# Microglia Play a Major Role in Direct Viral-Induced Demyelination

Authors: Dhriti Chatterjee; Kaushiki Biswas; Soma Nag; S. G. Ramachandra; Jayasri Das Sarma
Journal: Clinical and Developmental Immunology
 (2013)

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## Abstract

Microglia are the resident macrophage-like populations in the central nervous system (CNS). Microglia remain quiescent, unable to perform effector and antigen presentation (APC) functions until activated by injury or infection, and have been suggested to represent the first line of defence for the CNS. Previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (MHV) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (MS). Current studies revealed that MHV infection is associated with the pronounced activation of microglia during acute inflammation, as evidenced by characteristic changes in cellular morphology and increased expression of microglia-specific proteins, Iba1 (ionized calcium-binding adaptor molecule 1), which is a macrophage/microglia-specific novel calcium-binding protein and involved in membrane ruffling and phagocytosis. During chronic inflammation (day 30 postinfection), microglia were still present within areas of demyelination. Experiments performed inex vivo spinal cord slice culture and in vitro neonatal microglial culture confirmed direct microglial infection. Our results suggest that MHV can directly infect and activate microglia during acute inflammation, which in turn during chronic inflammation stage causes phagocytosis of myelin sheath leading to chronic inflammatory demyelination.


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## Body

## 1. Introduction

Microglia are specialized macrophages of the CNS that constitute 5–20% of total glial cells in rodents, depending on the specific neuroanatomical region of the CNS. Microglia are distinguished from neuron as well as glial cells, such as astrocytes and oligodendrocytes, by their origin, morphology, gene expression pattern, and function. While neuron and conventional glial cells are neuroectodermal in origin, microglia are of haematopoietic origin and act as primary responding cells for pathogen infection and injury like monocytes/macrophages in peripheral tissues. Microglia exhibit several features that distinguish them from other populations of macrophages, such as their “ramified” branches that emerge from the cell body and communicate with surrounding neurons and other glial cells. Microglia can rapidly respond to infectious and traumatic stimuli and adopt a “phagocytotic” nature. Activated microglia are known to produce many proinflammatory mediators including cytokines, chemokines, reactive oxygen species (ROS), and nitric oxide which mainly contribute to the clearance of pathogens or infections. However, prolonged or unwarranted microglial cell activation may result in pathological forms of inflammation which can lead to several neuroinflammatory conditions of the nervous system.  Microglia-mediated innate immune response in the CNS is now considered to be potentially one of the major pathogenic factors in a number of CNS neuroinflammatory diseases that lack the prominent leukocytic infiltrates of adaptive immune responses [1]. Neuroinflammation is associated with many neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS) [2]. While AD, PD, and ALS are commonly known to be neurodegenerative disease with underlying neuroinflammatory mechanisms, MS is one of the major chronic inflammatory CNS diseases in humans with heterogeneous (chronic/remitting) clinical presentations and course [3, 4].MS is believed to be an autoimmune inflammatory demyelinating disease in which exposure of genetically predisposed people to environmental factors triggers a breakdown in T-cell tolerance to myelin antigens. Demyelination is a complex process, and while the precise mechanisms of this pathology are unclear, inflammatory demyelination is thought to be the result of adaptive immune-mediated responses to myelin antigens in the myelin sheaths of axons and/or in the myelin-forming oligodendrocytes. Most studies have focused on the pathogenic role of myelin-specific CD4+ T cells because of the relatively strong association of susceptibility to MS with major histocompatibility complex (MHC) class II alleles [5, 6]. There is also increasing recognition of the potential importance of CD8+ T cells in the pathogenesis of demyelination [7, 8].  However, the contribution of innate immune cells in mediating MS pathogenesis has been recently gained attention, as several studies demonstrated the role of various innate immune cells in mediating MS pathogenesis, in particular, the potential anti-inflammatory or proinflammatory function of microglial cells along with its physical  interaction with myelin [9–11]. For long time, microglia were known to be present in the chronic inflammatory demyelinating plaque to remove myelin from the dead sick neuron in MS patients but the emerging recognition of microglia as CNS resident immune cells and their role in CNS health and diseases stimulated substantial efforts to redefine the role and function of microglia in the regulatory mechanisms of demyelination.MS is best studied in some experimental models such as experimental autoimmune encephalitis (EAE), Theiler’s murine encephalomyelitis (TMEV), and mouse hepatitis virus- (MHV-) induced neuroinflammation. Virtually, all types of adaptive immune response have been proposed to play important roles in the pathogenesis of EAE [4, 12], TMEV [13], and a neurotropic strain of mouse hepatitis virus (MHV); MHV-JHM [14, 15], mimicking the pathogenesis of the MS.Upon intracranial (i.c.) infection of neurotropic MHVs, acute meningoencephalitis (with or without hepatitis) is the major pathologic process (see Supplementary Figure 1 available online athttp://dx.doi.org/10.1155/2013/510396) [16]. Natural and genetically constructed recombinant MHV strains (generated by targeted RNA recombination) with differential pathological properties were used in several studies to understand the mechanisms of demyelination and concomitant axonal loss [17–20]. The outcome and degree of MHV-induced disease are dependent on several factors, including the age and strain of the mouse, the strain of MHV, and the route of virus inoculation. Even very closely related strains of MHV differ in pathogenic properties. Some strains of MHV are purely hepatotropic (e.g., MHV-2) [21]; some are primarily neurotropic (e.g., JHM, MHV-4, an isolate of JHM) [15, 22]; while others (e.g., MHV-A59 and MHV3) [16, 23] are both hepatotropic and neurotropic. Viral titer reaches its peak at days 3 and 5 postinfection (p.i.) [21]. Infectious virus is cleared within the first 10–14 days; however, at this time mice begin to develop demyelination, either clinical or accompanied by chronic hind limb paralysis [16, 24]. Both MHV-JHM and MHV-A59 cause inflammatory demyelination in the brain and spinal cord whereas MHV3 only causes vasculitis [23, 25]. It was formerly believed that in primary MHV-induced demyelination neuronal axons remain relatively preserved. Recently, it has been shown that axonal damage is, in large part, immune mediated in MHV-infected mice and occurs concomitantly with demyelination. Concurrent axonal loss and demyelination have recently also been observed with S protein recombinant demyelinating strain-infected mouse spinal cord [20].Evidence from highly neurovirulent JHM strains of MHV suggests that MHV-induced demyelination is primarily immune mediated [26, 27]. Clearance of infectious virus is mediated by both cytolytic and cytokine-mediated mechanisms and microglia, and T cells modulate pathologic changes. Demyelination can be prevented in JHM-infected lymphocyte-deficient (RAG-/-) mice [28]. However, demyelination will occur upon transfer of splenocytes from immunocompetent mice to RAG-/- mice [28]. It has also been shown by depletion and transfer studies in the JHM model that CD8+ T cells can induce demyelination. These studies suggest that an intact adaptive immune system is required to promote demyelination in JHM-MHV infection. Contrary to these findings, demyelination, induced by MHV-A59, has been shown to develop in adult immunocompromised mice lacking B and T cells [29]. It has also been demonstrated that the depletion of CD4+ or CD8+ T cells after the acute stage of infection does not reduce demyelination [28, 30]. Indeed, MHV-A59 or its isogenic spike protein (host-attachment protein) recombinant strain, RSA59 [19, 20, 31], induces a MS-like disease in mice mediated by microglia, along with a small population of T cells. The mechanism of demyelination is at least, in part, due to macrophage-mediated myelin stripping, with some direct axonal injury as well as without involving the conventional αβT cells. In our current studies, we have used RSA59 infection in vivo, in vitro, and ex vivo as a model to understand whether MHV can directly infect CNS resident microglia and the mechanism of microglial activation in the induction of chronic demyelination.


## 2. Material and Methods

### 2.1. Ethics Statement

Use of animals and all experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the Indian Institute of Science Education and Research Kolkata and Indian Institute of Science, Bangalore India. Animal protocols adhered to the guidelines of the CPCSEA, India.


