. This induces an intense pro-inflammatory response by the secretion of mediators like IL-6, TNF-α, IL-1β, and MCP-1 [61]. Also, in HIV encephalitis, many microglia genes undergo significant changes, including immune activation and function, kinases, phosphatases, and pro-/ anti-apoptotic and neurotrophic factors, which indicates that microglia functions are compromised and skewed towards pro-inflammation [99].

Thus, it can be ascertained that, microglia are not only important to maintain homeostasis in the CNS but are also critical for responding to injury, infection, and neurodegeneration. Often microglia act quickly in response to injury but with varied stimuli received, their activation profile can differ and may result in harmful or beneficial effects. It is true that viral encephalitis has caused high morbidity, which is a grave concern, but it is also imperative that research on microglia and viral encephalitis can provide new and efficient targets for treatment. Considering the unique response of microglia with different viral infections and at different stages of encephalitis, it is needless to say that the current research on microglia and viral encephalitis remains is at a nascent state. Depending on the type of encephalitis, careful fine-tuning of microglial activation has the potential to improve the therapeutic effect of encephalitis, its prognosis and also reduce the sequelae of encephalitis.

### **5. Encephalitis caused by several other neurotropic virus families**

Many viruses from numerous virus families in different geographical areas can induce immediate or delayed neuropathological manifestations in humans and animals [18, 100]. Infection by neurotropic viruses and their resultant immune response has the potential to irreversibly disrupt the complex structural and functional dynamics of the central nervous system, frequently leaving the patient with a poor or fatal prognosis. The incidences of virus-induced CNS disorder are significantly higher than the damage caused by other pathogens.

Members of several virus families are known to be neurotropic, e.g., herpes family viruses, flaviviruses, paramyxoviruses, alphaviruses, bunyaviruses, orthomyxoviruses, arenaviruses, enteroviruses, rhabdoviruses, coronaviruses, and picornaviruses. Specifically, some of the viruses from these families are viruses like SARS-CoV, MERS-CoV, herpes simplex virus, poliovirus, West Nile virus (WNV), Chikungunya virus (CHIKV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), La Crosse encephalitis (LACV), Epstein-Barr virus (EBV), measles, and mumps viruses, among many others [100, 101]. These viruses have been frequently associated with significant encephalitis, as well as meningitis and myelitis in the CNS. The clinical disease outcome of the CNS virus infection depends on several factors, like the host immune status, viral genomic constitution, and other environmental factors [100]. There is also evidence suggesting coronaviruses such as α-CoVs (NL63 and 229E) and the β-CoVs (OC43, HKU1) being positive-sense single-stranded, enveloped RNA viruses,

#### *Neurotropic Virus-Induced Meningoencephalomyelitis DOI: http://dx.doi.org/10.5772/intechopen.102674*

can induce numerous neurological manifestations along with systemic inflammations in humans. A very recent outbreak of human β coronavirus SARS-CoV-2 is primarily known for its ARDS and paramount evidence suggest that it may enter the brain via olfactory route or can enter through the lung brain axis or gut-brain axis and is also known to cause meningitis, encephalitis, and demyelination [102–104].

The etiology of CNS infections induced by viruses can also depend on and vary across variable geographical locations. For example, reports have shown that herpes simplex viruses are the most common pathogens observed among both children and adults in the United States (US), Australia, and Italy. In contrast, in Southeast Asian countries like southern Vietnam, the Japanese encephalitis virus has been shown to be one of the most frequent inducers of viral encephalitis, especially among children. Enteroviruses have been commonly isolated to be involved in causing encephalitis in several parts of India. At the same time, HSVs have been observed to be more prevalent in the eastern parts of India, both among adults as well as children. Furthermore, the virulence of the viruses also varies geographically [105]. Therefore, understanding the etio-biology and epidemiology of neurotropic viruses is paramount in designing the targeted intervention.

