key: cord-0004937-ajf9zig0
authors: Ray, N. B.; Power, C.; Lynch, W. P.; Ewalt, L. C.; Lodmell, D. L.
title: Rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes
date: 2014-03-07
journal: Arch Virol
DOI: 10.1007/s007050050136
sha: 5d513f9201701b2dd0913b01c3813eb88c2f43e5
doc_id: 4937
cord_uid: ajf9zig0

Recent studies have reported the detection of rabies viral antigens and virions in astrocytes and microglia of rabies-infected animals. As a first step toward understanding whether these glial cells may be involved in rabies virus replication, persistence, and/or pathogenesis, we explored their potential to be infected in vitro. Primary cultures of murine, feline, and human microglia and astrocytes were infected with several different rabies viruses: two unpassaged street virus isolates, a cell culture-adapted strain, and a mouse brain-passaged strain. Infection, as determined by immunofluorescence, was detected in 15 of the 16 (94%) virus-glial cell combinations. Replication of infectious virus, determined by infectivity assay, was detected in 7 of the 8 (88%) virus-cell combinations. These results show that astrocytes and microglia can be infected by rabies viruses, suggesting that they may have a potential role in disease, perhaps contributing to viral spread, persistence and/or neuronal dysfunction.

Summary. Recent studies have reported the detection of rabies viral antigens and virions in astrocytes and microglia of rabies-infected animals. As a ®rst step toward understanding whether these glial cells may be involved in rabies virus replication, persistence, and/or pathogenesis, we explored their potential to be infected in vitro. Primary cultures of murine, feline, and human microglia and astrocytes were infected with several different rabies viruses: two unpassaged street virus isolates, a cell culture-adapted strain, and a mouse brain-passaged strain. Infection, as determined by immuno¯uorescence, was detected in 15 of the 16 (94%) virus-glial cell combinations. Replication of infectious virus, determined by infectivity assay, was detected in 7 of the 8 (88%) virus-cell combinations. These results show that astrocytes and microglia can be infected by rabies viruses, suggesting that they may have a potential role in disease, perhaps contributing to viral spread, persistence and/or neuronal dysfunction. * Rabies viruses have been well documented to primarily infect neurons [19, 30, 33] . In addition, a number of studies have reported rabies viral antigens and virions in human [1, 13, 29] and murine astrocytes [10, 18, 23] , and in murine rami®ed microglial cells [27] , begging the question of the role of these cells in viral replication and persistence, as well as pathogenesis. Schneider [23] reported that glial cells of the pia mater appear to be involved in replication, and Matsumoto [18] indicated that astrocytes support virus replication in vivo. Tsiang also detected a minimal amount of virus when he infected a cloned mouse glioma cell line, but he did not detect virus when he attempted to infect primary glial cell cultures composed primarily of astrocytes [30] . Thus, it has not been clearly established whether rabies viruses productively infect microglia and astrocytes, leaving unanswered the question of whether these cells are susceptible to infection or simply absorb or phagocytose virus or cellular debris from infected neurons.

Evidence suggests that macrophages, which are related to microglial cells, and astrocytes play a critical role in many viral infections by supporting viral replication and sequestering viruses [2, 5, 6, 9, 11] . We have previously reported evidence for rabies virus replication and persistence in murine bone marrow-derived macrophages and in human macrophage-like cells [22] . It also has been shown that microglial cells and astrocytes infected by viruses, including vesicular stomatitis virus (VSV), may be involved in the disease process via secretion of cytokines and neurotoxins [3, 14, 32, 34] , including nitric oxide (NO) [28] . In the present study, as an initial step toward evaluation of the potential involvement of these glial cells in rabies virus infections, we have directly examined the ability of different rabies virus strains and isolates to infect and replicate in primary cultures of microglia and astrocytes.

Four rabies viruses were used: 1) The Evelyn-Rokitnicki-Abelseth (ERA) strain, which had been adapted to tissue culture through multiple serial passages in CER cells [15] ; 2) a street isolate (SRV) of bat origin [16] that had been serially passaged six times intracranially (ic) in mice; 3) an unpassaged isolate from a sheep brain; and 4) an unpassaged isolate from a skunk brain.