### 2.2. Virus

RSA59 an isogenic recombinant demyelinating strain of MHV-A59, where the spike gene (encodes virus host-attachment protein), was exchanged by MHV-A59 spike gene only in the background of MHV-A59 gene by targeted RNA recombination as described in our previous studies [18, 19]. This recombinant strain also expresses enhanced green fluorescence protein (EGFP) [32] for easy detection of viral particle by EGFP fluorescence.To engineer the targeted recombinant strains, molecularly cloned vector pMH54 [33, 34], which contains the entire 3′ end of the genome from MHV-A59, was used for construction of the recombinant viruses. RSA59 and RSMHV2 are isogenic except the spike protein. RSA59 strain is expressing the MHV-A59 spike in the MHV-A59 background, whereas RSMHV2 strain is expressing the MHV-2 spike in the MHV-A59 background [18]. Furthermore, EGFP gene was inserted into the MHV genome in place of the nonessential gene 4 in both RSA59 and RSMHV2 [19].In order to replace gene 4 with the EGFP gene, pMH54 was modified by the introduction of a SalI site 42 nucleotides downstream of the intergenic sequence for gene 4a and a NotI site 102 bp upstream of the stop codon for gene 4b, using the Quick Change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). (These are coding-silent nucleotide changes.) The coding sequence of EGFP was cleaved from the pEGFP-N1 vector (Clontech, Palo Alto, CA, USA) using SalI and NotI and inserted in the place of the SalI/NotI fragment of pMH54. The resulting plasmid contains 760 bp of non-MHV sequence, including the 722-bp EGFP open-reading frame, replacing the entire gene 4a and the rest 213 bp of gene 4b.Previous studies reported with JHM strain revealed that the interruption of the ORF 4 did not alter the neurovirulence neitherin vivo nor the replication in vitro [35]. Targeted recombination was used to select MHV isolates with stable and efficient expression of the gene encoding EGFP to facilitate the in vivo detection of virus in the mouse CNS as well as to trace the viral entry and spread in tissue culture. The viruses replicated with similar kinetics as wild-type virus both in tissue culture and in the mouse CNS. They caused similar encephalitis and demyelination in animals as the wild-type virus or their recombinant strains; however, they were somewhat attenuated in virulence [19].


### 2.3. Inoculation of Mice

Four-week-old, ten MHV-free, C57BL/6 (B6) mice (Jackson Laboratory, obtained from IISc, Bangalore, India) were inoculated intracranially with 50% LD50 dose of RSA59 strain (20,000 PFU) as described previously [19, 32]. Mice were monitored daily for signs of disease. Three mock-infected controls were inoculated similarly but with an uninfected cell lysate at a comparable dilution.  Three mice were sacrificed in between days 5, 6, or 7 (period for peak of inflammation postinfection for routine paraffin-based histopathological analysis), and the other three were used for frozen sections. The rest of the infected mice were sacrificed at day 30 postinfection for routine paraffin-based histopathological analysis. Cervical, thoracic, and lumbar regions of spinal cord were successively processed, and 4 quadrants (dorsal/posterior column, anterior column, and two anterior horns) from two separate sections of each spinal cord level were examined.


### 2.4. Histopathology

At 5, 6, or 7 and 30 days postinfection, brain and spinal cord tissues were harvested from both mock infected and RSA59-infected mice. For routine paraffin sectioning, brain and spinal cord tissues were postfixed in 4% PFA for overnight. Fixed tissues were processed and 5 micron thin sections were prepared for routine CNS pathology, whereas frozen sections tissues were postfixed with 4% PFA for 4−6 hours and then transferred in 4% sucrose solution for 4–6 hours and in 20% sucrose solution for 16–24 hours and mounted in Cryomatrix (Thermo Shandon). Ten micron thin sections were prepared for frozen tissue immunofluorescence. The paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) to determine the inflammation, whereas Luxol Fast Blue (LFB) staining was used to detect the loss of myelin sheath. All slides are coded and read in blind manner.


### 2.5. Immunohistochemical Analysis

Serial sections from brain and spinal cord were stained by the avidin-biotin-immunoperoxidase technique (Vector Laboratories) using 3,3-diaminobenzidine as substrate and a 1 : 100 dilution of anti-Iba1 (Wako, Richmond, VA, USA), 1 : 100 dilution of anti-CD45 (LCA; leukocyte common antigen, LY-5, BD Pharmingen), anti-Iba1 (Wako, Richmond, VA, USA), or CD3 (Dako; Carpinteria, CA, USA), and 1 : 20 dilution of monoclonal antibody directed against the nucleocapsid protein (N) of MHV-JHM (monoclonal antibody clone 1-16-1 (kindly provided by Julian Leibowitz))  as primary antibodies. Control slides from mock-infected mice were incubated in parallel.


### 2.6. Immunofluorescence and Fluorescence Microscopy

Cryosections from the spinal cord tissues were washed with PBS at room temperature in a humidified chamber, incubated for 10 min at room temperature with 1 mg/mL NaBH4 in PBS to reduce autofluorescence, washed, incubated for 1 h at room temperature with 1 M glycine in PBS to reduce nonspecific cross-linking, and then washed subsequently with PBS, PBS with 0.5% Triton X-100 (TX), and PBS with TX and 2% goat serum (GS). The sections were incubated overnight at 4°C with a 1 : 100 dilution of a rabbit anti-Iba1 antibody diluted in PBS with TX and GS, washed, and then incubated with a secondary antiserum diluted into PBS with GS for 2 hrs at 37°C. All incubations were carried out in a humidified chamber. Viral antigen was detected by EGFP in a fluorescein isothiocyanate channel [19]. Control slides were incubated in parallel with preimmune rabbit sera, and sections from mock-infected mice were incubated with secondary antibodies only. Tissue sections were sequentially washed with PBS plus TX and with PBS and mounted and visualized by IX-81 fluorescence microscopy with a 40x UPlanApo objective, with the iris diaphragm partially closed to limit the contribution of out-of-plane fluorescence, and with filter packs suitable for green fluorescence and red fluorescence. Images were acquired with a Hamamatsu Orca-1 charge-coupled device camera and Image-Pro image analysis software (Media Cybernetics, Silver Spring, MD, USA).


### 2.7. Transmission Electronic Microscopic Observation for the Accumulation of Microglia in the Demyelinating Plaque during Chronic Inflammatory Stage of RSA59 Infection

To further characterize the presence of microglia in the chronic demyelinating plaque at the ultrastructural level, mice were anesthetized, perfused with 4% PFA, and spinal cords from mock-infected and RSA59-infected were harvested and fixed overnight in 2% glutaraldehyde as described earlier [20]. Samples for transmission electron microscopy (TEM) were postfixed with 1% osmium tetroxide, dehydrated, and flat-embedded in Poly-Bed 812 epoxy resin (Polysciences). Half micrometer thick sections were cut from the lesional epicenter, stained with toluidine blue, and examined by light microscopy. Ultrathin TEM sections (600 Å) were cut from representative foci of demyelination from the toluidine blue-stained semithin sections and mounted on 200 mesh copper grids, stained with uranyl acetate and bismuth subnitrate, and viewed under a JEOL JEM 1010.


### 2.8.Ex Vivo Slice Culture

Four-week-old, MHV-free, C57BL/6 were perfused transcardially with sterile PBS. Spinal cord was harvested and washed with PBS containing 1% penicillin/streptomycin (Pen/Strep). The spinal cord was then embedded in 2% agarose mould, and 200 micron thick cross-sections were prepared by Vibratome (Leica Vibrating Blade Microtome; VT1200S). The slices were washed twice with PBS containing 1% Pen/Strep. The slices were then transferred to a 24-well plate with one slice in each well. 500μL of DMEM containing 10% FBS, 1% Pen/Strep, and 1% L-Glutamine were added in each well and incubated overnight with 5% CO2.


### 2.9. Viral Infection and Staining of Spinal Cord Slices

After 24 hrs of explantation, slices were infected with RSA59 at 20,000 PFU (Half of the LD50 dose) in low serum (2%) containing medium for 1 hr and then washed with PBS to remove the unbound viruses, and 10% serum containing medium were added to the infected culture and the cultures were maintained for 72 hrs. At 24 hrs, 48 hrs, and 72 hrs of postinfection, slices were processed for immunostaining with anti-Iba1 antibody, and EGFP fluorescence was used to detect viral antigen. Briefly, at different times postinfection slices were washed gently with PBS and fixed with 4% PFA for 2 hours. Postfixed slices were washed with PBS, permeabilized with 0.5% Triton X-100 for 5 mins, and blocked with 1% goat serum for 1 hr at RT followed by overnight incubation with anti-Iba1 antibody. For better staining, next day antibody solution was replaced with fresh antibody and incubated at 4°C for additional 16–20 hrs. Slices were washed to remove any unbound antibody and then labelled with TRITC conjugated goat anti-rabbit IgG for 16 hrs. Labelled slices were then washed to remove any unbound fluorescent tagged antibody and then mounted in Vectashield (Vector Laboratories, CA, USA with DAPI and observed in Zeiss Confocal Microscope (LSM710). Images were acquired and processed by using Zen2010 software (Carl Zeiss).