Therefore, based on epidemiological prevalence and episodic occurrence evidence as well as the employment of these viruses as experimental model systems for human disorders, this book chapter will also briefly discuss some of the other viruses.

Flaviviruses are the enveloped, single-stranded, positive-sense RNA viruses that consist of the world's most clinically critical viruses like the following species: Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and Powassan encephalitis virus (POWV), as well as other mosquito-borne viruses, like Dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Zika virus (ZIKV) [106, 107]. JEV is highly prevalent in the Southeast Asian countries as well as the Indian subcontinent, affecting infants and children, and can also be transmitted to the fetus during pregnancy [108, 109]. The incidences of Tick-borne encephalitis (transmitted to humans by the bites of ticks) are progressively expanding in European and Asian countries with severe neurological complications [110]. WNV infection is endemic in temperate and tropical regions throughout the world, triggering yearly outbreaks of encephalitis [111]. St. Louis encephalitis virus (SLEV) is found predominantly in North, Central, and South America and accounts for nearly 35–60% of meningitis in all symptomatic cases in children [112, 113]. Zika virus is also an emerging pathogen with substantial clinical impact significantly on the CNS, as reported, in the form of severe congenital malformations (microcephaly) and neurological complications, mainly Guillain-Barré syndrome (GBS) [114]. It has shown explosive outbreaks in African, South, and Central American countries [112, 115].

Alphaviruses like Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and Venezuelan equine encephalitis virus (VEEV), as well as the Mayaro virus (MAYV), Una virus (UNAV), and Chikungunya virus (CHIKV) are the small, enveloped viruses with a single-stranded, positive-sense RNA [100, 113]. The most critical neurotropic alphavirus is VEEV, which induced many outbreaks in South, Central, and North America [116]. CHIKV has also caused severe neurological complications in humans at all age groups, especially in infants, in Europe, Asia, and Africa [117, 118]. EEEV can also induce encephalitis in humans in about 50–75% of the cases [8, 119].

Herpes family viruses which are double-stranded DNA viruses, have been commonly associated with severe encephalitis and meningitis in the CNS and have been distributed globally. The members of herpes family viruses that are shown to be neurotropic include HSV types 1 and 2, varicella-zoster, Epstein-Barr virus, and cytomegalovirus [120, 121]. Both children and immunocompromised individuals are most vulnerable to herpes simplex meningoencephalitis [120]. Another critical property to herpes family viruses, especially varicella-zoster virus, is reactivation [122]. Primary infection with VZV during childhood induces chickenpox, but the virus becomes latent in the spinal and cranial ganglia. However, deteriorating cellular immunity with senescence or immunocompromised conditions may lead to virus reactivation that promotes zoster (shingles) [123, 124].

Paramyxoviruses that induce neurological diseases are from genera Rubulavirus (consisting of the mumps virus), which is neurotropic [125]; Morbillivirus genera (consisting of measles virus) [126] and Henipavirus with the emerging Nipah virus (NiV) being one of the neurotropic variants [127]. These are single-stranded, nonsegmented RNA viruses [128]. Measles virus-induced encephalitis is one of the leading causes of morbidity and mortality in the developing world [129]. Nipah virus is one of the emerging viruses that present with numerous cases of acute encephalitis in humans [127, 130].

Lymphocytic choriomeningitis virus (LCMV) belonging to the family Arenaviridae is an enveloped, single-stranded RNA virus. Although its primary host is mice, it is also present in other rodents and has the ability to infect humans, especially laboratory workers, pet owners, and individuals living in impoverished conditions. It is predominant in Europe, Asia, American continents, and Africa [131–133].

Picornaviruses are single-stranded, non-enveloped RNA viruses encompassing enteroviruses (echoviruses, coxsackieviruses) and parechoviruses (PeVs) pathogenic against humans. Infants and children are highly susceptible to human pathogenic picornaviruses that induce aseptic meningitis and meningoencephalitis [134, 135]. It is highly predominant in the UK, Ireland, the US and some Southeast Asian countries [105, 136, 137].
