Macrophages represent an essential part of innate immunity, and the viral infection of macrophages results in the release of multiple proinflammatory mediators, such as nitric oxide (NO), cytokines, and chemokines. This study was undertaken to define the molecular mechanism of macrophage activation in response to rabies virus (RV) infection. In RAW264 murine macrophage cells, a well-characterized macrophage model, RV replication was strictly restricted, whereas cell proliferation was significantly enhanced upon RV inoculation. Transcriptional analyses for the expression of inducible forms of NO synthase (iNOS), cytokines, and chemokines revealed that RV virions potentiate the gene expression of iNOS and CXC chemokine ligand 10 (CXCL10), a major chemoattractant of T helper cell type 1. However, RV stimulation had little or no effect on the expression profiles of proinflammatory cytokines and other types of chemokines. In macrophages stimulated with UV-inactivated RV virions, as well as infectious viruses, the phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, members of the mitogen-activated protein kinase family, was significantly induced. Specific inhibitors of MAPK/ERK kinase reduced the RV-induced production of NO and CXCL10. Furthermore, the RV-induced activation of the ERK1/2 pathway was severely impaired by the neutralization of the endosomal and lysosomal pH environment with lysosomotropic agents, indicating that endocytosis is a key step leading to the activation of ERK1/2 signaling. Taken together, these results suggest that the ERK1/2-mediated signaling pathway plays a cardinal role in the selective activation of macrophages in response to RV virions, thereby regulating cellular functions during virus infection.Rabies virus (RV) is a negative-strand RNA virus belonging to the Rhabdoviridae family, genus Lyssavirus. Most RV strains are highly neurotropic, which usually causes a fatal infection in warm-blooded animals, and viral replication primarily occurs in neurons as a cellular target (43). It has been reported that RV replicates in muscle cells prior to the invasion of the peripheral nervous system and central nervous system (CNS) in vivo (62). In vitro, it is known that a highly neurovirulent RV strain, challenge virus standard (CVS), and an attenuated strain, high egg passage (HEP)-Flury (hereafter called HEP), infect a variety of cell types, including nonneuronal cells (26,82,84,85).It is well known that infection of experimental animals with nonpathogenic RV strains triggers a strong antiviral immune response (90). Recent studies have demonstrated that the strong antiviral immune response elicited by attenuated RV infection is closely related to the induction of apoptosis in infected cells due to the expression of viral glycoprotein (61,76). Several groups have attempted to develop vaccine vehicles using RV-based vectors. It has been reported that the RV vectors expressing foreign antigens induce strong humoral and cellular responses against other kinds of viral pathogens, s...

Following virus infection of the central nervous system, microglia, the ontogenetic and functional equivalents of macrophages in somatic tissues, act as sources of chemokines, thereby recruiting peripheral leukocytes into the brain parenchyma. In the present study, we have systemically examined the growth characteristics of rabies virus (RV) in microglia and the activation of cellular signaling pathways leading to chemokine expression upon RV infection. In RV-inoculated microglia, the synthesis of the viral genome and the production of virus progenies were significantly impaired, while the expression of viral proteins was observed. Transcriptional analyses of the expression profiles of chemokine genes revealed that RV infection, but not exposure to inactivated virions, strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. RV infection triggered the activation of signaling pathways mediated by mitogen-activated protein kinases, including p38, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase, and nuclear factor B (NF-B). RV-induced expression of CXCL10 and CCL5 was achieved by the activation of p38 and NF-B pathways. In contrast, the activation of ERK1/2 was found to down-regulate CCL5 expression in RV-infected microglia, despite the fact that it was involved in partial induction of CXCL10 expression. Furthermore, NF-B signaling upon RV infection was augmented via a p38-mediated mechanism. Taken together, these results indicate that the strong induction of CXCL10 and CCL5 expression in microglia is precisely regulated by the activation of multiple signaling pathways through the recognition of RV infection.

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Rabies virus (RV) deficient in the P gene was generated by reverse genetics from cDNA of HEP-Flury strain lacking the entire P gene. The defective virus was propagated and amplified by rescue of virus, using a cell line that complemented the functions of the deficient gene. The P gene-deficient (def-P) virus replicated its genome and produced progeny viruses in the cell lines that constitutively expressed the P protein, although it grew at a slightly retarded rate compared to the parental strain. In contrast, no progeny virus was produced in the infected host when the def-P virus-infected cells that did not express the P protein. However, we found that the def-P virus had the ability to perform primary transcription (by the virion-associated polymerase) in the infected host without de novo P protein synthesis. The def-P virus was apathogenic in adult and suckling mice, even when inoculated intracranially. Inoculation of def-P virus in mice induced high levels of virus-neutralizing antibody (VNA) and conferred protective immunity against a lethal rabies infection. These results demonstrate the potential utility of gene-deficient virus as a novel live attenuated rabies vaccine.

List of Abbreviations:AIDS, acquired immunodeficiency syndrome; BKV, BK virus; BKVAN, BK virus-associated nephropathy; CPE, cytopathic effect; DAB, 3,3 -diaminobenzidine tetrahydrochloride; FBS, fetal bovine serum; HA, hemagglutination; HIV-1, human immunodeficiency virus-1; JCV, JC virus; LTR, long terminal repeat; luc, luciferase; MEM, minimum essential medium; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; pLTR-luc, HIV-1 LTR-dependent reporter gene; PML, progressive multifocal leukoencephalopathy; PML-type JCV, JCV isolated from the brain of PML patients; pRSV-β gal, RSV promoter-dependent expression vector for β-galactosidase; RLU, relative light units; RT, reverse transcriptase; SV40, simian virus 40.replication origin and agnoprotein gene in JCV genomic DNA. It is well known that the regulatory region of JCV isolated from PML-type JCV contains hypervariable sequences (2).