### 2.10. Isolation of Mixed Glial Cells from Neonatal Mice Brain

Primary cultures of mixed glia from day 0 to day 3 newborn mice were prepared as described previously [36]. Briefly, following the removal of meninges, brain tissues were minced and incubated in a rocking water bath at 37°C for 30 min in Hanks Balanced Salt Solution (HBSS, GIBCO) in the presence of 300 μg/mL of DNaseI (Sigma) and 0.25% trypsin (Sigma). Enzyme-digested-dissociated cells were triturated with 0.25% of fetal calf serum (FCS), followed by a wash and centrifugation (300 ×g for 10 min). The pellet was resuspended in HBSS, passed through a 70 micron nylon mesh, followed by a second wash and centrifugation (300 ×g for 10 min). Following dilutions with astrocyte-specific medium (Dulbecco’s essential medium containing 1% penicillin-streptomycin, 0.2 mM l-glutamine, and 10% FCS), cells were plated and allowed to adhere for 1 day in a humidified CO2 incubator at 37°C. After 24 hrs, any nonadherent cells were removed and fresh astrocyte-specific medium was added. Adherent cells were maintained in astrocyte-specific medium for 10 days.


### 2.11. Isolation and Enrichment of Microglia from Mixed Glial Culture

After establishment of the mixed glia culture, feeding was stopped for 10 days to allow for significant microglial growth on top of the astrocyte monolayer. The microglia population peaked at 12–14 days in these cultures. To remove any cells adherent to the astrocyte monolayer, microglia-enriched cultures were thoroughly agitated in an orbital incubator shaker (200 rpm for 40 min at 37°C). Immediately following agitation, all cells suspended in the culture medium were collected and centrifuged at 300 ×g for 5 min at 4°C. The cell pellet was resuspended and diluted with fresh astrocyte-specific medium bringing the cells to a final concentration of 8 × 105 cells/mL; 1 mL was added to each well of a two-well CC2-treated chamber slide (specifically made for primary cell culture; NUNC) or 2 mL/well of a six-well plate. After 30 min, any non-adherent cells were discarded and adherent cells were maintained in fresh astrocyte-specific medium until infected with a medium change every 3-4 days.


### 2.12. Characterization of Microglia in a Mixed Glial Culture

To examine different cell types in a given culture, primary antibodies directed against cell-specific antigens were used to determine the presence and/or purity of each of the major glial cell types as described previously. Microglia were labelled with biotinylated anti-mouse CD11b (Chemicon, diluted 1 : 100 in F-12 nutrient medium) followed by Cy3-streptavidin (Jackson Immunoresearch, diluted 1 : 200 in F-12). Astrocytes were labelled with polyclonal rabbit anti-glial fibrillary acidic protein (anti-GFAP; DAKO) followed by either goat anti-rabbit Alexa488 (Molecular Probes), Cy2, or FITC (Jackson Immunoresearch) secondary antibodies. Before processing for double-label immunofluorescence microscopy, cells were washed in F-12 nutrient medium. Cells were incubated with primary antibodies to the surface markers CD11b at room temperature followed by three 2 min washes with F-12. Cells were then incubated with fluorescently coupled secondary antibodies for 30 min followed by three washes with PBS containing Ca++/Mg++. Surface-labelled cells were fixed for 10 min in 4% paraformaldehyde followed by PBS washes, permeabilized with PBS/TX (PBS with Ca++/Mg++, 0.5% Triton-X) for 5 min, and successively washed with PBS/TX/GS (PBS with Ca++/Mg++, 0.5% Triton-X, 2.0% normal goat serum) three times for 5 min each. Cells were incubated for 30 min with the astrocytic marker GFAP, washed three times with PBS/TX, labelled with an appropriate secondary antibody, and stained with DAPI (1 : 500 diluted in PBS without Ca++/Mg++ from 5 μg/mL stock solutions) for 5 min. Cells were then washed, mounted using Vectashield (Vector Laboratories), and visualized by fluorescence microscopy (Olympus I X-80) with a 20 PlanApo objective (1.0 numerical aperture). Images were acquired with a Hamamatsu ORCA CCD Camera and data were analyzed by using Image-Pro software.


### 2.13. Infection of Primary Microglial Cells with RSA59

On day 2 after seeding, neonatal microglial cultures were infected at a multiplicity of infection (MOI) of 2 : 1 with RSA59 or mock-infected with noninfected cell lysate. After allowing viral adsorption for 1 hr, cells were washed and placed in fresh media without virus. At 6, 12, and 24 hrs after infection, cultures were examined by microscopy for EGFP fluorescence.


### 2.14. RSA59 Growth Curve

Confluent monolayers of L2 cells were infected with undiluted and 1 : 2 diluted culture supernatant collected from thein vitro infected microglia and incubated for 1 hr at 37°C. Following adsorption, the cells were washed with Tris-Buffer saline 3 times and then fed with DMEM with 20% FBS mixed with 0.4% agarose for overlaying. 48 hours postinfection, culture was subjected for plaque count [32].


## 2.1. Ethics Statement

Use of animals and all experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the Indian Institute of Science Education and Research Kolkata and Indian Institute of Science, Bangalore India. Animal protocols adhered to the guidelines of the CPCSEA, India.


## 2.2. Virus

RSA59 an isogenic recombinant demyelinating strain of MHV-A59, where the spike gene (encodes virus host-attachment protein), was exchanged by MHV-A59 spike gene only in the background of MHV-A59 gene by targeted RNA recombination as described in our previous studies [18, 19]. This recombinant strain also expresses enhanced green fluorescence protein (EGFP) [32] for easy detection of viral particle by EGFP fluorescence.To engineer the targeted recombinant strains, molecularly cloned vector pMH54 [33, 34], which contains the entire 3′ end of the genome from MHV-A59, was used for construction of the recombinant viruses. RSA59 and RSMHV2 are isogenic except the spike protein. RSA59 strain is expressing the MHV-A59 spike in the MHV-A59 background, whereas RSMHV2 strain is expressing the MHV-2 spike in the MHV-A59 background [18]. Furthermore, EGFP gene was inserted into the MHV genome in place of the nonessential gene 4 in both RSA59 and RSMHV2 [19].In order to replace gene 4 with the EGFP gene, pMH54 was modified by the introduction of a SalI site 42 nucleotides downstream of the intergenic sequence for gene 4a and a NotI site 102 bp upstream of the stop codon for gene 4b, using the Quick Change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). (These are coding-silent nucleotide changes.) The coding sequence of EGFP was cleaved from the pEGFP-N1 vector (Clontech, Palo Alto, CA, USA) using SalI and NotI and inserted in the place of the SalI/NotI fragment of pMH54. The resulting plasmid contains 760 bp of non-MHV sequence, including the 722-bp EGFP open-reading frame, replacing the entire gene 4a and the rest 213 bp of gene 4b.Previous studies reported with JHM strain revealed that the interruption of the ORF 4 did not alter the neurovirulence neitherin vivo nor the replication in vitro [35]. Targeted recombination was used to select MHV isolates with stable and efficient expression of the gene encoding EGFP to facilitate the in vivo detection of virus in the mouse CNS as well as to trace the viral entry and spread in tissue culture. The viruses replicated with similar kinetics as wild-type virus both in tissue culture and in the mouse CNS. They caused similar encephalitis and demyelination in animals as the wild-type virus or their recombinant strains; however, they were somewhat attenuated in virulence [19].


## 2.3. Inoculation of Mice

Four-week-old, ten MHV-free, C57BL/6 (B6) mice (Jackson Laboratory, obtained from IISc, Bangalore, India) were inoculated intracranially with 50% LD50 dose of RSA59 strain (20,000 PFU) as described previously [19, 32]. Mice were monitored daily for signs of disease. Three mock-infected controls were inoculated similarly but with an uninfected cell lysate at a comparable dilution.  Three mice were sacrificed in between days 5, 6, or 7 (period for peak of inflammation postinfection for routine paraffin-based histopathological analysis), and the other three were used for frozen sections. The rest of the infected mice were sacrificed at day 30 postinfection for routine paraffin-based histopathological analysis. Cervical, thoracic, and lumbar regions of spinal cord were successively processed, and 4 quadrants (dorsal/posterior column, anterior column, and two anterior horns) from two separate sections of each spinal cord level were examined.


## 2.4. Histopathology

At 5, 6, or 7 and 30 days postinfection, brain and spinal cord tissues were harvested from both mock infected and RSA59-infected mice. For routine paraffin sectioning, brain and spinal cord tissues were postfixed in 4% PFA for overnight. Fixed tissues were processed and 5 micron thin sections were prepared for routine CNS pathology, whereas frozen sections tissues were postfixed with 4% PFA for 4−6 hours and then transferred in 4% sucrose solution for 4–6 hours and in 20% sucrose solution for 16–24 hours and mounted in Cryomatrix (Thermo Shandon). Ten micron thin sections were prepared for frozen tissue immunofluorescence. The paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) to determine the inflammation, whereas Luxol Fast Blue (LFB) staining was used to detect the loss of myelin sheath. All slides are coded and read in blind manner.