Murine, feline, and human glial cultures were used to examine the infection of glial cells from different species. Murine mixed glial cell cultures were prepared from brains of neonatal IRW mice by the method of Giulian and Baker [7] and incubated in RPMI-1640 medium (Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan, Utah) (RPMI-10), as previously described [17] . Microglia that were shaken off mixed glial cultures were incubated in a 50:50 mixture of RPMI-10 medium and conditioned RPMI-10 medium that had been harvested from one week old mixed glial cultures. Feline and human glial cultures were prepared in the same way with the exception that the cells were incubated in Dulbecco's MEM medium supplemented with 20% fetal calf serum. Mixed glial cultures were prepared from neonatal and adult cat brains, and human brain tissue derived from 17 week-old fetuses or adults during operative procedures for deep CNS lesions requiring the removal of healthy tissues [21] . Human fetal astrocyte cell cultures were prepared from mixed glial cultures as described [20] .

Microglia and astrocytes were identi®ed with speci®c reagents. Microglia were identi®ed by their ability to endocytose¯uorescent-labeled DiI-AcLDL [7] as well as by immuno¯uorescent staining with speci®c reagents including rat-anti-Mac-1 antibody for murine cells [22] , rabbit anti-human ferritin antibody for feline microglia [4] , and anti-human macrophage antibody CD68 [8] for human microglia. Murine microglia were also identi®ed by non-speci®c esterase staining and a phagocytosis assay [22] . Astrocytes of the three species were identi®ed with rabbit anti-bovine glial ®brillary acidic protein (GFAP) antibody [4] . All glial cultures were free of ®broblasts as determined by staining with anti-human ®bronectin antibody [20] . Rabies virus-infected cells were identi®ed by double immuno¯uorescent labeling as previously described [22] . Because infected cells did not deteriorate during the time course of these experiments, microglia containing viral antigens were considered to be infected and not to have phagocytosed and sequestered infected cell debris.

We ®rst examined the ability of rabies viruses to infect and replicate in neonatal murine glial cultures that were greater than 95% microglia. Infection of cells with either the tissue culture-adapted ERA strain, or mouse brainadapted virus resulted in substantial virus replication, evident by 48 h postinfection and increasing in titer throughout the course of the study (up to 192 h) (Fig. 1A) . The infected cells remained phagocytic, but appeared to phagocytose fewer Fluorescebrite (Polysciences, Inc., Warrington, PA) beads per cell than uninfected cells ( Fig. 2A) . Infected cells also were detected by double immuno¯uorescent staining ( Fig. 2B and 2C) . Unpassaged street viruses also infected murine microglia (Table 1 ). In addition, double immuno¯uorescent staining revealed that the ERA strain infected astrocytes present in the mixed glial cultures (Table 1 and Fig. 3) .

We next examined the ability of rabies viruses to infect and replicate in feline glial cells. Both the neonatal and adult cultures were greater than 80% virus was assayed on CER cells using a¯uorescent focus assay [24] microglia. The ERA strain of virus replicated well in adult microglia, with a greater than 100-fold increase in virus titer between 24 and 120 h. The mouse brain-passaged isolate replicated in neonatal microglia (greater than 10-fold increase in titer), but replication was not detected until 120 h after infection. In contrast, this isolate did not infect adult feline microglia (Fig. 1B) , a situation that may have been due to the state of activation and/or differentiation of the cells [22] . Using double immuno¯uorescent staining, it also was determined that the ERA strain infected the few astrocytes that were present in the microglia cultures (Table 1) . Lastly, we examined the ability of the viruses to infect and replicate in cultures of human glial cells. The ERA strain and the mouse-passaged virus replicated well in adult glial cultures that were greater than 80% microglia, with a greater than 1000-fold and a greater than 10-fold increase in titer, respectively, between 24 and 168 h postinfection (Fig. 1C) . The ERA strain also replicated well in fetal glial cultures that were greater than 98% astrocytes, with a greater than 100-fold increase in viral titer between 24 and 144 h postinfection (Fig. 1C) . Double immuno¯uorescent staining revealed that unpassaged street virus isolates and the mouse passage isolate also infected the human fetal astrocytes (Table 1) .

