*3.1.1 Comparative studies between spike gene recombinants murine coronaviruses RSA59 and RSMHV2 to understand the genomic control of meningioencephalomyelitic properties*

Using targeted RNA recombination, two isogenic spike protein recombinant strains of MHV, RSA59, and RSMHV2 (background is from demyelinating strain MHV-A59) were generated. For RSA59, the spike was taken from the parental demyelinating strain MHV-A59, and RSMHV2 had the spike from the parental nondemyelinating MHV-2 strain. Enhanced green fluorescent protein (EGFP) was also inserted in the recombinants by replacing the nonessential gene 4a and part of 4b in the MHV-A59 genome by heterologous targeted recombination [34].

Comparative studies between the two recombinants revealed similar pathology to their parental strains for RSA59 and RSMHV2, respectively. Intracerebral (IC) inoculation of RSA59 in 4-week-old C57BL/6 mice caused acute hepatitis, neuroinflammation comprising of meningitis, encephalitis, myelitis, and chronic demyelination and axonal loss, characterized by lymphocytic infiltrates and microglial nodules with focal neuronophagia associated lepto-meningitis [29, 35]. IC inoculation of RSMHV2 caused acute stage hepatitis, meningitis, and encephalitis but no myelitis or chronic demyelination. The encephalitis was indeed more robust compared to RSA59. MHV-2 does not induce encephalitis; it cannot even enter the brain parenchyma and restricts to the meninges inducing meningitis alone [34].

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

Both RSA59 and RSMHV2 showed similar infection in the brain where they successfully infected and replicated in meninges, the site of inoculation (near the lateral geniculate nucleus), ventral striatum/basal forebrain, hippocampus, and brainstem, and infect the neurons, astrocytes, microglia, and oligodendrocytes in the brain. However, they show different tropism in the spinal cord. DM infects the grey matter neurons and takes an axonal route to be released at the nerve end, whereas in the white matter, it preferentially infects oligodendrocytes [36]. By day 7 p.i. most of the viruses have traversed to the spinal cord white matter. Though NDM can also infect the neurons in the grey matter, they fail to infect the white matter oligodendrocytes [35, 36] due to their inefficiency to translocate through neurites and fusion at the nerve end [37]. The difference in the disease outcome can be attributed to their differential spinal cord tropism and persistence. Thus, it can be inferred that the combined action of spike mediated axon transport of DM strain to evade the heightened immune response and ability to infect the white matter oligodendrocytes and persist in the white matter could be the key to inducing demyelination and axonal loss during the chronic phase of neuroinflammation. DM strain-induced axonal loss and myelin loss are associated with profuse accumulation of macrophages filled with myelin debris within the demyelinating plaques which is not observed in NDM strain infection. High resolution TEM microscopy revealed that microglia/macrophages are indeed responsible for direct myelin stripping, leading to demyelination [29, 35, 38].

It is important to note that neuroinflammation and encephalitis in MHV infection is accompanied with pronounced activation of CNS resident immune cells, microglia, and astrocytes. Upon activation, they take their characteristic activated phenotype and start expressing microglia/macrophage-specific protein Iba1 (ionized calcium-binding adaptor molecule 1), which promotes ruffling and phagocytosis [20, 38]. Detailed Affymetrix microarray analysis revealed that both RSA59 and RSMHV2 initiate innate immune responses during the acute phase with the expression of chemokines like CXCL10, CXCL9, CCL5, and CCL12 and other CD molecules that represent activation of microglia/macrophages [39]. Results also showed the induction of antiviral host response represented by the expression of perforins and IFN gamma signaling genes. Together, the acute stage innate-immune responses and encephalitis were comparable in both RSA59 and RSMHV2 infection [39].

The inflammatory responses gradually declined in RSMHV2 infection following virus clearance, but RSA59 chronic infection showed persistent microglia in the demyelinating plaques and the production of microglia-associated inflammatory mediators. Studies have shown that IFN responses can promote phagolysosomes maturation and autophagy in the persistently activated microglia/macrophages, which can promote myelin sheath engulfment leading to demyelination. This evidence demonstrated that RSA59 induced demyelination could occur through innate immune neuroinflammation denoted by meningioencephalomyelitis triggered during the acute infection stage. Although innate immune responses contribute partially towards controlling of initial virus spread, virus-specific T cell effector functions are essential to eliminate the infectious virus load during most acute infections. Control of m-CoV spread requires the functioning of both CD4+ and CD8+ T cells [40]. CD8+ T cells are the primary effectors but require support from CD4+ T cells. A recent study in CD4−/− mice showed impaired RSA59 clearance, despite the presence of functional CD8<sup>+</sup> T cells, demonstrating the importance of CD4<sup>+</sup> T cells for the efficient functioning of CD8+ T responses [41].