## 2.5. Immunohistochemical Analysis

Serial sections from brain and spinal cord were stained by the avidin-biotin-immunoperoxidase technique (Vector Laboratories) using 3,3-diaminobenzidine as substrate and a 1 : 100 dilution of anti-Iba1 (Wako, Richmond, VA, USA), 1 : 100 dilution of anti-CD45 (LCA; leukocyte common antigen, LY-5, BD Pharmingen), anti-Iba1 (Wako, Richmond, VA, USA), or CD3 (Dako; Carpinteria, CA, USA), and 1 : 20 dilution of monoclonal antibody directed against the nucleocapsid protein (N) of MHV-JHM (monoclonal antibody clone 1-16-1 (kindly provided by Julian Leibowitz))  as primary antibodies. Control slides from mock-infected mice were incubated in parallel.


## 2.6. Immunofluorescence and Fluorescence Microscopy

Cryosections from the spinal cord tissues were washed with PBS at room temperature in a humidified chamber, incubated for 10 min at room temperature with 1 mg/mL NaBH4 in PBS to reduce autofluorescence, washed, incubated for 1 h at room temperature with 1 M glycine in PBS to reduce nonspecific cross-linking, and then washed subsequently with PBS, PBS with 0.5% Triton X-100 (TX), and PBS with TX and 2% goat serum (GS). The sections were incubated overnight at 4°C with a 1 : 100 dilution of a rabbit anti-Iba1 antibody diluted in PBS with TX and GS, washed, and then incubated with a secondary antiserum diluted into PBS with GS for 2 hrs at 37°C. All incubations were carried out in a humidified chamber. Viral antigen was detected by EGFP in a fluorescein isothiocyanate channel [19]. Control slides were incubated in parallel with preimmune rabbit sera, and sections from mock-infected mice were incubated with secondary antibodies only. Tissue sections were sequentially washed with PBS plus TX and with PBS and mounted and visualized by IX-81 fluorescence microscopy with a 40x UPlanApo objective, with the iris diaphragm partially closed to limit the contribution of out-of-plane fluorescence, and with filter packs suitable for green fluorescence and red fluorescence. Images were acquired with a Hamamatsu Orca-1 charge-coupled device camera and Image-Pro image analysis software (Media Cybernetics, Silver Spring, MD, USA).


## 2.7. Transmission Electronic Microscopic Observation for the Accumulation of Microglia in the Demyelinating Plaque during Chronic Inflammatory Stage of RSA59 Infection

To further characterize the presence of microglia in the chronic demyelinating plaque at the ultrastructural level, mice were anesthetized, perfused with 4% PFA, and spinal cords from mock-infected and RSA59-infected were harvested and fixed overnight in 2% glutaraldehyde as described earlier [20]. Samples for transmission electron microscopy (TEM) were postfixed with 1% osmium tetroxide, dehydrated, and flat-embedded in Poly-Bed 812 epoxy resin (Polysciences). Half micrometer thick sections were cut from the lesional epicenter, stained with toluidine blue, and examined by light microscopy. Ultrathin TEM sections (600 Å) were cut from representative foci of demyelination from the toluidine blue-stained semithin sections and mounted on 200 mesh copper grids, stained with uranyl acetate and bismuth subnitrate, and viewed under a JEOL JEM 1010.


## 2.8.Ex Vivo Slice Culture

Four-week-old, MHV-free, C57BL/6 were perfused transcardially with sterile PBS. Spinal cord was harvested and washed with PBS containing 1% penicillin/streptomycin (Pen/Strep). The spinal cord was then embedded in 2% agarose mould, and 200 micron thick cross-sections were prepared by Vibratome (Leica Vibrating Blade Microtome; VT1200S). The slices were washed twice with PBS containing 1% Pen/Strep. The slices were then transferred to a 24-well plate with one slice in each well. 500μL of DMEM containing 10% FBS, 1% Pen/Strep, and 1% L-Glutamine were added in each well and incubated overnight with 5% CO2.


## 2.9. Viral Infection and Staining of Spinal Cord Slices

After 24 hrs of explantation, slices were infected with RSA59 at 20,000 PFU (Half of the LD50 dose) in low serum (2%) containing medium for 1 hr and then washed with PBS to remove the unbound viruses, and 10% serum containing medium were added to the infected culture and the cultures were maintained for 72 hrs. At 24 hrs, 48 hrs, and 72 hrs of postinfection, slices were processed for immunostaining with anti-Iba1 antibody, and EGFP fluorescence was used to detect viral antigen. Briefly, at different times postinfection slices were washed gently with PBS and fixed with 4% PFA for 2 hours. Postfixed slices were washed with PBS, permeabilized with 0.5% Triton X-100 for 5 mins, and blocked with 1% goat serum for 1 hr at RT followed by overnight incubation with anti-Iba1 antibody. For better staining, next day antibody solution was replaced with fresh antibody and incubated at 4°C for additional 16–20 hrs. Slices were washed to remove any unbound antibody and then labelled with TRITC conjugated goat anti-rabbit IgG for 16 hrs. Labelled slices were then washed to remove any unbound fluorescent tagged antibody and then mounted in Vectashield (Vector Laboratories, CA, USA with DAPI and observed in Zeiss Confocal Microscope (LSM710). Images were acquired and processed by using Zen2010 software (Carl Zeiss).


## 2.10. Isolation of Mixed Glial Cells from Neonatal Mice Brain

Primary cultures of mixed glia from day 0 to day 3 newborn mice were prepared as described previously [36]. Briefly, following the removal of meninges, brain tissues were minced and incubated in a rocking water bath at 37°C for 30 min in Hanks Balanced Salt Solution (HBSS, GIBCO) in the presence of 300 μg/mL of DNaseI (Sigma) and 0.25% trypsin (Sigma). Enzyme-digested-dissociated cells were triturated with 0.25% of fetal calf serum (FCS), followed by a wash and centrifugation (300 ×g for 10 min). The pellet was resuspended in HBSS, passed through a 70 micron nylon mesh, followed by a second wash and centrifugation (300 ×g for 10 min). Following dilutions with astrocyte-specific medium (Dulbecco’s essential medium containing 1% penicillin-streptomycin, 0.2 mM l-glutamine, and 10% FCS), cells were plated and allowed to adhere for 1 day in a humidified CO2 incubator at 37°C. After 24 hrs, any nonadherent cells were removed and fresh astrocyte-specific medium was added. Adherent cells were maintained in astrocyte-specific medium for 10 days.


## 2.11. Isolation and Enrichment of Microglia from Mixed Glial Culture

After establishment of the mixed glia culture, feeding was stopped for 10 days to allow for significant microglial growth on top of the astrocyte monolayer. The microglia population peaked at 12–14 days in these cultures. To remove any cells adherent to the astrocyte monolayer, microglia-enriched cultures were thoroughly agitated in an orbital incubator shaker (200 rpm for 40 min at 37°C). Immediately following agitation, all cells suspended in the culture medium were collected and centrifuged at 300 ×g for 5 min at 4°C. The cell pellet was resuspended and diluted with fresh astrocyte-specific medium bringing the cells to a final concentration of 8 × 105 cells/mL; 1 mL was added to each well of a two-well CC2-treated chamber slide (specifically made for primary cell culture; NUNC) or 2 mL/well of a six-well plate. After 30 min, any non-adherent cells were discarded and adherent cells were maintained in fresh astrocyte-specific medium until infected with a medium change every 3-4 days.


## 2.12. Characterization of Microglia in a Mixed Glial Culture

To examine different cell types in a given culture, primary antibodies directed against cell-specific antigens were used to determine the presence and/or purity of each of the major glial cell types as described previously. Microglia were labelled with biotinylated anti-mouse CD11b (Chemicon, diluted 1 : 100 in F-12 nutrient medium) followed by Cy3-streptavidin (Jackson Immunoresearch, diluted 1 : 200 in F-12). Astrocytes were labelled with polyclonal rabbit anti-glial fibrillary acidic protein (anti-GFAP; DAKO) followed by either goat anti-rabbit Alexa488 (Molecular Probes), Cy2, or FITC (Jackson Immunoresearch) secondary antibodies. Before processing for double-label immunofluorescence microscopy, cells were washed in F-12 nutrient medium. Cells were incubated with primary antibodies to the surface markers CD11b at room temperature followed by three 2 min washes with F-12. Cells were then incubated with fluorescently coupled secondary antibodies for 30 min followed by three washes with PBS containing Ca++/Mg++. Surface-labelled cells were fixed for 10 min in 4% paraformaldehyde followed by PBS washes, permeabilized with PBS/TX (PBS with Ca++/Mg++, 0.5% Triton-X) for 5 min, and successively washed with PBS/TX/GS (PBS with Ca++/Mg++, 0.5% Triton-X, 2.0% normal goat serum) three times for 5 min each. Cells were incubated for 30 min with the astrocytic marker GFAP, washed three times with PBS/TX, labelled with an appropriate secondary antibody, and stained with DAPI (1 : 500 diluted in PBS without Ca++/Mg++ from 5 μg/mL stock solutions) for 5 min. Cells were then washed, mounted using Vectashield (Vector Laboratories), and visualized by fluorescence microscopy (Olympus I X-80) with a 20 PlanApo objective (1.0 numerical aperture). Images were acquired with a Hamamatsu ORCA CCD Camera and data were analyzed by using Image-Pro software.