In summary, primary glial cell cultures from three different species were infected with several different rabies virus strains and isolates in 15 of the 16 (94%) virus-glial cell combinations tested. In addition, productive viral replication was detected in glial cells of all three species in 7 of the 8 (88%) combinations tested. The fact that mouse-passaged and unpassaged street viruses infected and replicated in these glial cells suggests that this was not a virus-cell passage-adaptation laboratory phenomenon. Furthermore, the viral replication in primary microglia and astrocytes suggests that the previous detection of rabies viral antigens and virions within these cells may have been infectious virus rather than phagocytosed debris or absorbed virus from infected neurons [27] . Natural anatomical relations and cellular interactions within the central nervous system are known to be important for rabies virus infection of The arrow identi®es a cell that was infected, but did not stain with GFAP glial cells in vivo [1] , possibly making infection of relatively pure cultures of primary glial cells dif®cult. This situation may explain why others failed to detect rabies challenge virus strain (CVS) antigens in cultures of primary neonatal murine astrocytes 18 to 72 h postinoculation [30] .

We can only hypothesize on the role of glial cells in rabies virus infections. Our data suggest that microglial cells and astrocytes support viral replication independent of neuronal infection, possibly contributing to viral spread or persistence of virus at the site of exposure in infections of extended incubation periods [25] . Glial cells may also be involved in rabies virus pathogenesis by affecting the physiological health of neurons via the release of cytokines or neurotoxins. Support for this involvement has been shown recently in rabiesinfected mice and rats. In these animals, increased levels of inducible nitric oxide synthase (iNOS), which generates NO in microglial cells and astrocytes [26] , relate to or directly correlate with the neuronal dysfunction that underpins clinical rabies disease [12, 31] . Clearly, the infection of glial cells by rabies viruses and their potential role in viral replication, persistence or pathogenesis is intriguing and merits further investigation.

Rabies encephalitis: immunohistochemical investigations

Growth kinetics of human cytomegalovirus are altered in monocyte-derived macrophages

On the cellular source and function of interleukin 6 produced in the central nervous system in viral diseases

Diffuse microgliosis associated with cerebral atrophy in the acquired immunode®ciency syndrome

Slow, persistent replication of lentiviruses: role of tissue macrophages and macrophage precursors in bone marrow

Tropism of sheep lentiviruses for monocytes: susceptibility to infection and virus gene expression increase during maturation of monocytes to macrophages

Characterization of ameboid microglia isolated from developing mammalian brain

Ultrastructural location of major histocompatibility complex (MHC) class II positive perivascular cells in histologically normal human brain

Human cytomegalovirus productively infects primary differentiated macrophages

Cell to cell transmission of virus in the central nervous system. II. Experimental rabies in mouse

In vivo selection of lymphocytetropic and macrophage-tropic variants of lymphocytic choriomeningitis virus during persistent infection

In vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases

Rabies encephaloradiculomyelitis. Case report

Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus

In¯uence of cell type and virus upon lysis of rabies virus-infected cells by antibody and complement

Genetic control of resistance to rabies

Microglial infection by a neurovirulent murine retrovirus results in defective processing of envelope protein and intracellular budding of virus particles

Electron microscope studies of rabies virus in mouse brain

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Appearance of inducible nitric oxide synthase in the rat central nervous system after rabies virus infection and during experimental allergic encephalomyelitis but not after peripheral administration of endotoxin

Macrophage-and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immune de®ciency syndrome

Rabies viruses ± pathogenesis and immunity

Cytokine-gene expression in measles-infected adult human glial cells

We thank F. Murphy, J, Portis, S. Priola, and K. Hasenkrug for critical review of this manuscript. We also thank Bob Evans and Gary Hettrick for graphics art assistance and I. C. Rodriguez for computer assistance.

Received October 21, 1996