The distinct cause-effect relationship-driven studies demonstrate that RSA59 infection can be instrumental towards understanding a direct CNS resident immune cell-mediated as well as antibody-mediated encephalomyelitis and demyelination pathologies.

Both DM and NDM strains show a reduction in the expression of genes responsible for innate immune response, and this reduction is more pronounced in the NDM strain-infected mice. In contrast, the genes involved in adaptive immune cell response are upregulated only in DM strain, specifically during the chronic stage of spinal cord infection. A significant upregulation of genes involved in T helper cell signaling pathways, B-cell development, and communication between innate and adaptive immune cells as well as of the expression of IgG genes are observed in the DM strain infection leading to chronic pathology but not in NDM strain [42].

While MHV infection in mouse is a prototype to understand the cellular and molecular consequences of encephalitis and demyelination, Theiler's murine encephalomyelitis virus (TMEV) in SJL mice also serves as another excellent experimental model of MS because of its histopathological and immunological similarities with MS.

#### **3.2 Theiler's murine encephalomyelitis virus (TMEV) induced encephalitis**

TMEV is a non-enveloped, single-stranded positive-sense RNA virus belonging to the family Picornaviridae and also used as a model to understand the immunemediated mechanism of demyelination [43, 44].

Upon intracranial (i.c.) infection in SJL mice induces characteristic polioencephalomyelitis in the CNS. In a biphasic CNS disease, first, during the acute phase immune cells including the CD4+ T cells and CD8+ T cells infiltrate the CNS in response to profuse virus replication, and inflammation [45]. They exert rather protective effects by helping clear the virus from the grey matter and result in immune-mediated encephalomyelitis [46]. During the chronic phase, TMEV persistently infects glial cells and macrophages in the white matter, further, there is an infiltration of leukocytes, including macrophages, TMEV-specific T cells and antibodies. The immune effectors (CD4+ and CD8+ T cells) that exerted protective functions during the acute phase exert detrimental effects in the chronic phase by participating in epitope spreading to myelin antigens resulting in severe immune pathology in the white matter of the spinal cords, which causes demyelination [47]. White matter lesions harbor monocytes/macrophages and a few B cells in addition to the T lymphocytes [48]. Initially, the CD4+ T cells recognized the abundant myelin protein PLP, but later, the CD4+ T cells subsets start to recognize subdominant myelin protein epitopes and led to autoreactivity [47]. Additionally, the CD8+ T cells have also been suggested to function as autoreactive cytotoxic cells or regulatory cells in TMEV infection [49]. TMEV induced CNS pathology is immunologically mediated like in MS, wherein MHC plays an important role, and substantial similarities exist in neuropathology, including axonal damage and remyelination [43]. Also, like MS, T-cell apoptosis is less in TMEV induced disease [50].

Cytokines are known to play important functions in the induction and regulation of immune responses against the neurotropic virus. Similarly, the cytokine production by the CNS resident cells astrocytes and microglia, as well as the peripheral immune cells such as T cells and macrophages heavily, influence the encephalitic response induced by TMEV infection [51, 52]. Studies have shown that a critical balance between the inflammatory cytokines governing the propagation of antiviral response to clear the virus during early infection and controlling of immune

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

pathology and establish homeostasis during the late stage. That being said, no particular cytokine pattern is yet established and successfully associated with resistance or susceptibility to TMEV-induced encephalitis and demyelination by the different strains of the virus. A study compared the cytokine response between TMEV DA-infected susceptible (SJL) and resistant (B6) mice. Results showed a high expression of proinflammatory cytokines (IFN-γ, TNF-α, IL-1, IL-2, and IL-6) and low levels of anti-inflammatory cytokines (IL-4, IL-5, and IL-10) in the brains of both SJL and B6 mice during the early acute phase decreasing thereafter [51]. However, only in the SJL mice after a peak of the inflammatory response during day 8–12 p.i. in the brain with a minimum recorded during days 20–25 p.i., the second wave of inflammatory cytokine production was observed later in the spinal cord, which could explain the inflammatory demyelination only in the SJL mice. TGF-β, an important antiinflammatory cytokine, has shown a significantly higher upregulation in SJL mice compared to B6 [51]. TGF-β can specifically inhibit the cytotoxic T lymphocyte (CTL) response [53] which is noted to be significantly impaired in the SJL mice resulting in reduced TMEV clearance and persistence, leading to virus-mediated encephalitis and demyelination.