## 2.13. Infection of Primary Microglial Cells with RSA59

On day 2 after seeding, neonatal microglial cultures were infected at a multiplicity of infection (MOI) of 2 : 1 with RSA59 or mock-infected with noninfected cell lysate. After allowing viral adsorption for 1 hr, cells were washed and placed in fresh media without virus. At 6, 12, and 24 hrs after infection, cultures were examined by microscopy for EGFP fluorescence.


## 2.14. RSA59 Growth Curve

Confluent monolayers of L2 cells were infected with undiluted and 1 : 2 diluted culture supernatant collected from thein vitro infected microglia and incubated for 1 hr at 37°C. Following adsorption, the cells were washed with Tris-Buffer saline 3 times and then fed with DMEM with 20% FBS mixed with 0.4% agarose for overlaying. 48 hours postinfection, culture was subjected for plaque count [32].


## 3. Results

### 3.1. CNS Pathology of RSA59

To confirm the RSA59-induced CNS inflammation, brain and spinal cord sections from day 7 (peak of inflammation) and day 30 (peak of demyelination) postinfected mice were stained with H&E or LFB and examined. RSA59-induced meningitis (Supplementary Figure 1(a)), and encephalomyelitis (accumulation of inflammatory cell and perivascular cuffing) (Supplementary Figures1(b) and 1(c)) were observed as shown previously [18, 19] (Supplementary Figure 1; these data are partly published but for the ready information compiled in one figure.). To characterize inflammatory cell types, brain sections from day 7 postinfection were stained immunohistochemically with anti-CD45 (leukocyte common antigen (LCA)), anti-CD11b and/or anti-Iba1 (macrophage/microglial marker), or anti-CD3 (pan T-cell marker) (data not shown). The majority of inflammatory cells in RSA59-infected brains were immunoreactive for both LCA (Supplementary Figure 1(d)) and CD11b (Supplementary Figure 1(e)) and Iba1 (Supplementary Figure 1(f)). Some CD3-stained infiltrating T cells were also found (data not shown), although nonspecific background staining of neurons with available anti-CD3 antibodies made quantification difficult. No CD4- and CD19-positive cells but few CD8-positive cells were observed in the inflamed brain and spinal cord sections in RSA59-infected mice (data not shown). Demyelination was observed by LFB staining as early as day 7 as examined (Supplementary Figure 1(h)) and it reaches its peak at day 30 postinfection (Supplementary Figure 1(i)) as observed earlier [20, 31]. LFB-stained spinal cord section showed no myelin loss (Supplementary Figure 1(g)). Together, the data indicate that RSA59 causes meningoencephalitis and demyelination. CNS inflammation consists of a mixed population of inflammatory cells, predominantly macrophages/microglia as well as a smaller population of T lymphocytes as shown previously [17, 20, 37].


### 3.2. Direct Infection of CNS Resident Microglia in RSA59 Infection during Acute Inflammation

Previously, it has been demonstrated that neurotropic strains of MHV can directly infect different neural cell types [16, 19, 38, 39] but there is no evidence whether neurotropic strain can directly infect microglia or only acquire activity indirectly due to the infection of other neural cell types. In order to determine the tropism of RSA59 in CNS resident microglia, four-week-old, MHV-free, C57BL/6 (B6) mice (Jackson Laboratory) were inoculated intracranially with RSA59. Mice were sacrificed at the peak of inflammation (day 6), and the spinal cord sections were prepared for cryostat sectioning. Since RSA59 expresses EGFP, viral antigen was viewed directly by fluorescence microscopy. Identification of CNS resident microglia was performed by using Iba1 as a specific marker for microglia/macrophages [40]. While Iba1 immunofluorescence was observed in both gray and white matter, double fluorescence/immunofluorescence demonstrated dual labelling of EGFP (viral antigen) positive Iba1 positive microglia/macrophages were present only in the white matter of RSA59 infected mice (Figure 1). In the white matter, all the microglia (Iba1-positive) were not infected as shown by arrowheads (Figures 1(e) and 1(f)). In the control mock infected spinal cord section, no double fluorescent labelled cells were observed as expected (data not shown).Colocalization of EGFP-expressing cells (positive for viral antigen) and microglia/macrophages (CD11b) in spinal cord white matter (dorsal columns) of RSA59-infected mice. RSA59-infected mice (low magnification (a–c); high magnification (d–f)). EGFP fluorescence (green) identifies virus-infected cells (a, d) and anti-CD11b (red fluorescence) detects microglia/macrophages (b, e). Merged images (c, f) in RSA59-infected spinal cord show colocalization of EGFP in CD11b-positive cells in spinal cord white matter. Arrows mark CD11b-positive cells that are infected (EGFP-positive), whereas arrowheads mark CD11b-positive cells that are not infected (EGFP-negative).
(a)
(b)
(c)
(d)
(e)
(f)


### 3.3. Trafficking of Microglia from Gray Matter to the White Matter with Different Days of Postinfection of RSA59

Previous studies demonstrated that with time of postinfection viral antigen spread from gray matter to white matter [19] in the infected mice. This phenomenon is more prevalent in the spinal cord of infected mice as gray and white matter is clearly separated from white matter. Immunostained section demonstrated that the viral antigen is localized both in gray and white matter at day 7 postinfection (Figure 2(a)). At day 30 viral antigen is below the detection level, more specifically after day 10 postinfection (as observed) viral antigen is below the detection limit (data not shown) as discussed previously [18, 19]. To determine whether microglia also follow the trajectory of the viral spread at days 7 and 30 postinfection, spinal cord tissues sections were immunostained with anti-Iba1 antibody. While Iba1-positive cells were scattered in both gray and white matter at day 7 (Figure 2(b)), at day 30 postinfection Iba1-positive cells are mainly localized to the white matter (Figures 2(c) and 2(e)). At day 30, most of the Iba1 positive cells are present in the inflammatory demyelinating plaques as observed in the LFB-stained serial sections (Figures 2(d) and 2(f)). If any Iba1-positive cells are present in the gray matter, they are mainly reverted to their quiescent stage (as observed by morphology). Time-dependent trafficking of Iba1-positive cells demonstrate that microglia follow the track of viral antigen spread.Distribution of viral antigen and microglia in the spinal cord section of RSA59-infected mice at different days post infection. Mice were inoculated intracranially with RSA59 or mock-infected and sacrificed at day 7 and day 30 postinfection. Spinal cord sections were stained with antinucleocapsid antiserum and/or anti-Iba1 antisera. At day 7 postinfection, viral antigen is present both in gray and white matter but predominantly in the white matter (a). Similarly, at day 7 postinfection, Iba1-positive cells are scattered throughout the gray and white matter with a tendency of white matter accumulation similar to viral antigen (b). At day 30 post infection majority of the Iba1 positive cell-(c, e) are present in the demyelination plaque (LFB-stained section (d, f)). Day 30 spinal cord sections are shown from two different RSA59-infected mice. Arrows show accumulation of Iba1 in (c, e) and demyelinating plaque in (d, f). Arrow, indicate Iba1-positive cells in the demyelination plaque.
(a)
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(f)


### 3.4. High-Resolution Electron Micrographic Analysis Confirms the Presence of Vacuolated Microglia with Engulfed Myelin Debris in Demyelinating Plaque

Previously microglial accumulation was observed in the demyelinating plaque of RSA59 with an emphasis on the stripping of the myelin sheath [20]. To reemphasize on the accumulation of microglia in the demyelination plaque during chronic phase of the inflammation at ultrastructural level, semithin sections were cut at 1 micron intervals from five infected mice at day 30 post infection. Semithin sections were stained with toluidine blue. Control mock-infected mouse spinal cord was used to evaluate for background fixation and/or postfixation artefacts (Supplementary Figure 2(a)). RSA59-infected spinal cords showed significant myelin loss and accumulation of phagocytotic microglia within plaques as observed earlier (Supplementary Figures 2(b) and 2(c)). Representative foci of demyelination were selected from semithin sections, and 600 Å ultrathin sections from Poly-Bed embedded blocks were processed for TEM. High-resolution TEM images show accumulation of large number of microglia with no basement membrane which is the characteristic features of microglia/macrophages (Supplementary Figure 2(e)). Multiple vacuoles with myelin fragments were seen within the cytoplasm of the microglia in the plaque (Supplementary Figure 2(f)). No such microglial accumulation was observed in the control mock infected mice at high-resolution TEM images (Supple Figure 2(d)).


### 3.5. RSA59 Can Infect Microglia inEx Vivo Spinal Cord Explants Culture in Absence of Any Peripheral Inflammatory Cells

In vivo colocalization of Iba1 with EGFP-(viral antigen) positive cells demonstrated that RSA59 can directly infect microglia but that does not confirm that infected microglia were resident microglia because in intracranial (IC) inoculation blood brain barrier can be disrupted and blood monocytes/macrophages can migrate and acquire infection. To exclude this possibility, 200 micron thick explants cultures were prepared from the four-week-old, C57BL/6 mice spinal cord and infected with RSA59. At 24, 48 and 72 hrs of postinfection explants, slices were processed for fluorescent-immunostaining with anti Iba1-antibody and EGFP fluorescence was used to detect viral antigen. Confocal microscopic observation of double label immunofluorescence slices demonstrated that in absence of any peripheral immune cell, RSA59 can directly infect resident microglia. At 24 hrs post infection very few microglia were positive for viral antigen (EGFP-positive) (Figure 3(c)) but the number of double positive cells was increased with time as examined in 48 and 72 hrs (Figures 3(g) and 3(k)) which demonstrated that CNS resident microglia can directly acquire infection and become activated (by morphological analysis as number of processes increased and enlarged). Arrowheads in Figures 3(b), 3(c), 3(d), 3(f), 3(g), 3(h), 3(j), 3(k), and 3(l) showed that some of the resident microglia did not get infection. Control noninfected explant cultures were also immunolabeled with anti-Iba1 antibody (Figures 3(a), 3(e), and 3(i)) as microglia in vitro in culture system behave like activated macrophages due to perturbation of the culture system. Figures 3(d), 3(h), and 3(l) show a merged image of EGFP (viral antigen; green), Iba1 (microglia; red), and DAPI (nucleus; blue) and demonstrate the presence of viral antigen in the cell cytoplasm of microglia.RSA59 can directly infect CNS resident microglia in the spinal cord explants culture in absence of any peripheral immune cells. Two hundred micron thick slice cultures were prepared from 4-week old C57BL/6 mice. 24 hrs after postexplantation, cultures were infected with 20,000 PFU/mL of RSA59 and incubated for 24 hrs, 48 hrs, and 72 hrs. Mock-infected cultures were also maintained in the same culture condition for the same period post explantation.  24, 48, and 72 hrs postinfection mock and RSA59-infected slices were stained with Iba1. Iba1 staining (red fluorescence) shows CNS resident microglia in mock-infected (a, e, i) and RSA59-infected slice culture (b, f, j). Merged images (c, d, g, h, k, l) show colocalization of EGFP in Iba1-positive cells of RSA59 infected spinal cord white matter. EGFP fluorescence (green) identifies virus-infected cells, anti-Iba1 (red fluorescence) detects microglia and DAPI (blue fluorescence) stains nucleus in the merged images.
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(g)
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(l)Due to the thickness of the slices, clarity of the images was slightly compromised. RSA59 infection inex vivo explant cultures demonstrated that in the absence of peripheral immune cells CNS resident microglia can directly be infected.


### 3.6. RSA59 Can Infect Neonatal Microglia Cells and Forms Syncytia

In order to determine whether RSA59 can infect microglial cellsin vitro in absence of any neural cells, 2-day-old neonatal microglial cultures were infected at a multiplicity of infection (MOI) of 2 : 1 with RSA59 or mock infected with noninfected cell lysate. Microglia harvested in the cell suspension by the conventional shake-off method as described earlier [36] were 99±0.5% positive for CD11b staining (Figure 4(a)). Very few GFAP (astrocyte marker) positive cells were observed in the isolated microglia culture (data not shown). At 0, 6, 12, and 24 hrs after infection, cultures were examined by microscopy for EGFP fluorescence. At 0 and 6 hrs, no fluorescence was observed in the infected culture but at 12 hrs bright fluorescence started to appear denoting the presence of viral antigen in the microglia. At 12 hrs postinfection, infected microglia demonstrated stressed morphology and started to fuse with the neighbouring cells, and at 24 hrs postinfection, most of the infected cells were involved into large syncytia formation (Figure 4(c)) which is a characteristic of some enveloped RNA viruses and more specifically characteristic of MHV-A59 (parental strain of RSA59), an enveloped demyelinating strain of MHV [41]. Nucleus of the syncytia was very obvious as shown in Figure 4(b) by DAPI staining. In vitro experiment demonstrated that RSA59 can infect primary microglia in isolated culture and can also induce syncytia in primary microglia.Infection of microglia culture (free from astrocytes) with RSA59 demonstrates syncytia formation. Purified microglial cultures from the neonatal mixed glia were stained with anti-CD11b conjugated to Cy3 and anti-GFAP (astrocyte marker). Three different cultures were immunostained but the best representative culture is shown in (a). Immunostained culture demonstrates that majority of the isolated cells are microglia (positive for CD11b-Cy3) and negative for astrocytes (GFAP-negative). Purified cultures were infected with RSA59 at 2 : 1 M.O.I, and after 24 hrs postinfected cultures were observed under epifluorescence microscope. At 24 hrs postinfection, the majority of the cells were infected and most of the infected cells form syncytia (b, c). DAPI staining of infected cells shows the nucleus in the syncytia (b) and EGFP fluorescence demonstrates the RSA59 infection (c). Five culture plates were infected but the representative infected culture is shown here.
(a)
(b)
(c)


### 3.7. Viral Growth Curve

To demonstrate that the virus is replicating in the microglia, culture supernatant of infected microglia was assessed by routine plaque assay [32]. Routine plaque assay found very few plaques which were below the detection limit. But there, discrete syncytia was observed in the infected plates which denoted that the titer could be 30–50 PFU/mL.


## 3.1. CNS Pathology of RSA59

To confirm the RSA59-induced CNS inflammation, brain and spinal cord sections from day 7 (peak of inflammation) and day 30 (peak of demyelination) postinfected mice were stained with H&E or LFB and examined. RSA59-induced meningitis (Supplementary Figure 1(a)), and encephalomyelitis (accumulation of inflammatory cell and perivascular cuffing) (Supplementary Figures1(b) and 1(c)) were observed as shown previously [18, 19] (Supplementary Figure 1; these data are partly published but for the ready information compiled in one figure.). To characterize inflammatory cell types, brain sections from day 7 postinfection were stained immunohistochemically with anti-CD45 (leukocyte common antigen (LCA)), anti-CD11b and/or anti-Iba1 (macrophage/microglial marker), or anti-CD3 (pan T-cell marker) (data not shown). The majority of inflammatory cells in RSA59-infected brains were immunoreactive for both LCA (Supplementary Figure 1(d)) and CD11b (Supplementary Figure 1(e)) and Iba1 (Supplementary Figure 1(f)). Some CD3-stained infiltrating T cells were also found (data not shown), although nonspecific background staining of neurons with available anti-CD3 antibodies made quantification difficult. No CD4- and CD19-positive cells but few CD8-positive cells were observed in the inflamed brain and spinal cord sections in RSA59-infected mice (data not shown). Demyelination was observed by LFB staining as early as day 7 as examined (Supplementary Figure 1(h)) and it reaches its peak at day 30 postinfection (Supplementary Figure 1(i)) as observed earlier [20, 31]. LFB-stained spinal cord section showed no myelin loss (Supplementary Figure 1(g)). Together, the data indicate that RSA59 causes meningoencephalitis and demyelination. CNS inflammation consists of a mixed population of inflammatory cells, predominantly macrophages/microglia as well as a smaller population of T lymphocytes as shown previously [17, 20, 37].


## 3.2. Direct Infection of CNS Resident Microglia in RSA59 Infection during Acute Inflammation

Previously, it has been demonstrated that neurotropic strains of MHV can directly infect different neural cell types [16, 19, 38, 39] but there is no evidence whether neurotropic strain can directly infect microglia or only acquire activity indirectly due to the infection of other neural cell types. In order to determine the tropism of RSA59 in CNS resident microglia, four-week-old, MHV-free, C57BL/6 (B6) mice (Jackson Laboratory) were inoculated intracranially with RSA59. Mice were sacrificed at the peak of inflammation (day 6), and the spinal cord sections were prepared for cryostat sectioning. Since RSA59 expresses EGFP, viral antigen was viewed directly by fluorescence microscopy. Identification of CNS resident microglia was performed by using Iba1 as a specific marker for microglia/macrophages [40]. While Iba1 immunofluorescence was observed in both gray and white matter, double fluorescence/immunofluorescence demonstrated dual labelling of EGFP (viral antigen) positive Iba1 positive microglia/macrophages were present only in the white matter of RSA59 infected mice (Figure 1). In the white matter, all the microglia (Iba1-positive) were not infected as shown by arrowheads (Figures 1(e) and 1(f)). In the control mock infected spinal cord section, no double fluorescent labelled cells were observed as expected (data not shown).Colocalization of EGFP-expressing cells (positive for viral antigen) and microglia/macrophages (CD11b) in spinal cord white matter (dorsal columns) of RSA59-infected mice. RSA59-infected mice (low magnification (a–c); high magnification (d–f)). EGFP fluorescence (green) identifies virus-infected cells (a, d) and anti-CD11b (red fluorescence) detects microglia/macrophages (b, e). Merged images (c, f) in RSA59-infected spinal cord show colocalization of EGFP in CD11b-positive cells in spinal cord white matter. Arrows mark CD11b-positive cells that are infected (EGFP-positive), whereas arrowheads mark CD11b-positive cells that are not infected (EGFP-negative).
(a)
(b)
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(f)


## 3.3. Trafficking of Microglia from Gray Matter to the White Matter with Different Days of Postinfection of RSA59

Previous studies demonstrated that with time of postinfection viral antigen spread from gray matter to white matter [19] in the infected mice. This phenomenon is more prevalent in the spinal cord of infected mice as gray and white matter is clearly separated from white matter. Immunostained section demonstrated that the viral antigen is localized both in gray and white matter at day 7 postinfection (Figure 2(a)). At day 30 viral antigen is below the detection level, more specifically after day 10 postinfection (as observed) viral antigen is below the detection limit (data not shown) as discussed previously [18, 19]. To determine whether microglia also follow the trajectory of the viral spread at days 7 and 30 postinfection, spinal cord tissues sections were immunostained with anti-Iba1 antibody. While Iba1-positive cells were scattered in both gray and white matter at day 7 (Figure 2(b)), at day 30 postinfection Iba1-positive cells are mainly localized to the white matter (Figures 2(c) and 2(e)). At day 30, most of the Iba1 positive cells are present in the inflammatory demyelinating plaques as observed in the LFB-stained serial sections (Figures 2(d) and 2(f)). If any Iba1-positive cells are present in the gray matter, they are mainly reverted to their quiescent stage (as observed by morphology). Time-dependent trafficking of Iba1-positive cells demonstrate that microglia follow the track of viral antigen spread.Distribution of viral antigen and microglia in the spinal cord section of RSA59-infected mice at different days post infection. Mice were inoculated intracranially with RSA59 or mock-infected and sacrificed at day 7 and day 30 postinfection. Spinal cord sections were stained with antinucleocapsid antiserum and/or anti-Iba1 antisera. At day 7 postinfection, viral antigen is present both in gray and white matter but predominantly in the white matter (a). Similarly, at day 7 postinfection, Iba1-positive cells are scattered throughout the gray and white matter with a tendency of white matter accumulation similar to viral antigen (b). At day 30 post infection majority of the Iba1 positive cell-(c, e) are present in the demyelination plaque (LFB-stained section (d, f)). Day 30 spinal cord sections are shown from two different RSA59-infected mice. Arrows show accumulation of Iba1 in (c, e) and demyelinating plaque in (d, f). Arrow, indicate Iba1-positive cells in the demyelination plaque.
(a)
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(d)
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(f)


## 3.4. High-Resolution Electron Micrographic Analysis Confirms the Presence of Vacuolated Microglia with Engulfed Myelin Debris in Demyelinating Plaque

Previously microglial accumulation was observed in the demyelinating plaque of RSA59 with an emphasis on the stripping of the myelin sheath [20]. To reemphasize on the accumulation of microglia in the demyelination plaque during chronic phase of the inflammation at ultrastructural level, semithin sections were cut at 1 micron intervals from five infected mice at day 30 post infection. Semithin sections were stained with toluidine blue. Control mock-infected mouse spinal cord was used to evaluate for background fixation and/or postfixation artefacts (Supplementary Figure 2(a)). RSA59-infected spinal cords showed significant myelin loss and accumulation of phagocytotic microglia within plaques as observed earlier (Supplementary Figures 2(b) and 2(c)). Representative foci of demyelination were selected from semithin sections, and 600 Å ultrathin sections from Poly-Bed embedded blocks were processed for TEM. High-resolution TEM images show accumulation of large number of microglia with no basement membrane which is the characteristic features of microglia/macrophages (Supplementary Figure 2(e)). Multiple vacuoles with myelin fragments were seen within the cytoplasm of the microglia in the plaque (Supplementary Figure 2(f)). No such microglial accumulation was observed in the control mock infected mice at high-resolution TEM images (Supple Figure 2(d)).


## 3.5. RSA59 Can Infect Microglia inEx Vivo Spinal Cord Explants Culture in Absence of Any Peripheral Inflammatory Cells

In vivo colocalization of Iba1 with EGFP-(viral antigen) positive cells demonstrated that RSA59 can directly infect microglia but that does not confirm that infected microglia were resident microglia because in intracranial (IC) inoculation blood brain barrier can be disrupted and blood monocytes/macrophages can migrate and acquire infection. To exclude this possibility, 200 micron thick explants cultures were prepared from the four-week-old, C57BL/6 mice spinal cord and infected with RSA59. At 24, 48 and 72 hrs of postinfection explants, slices were processed for fluorescent-immunostaining with anti Iba1-antibody and EGFP fluorescence was used to detect viral antigen. Confocal microscopic observation of double label immunofluorescence slices demonstrated that in absence of any peripheral immune cell, RSA59 can directly infect resident microglia. At 24 hrs post infection very few microglia were positive for viral antigen (EGFP-positive) (Figure 3(c)) but the number of double positive cells was increased with time as examined in 48 and 72 hrs (Figures 3(g) and 3(k)) which demonstrated that CNS resident microglia can directly acquire infection and become activated (by morphological analysis as number of processes increased and enlarged). Arrowheads in Figures 3(b), 3(c), 3(d), 3(f), 3(g), 3(h), 3(j), 3(k), and 3(l) showed that some of the resident microglia did not get infection. Control noninfected explant cultures were also immunolabeled with anti-Iba1 antibody (Figures 3(a), 3(e), and 3(i)) as microglia in vitro in culture system behave like activated macrophages due to perturbation of the culture system. Figures 3(d), 3(h), and 3(l) show a merged image of EGFP (viral antigen; green), Iba1 (microglia; red), and DAPI (nucleus; blue) and demonstrate the presence of viral antigen in the cell cytoplasm of microglia.RSA59 can directly infect CNS resident microglia in the spinal cord explants culture in absence of any peripheral immune cells. Two hundred micron thick slice cultures were prepared from 4-week old C57BL/6 mice. 24 hrs after postexplantation, cultures were infected with 20,000 PFU/mL of RSA59 and incubated for 24 hrs, 48 hrs, and 72 hrs. Mock-infected cultures were also maintained in the same culture condition for the same period post explantation.  24, 48, and 72 hrs postinfection mock and RSA59-infected slices were stained with Iba1. Iba1 staining (red fluorescence) shows CNS resident microglia in mock-infected (a, e, i) and RSA59-infected slice culture (b, f, j). Merged images (c, d, g, h, k, l) show colocalization of EGFP in Iba1-positive cells of RSA59 infected spinal cord white matter. EGFP fluorescence (green) identifies virus-infected cells, anti-Iba1 (red fluorescence) detects microglia and DAPI (blue fluorescence) stains nucleus in the merged images.
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(l)Due to the thickness of the slices, clarity of the images was slightly compromised. RSA59 infection inex vivo explant cultures demonstrated that in the absence of peripheral immune cells CNS resident microglia can directly be infected.


## 3.6. RSA59 Can Infect Neonatal Microglia Cells and Forms Syncytia

In order to determine whether RSA59 can infect microglial cellsin vitro in absence of any neural cells, 2-day-old neonatal microglial cultures were infected at a multiplicity of infection (MOI) of 2 : 1 with RSA59 or mock infected with noninfected cell lysate. Microglia harvested in the cell suspension by the conventional shake-off method as described earlier [36] were 99±0.5% positive for CD11b staining (Figure 4(a)). Very few GFAP (astrocyte marker) positive cells were observed in the isolated microglia culture (data not shown). At 0, 6, 12, and 24 hrs after infection, cultures were examined by microscopy for EGFP fluorescence. At 0 and 6 hrs, no fluorescence was observed in the infected culture but at 12 hrs bright fluorescence started to appear denoting the presence of viral antigen in the microglia. At 12 hrs postinfection, infected microglia demonstrated stressed morphology and started to fuse with the neighbouring cells, and at 24 hrs postinfection, most of the infected cells were involved into large syncytia formation (Figure 4(c)) which is a characteristic of some enveloped RNA viruses and more specifically characteristic of MHV-A59 (parental strain of RSA59), an enveloped demyelinating strain of MHV [41]. Nucleus of the syncytia was very obvious as shown in Figure 4(b) by DAPI staining. In vitro experiment demonstrated that RSA59 can infect primary microglia in isolated culture and can also induce syncytia in primary microglia.Infection of microglia culture (free from astrocytes) with RSA59 demonstrates syncytia formation. Purified microglial cultures from the neonatal mixed glia were stained with anti-CD11b conjugated to Cy3 and anti-GFAP (astrocyte marker). Three different cultures were immunostained but the best representative culture is shown in (a). Immunostained culture demonstrates that majority of the isolated cells are microglia (positive for CD11b-Cy3) and negative for astrocytes (GFAP-negative). Purified cultures were infected with RSA59 at 2 : 1 M.O.I, and after 24 hrs postinfected cultures were observed under epifluorescence microscope. At 24 hrs postinfection, the majority of the cells were infected and most of the infected cells form syncytia (b, c). DAPI staining of infected cells shows the nucleus in the syncytia (b) and EGFP fluorescence demonstrates the RSA59 infection (c). Five culture plates were infected but the representative infected culture is shown here.
(a)
(b)
(c)


## 3.7. Viral Growth Curve

To demonstrate that the virus is replicating in the microglia, culture supernatant of infected microglia was assessed by routine plaque assay [32]. Routine plaque assay found very few plaques which were below the detection limit. But there, discrete syncytia was observed in the infected plates which denoted that the titer could be 30–50 PFU/mL.


## 4. Discussion

To understand the cellular mechanism of demyelination of neurotropic strain of MHV, prior studies in our laboratory have analyzed the detailed pathogenesis of recombinant MHV strain, RSA59 (demyelinating strain (DM)) and compared it with RSMHV2 (nondemyelinating strain (NDM)) that is isogenic except for the spike gene that encodes the virus-host-attachment spike glycoprotein [18, 20]. Both strains are capable of causing hepatitis, encephalitis, and meningitis. However, the two strains differ in their ability to induce subsequent demyelination and axonal loss [20]. Seven days post infection, RSA59 produces demyelination that is best observed in the spinal cord at day 30 postinfection (peak of inflammation). In contrast, RSMHV2 does not produce demyelination and only rarely demonstrates axonopathic changes in spinal cord white matter [20]. The inability of RSMHV2 to induce demyelination is due in part to a lack of transport of viral antigen (and the subsequent inflammatory reaction) to the white matter. Furthermore, in vivo and in vitro experiments demonstrate deficits in the ability of RSMHV2 to spread between neurons when compared to interneuronal spread by RSA59 [20]. RSA59-induced demyelination occurs in the setting of both axonal degeneration and macrophage mediated myelin stripping along intact axons [20]. While spike glycoprotein mediates spread of viral antigen to white matter through axonal transport, specific mechanisms leading to subsequent demyelination are not known. One plausible explanation is that MHV spreads intra-axonally within gray matter and when it reaches the white matter, viral particles may need to spread directly into oligodendrocytes, astrocyte, and microglia, using the spike protein, and can induce local oligodendroglial dystrophy and inflammation. Viral antigen in white matter axons may be sufficient to trigger an inflammatory response via microglial activation. Infected and activated microglia due to its intrinsic nature of chemotaxis can recruit more microglia to the site of inflammation and strip myelin from the damaged axon and successively cause demyelination.Our currentin vivo studies support this hypothesis that RSA59 can infect CNS resident microglia. The migration and activation of numerous microglia to the white matter during acute inflammation and the retention of microglia in the chronic inflammatory plaque reinforce the hypothesis that CNS resident microglia can be recruited to the region of local CNS injury. Ultrastructural morphology of microglia containing multiple vacuoles with myelin fragments in the cytoplasm in the demyelinating plaque further substantiate that CNS resident activated microglia can mediate myelin stripping and can successively mediate demyelination.As RSA59 spread intra-axonally, no colocalization was observed within the gray matter CNS resident microglia. If haematogenous propagation of peripheral monocytes/macrophages occurred to the CNS, one would expect more widespread distribution of activated microglia throughout the spinal cord which may not discriminate gray/white matter track. Furthermore, the delay in complete development of demyelination following partial resolution of encephalitis (up to 30 days after peak inflammation) documented in previous studies would not be expected [16, 18, 31]. Moreover, ex vivo colocalization of EGFP-positive cells with microglia confirmed that RSA59 can directly infect CNS resident microglia in absence of peripheral immune cells. In vitro infections of neonatal microglia demonstrate that RSA59 not only infects, but microglia can also forms syncytia which suggests that microglia supports RSA59 infection via cell-to-cell contact. Current combined in vivo, in vitro, and ex vivo explants culture studies established that the recruitment of microglia occurred from the CNS resident microglial pool rather than peripheral monocyte/macrophages.Our current studies are focused on the understanding of the innate immune mechanism of CNS resident microglia activation and maturation to perform phagocytotic activity.  Affymetrix microarray analyses for mRNA expression have revealed that expression of inflammatory mediators by MHV infected microglia, including chemokine and inflammatory cytokines. MHV infection of the mouse spinal cord was also associated with increased expression of genes involved in IFN signalling compared to mock-infected controls in the CNS. During chronic infection (day 30 postinfection), microglia are still present within areas of demyelination and microglia-associated inflammatory mediators are still produced which indicates that microglia are still active. Our results suggest that putative activated microglia and inflammatory mediators contribute to a local CNS microenvironment that eventually regulates viral replication and IFN-gamma production during acute phase of infection.  Sequentially, IFN-γ can activate microglia by promoting phagolysosomes maturation and activation (engulfment of the myelin sheath) leading to demyelination. Affymetrix microarray data warrants further confirmation.Viral infection in the CNS is classically recognized as inflammatory in nature, with meningeal perivascular and parenchymal infiltrates of peripheral leukocytes but RSA59 infection could be an exception where inflammation can proceed with CNS resident glial activation without involving the peripheral immune responses like Rabies virus infection [42], HIV infection [43], and prion diseases [44, 45]. In this perspective, it is tempting to speculate that the underlying mechanism of chronic myelin loss in MS could be a combination of persistence of myelin-related autoimmunogens that has escaped self-tolerance with persistence of activated CNS resident microglia which can mediate demyelination by phagocytised myelin.Microglia are known for their innate immune function for long time but the role of microglia in chronic inflammation opens a new episode in the field of glial biology of neuroinflammatory diseases. The concept of chronic inflammation as opposed to acute inflammation is more relevant in the context of understanding other CNS diseases, more specifically neurodegenerative diseases like Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, and Huntington’s disease. These neurodegenerative diseases lack the prominent infiltrates of mononuclear cells but the underlying mechanism of inflammation could be through activation of CNS resident microglia. Activation of CNS resident microglia in the context of chronic neuroinflammation as one of the underlying mechanism of neurodegeneration warrants further study. Microglia as the prime components of an intrinsic CNS resident immune system become a major focus in cellular neuroimmunology and, therefore, in neuroinflammation.


## 5. Conclusions

It has been known for long time that in absence of conventionalαβT cells microglia play a major role in neurotropic MHV-induced demyelination but the mechanism of infection and route of infection were not very clearly known for long time. Our current microglial tropism studies revealed that RSA59, an isogenic demyelinating strain of MHV, can infect and activate CNS resident microglia, and microglia can help to mediate demyelination by engulfing myelin debris. RSA59-induced neuroinflammatory models are helpful in understanding direct CNS cellular injury and demyelination that does not require an intact adaptive immune system. Understanding the role of direct CNS resident microglial infection and activation will shed some light on the pathogenesis of CNS inflammatory disease, not only infectious diseases but also chronic CNS disorders. The vision of CNS-resident-microglia-driven neuroinflammatory responses in RSA59 with neuropathological consequences has extended the avenue to explore the contribution of microglia in chronic neuroinflammatory CNS diseases.



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