It was found previously that induction of innate immunity, particularly chemokines, is an important mechanism of rabies virus (RABV) attenuation. To evaluate the effect of overexpression of chemokines on RABV infection, chemokines macrophage inflammatory protein 1␣ (MIP-1␣), RANTES, and IP-10 were individually cloned into the genome of attenuated RABV strain HEP-Flury. These recombinant RABVs were characterized in vitro for growth properties and expression of chemokines. It was found that all the recombinant viruses grew as well as the parent virus, and each of the viruses expressed the intended chemokine in a dose-dependent manner. When these viruses were evaluated for pathogenicity in the mouse model, it was found that overexpression of MIP-1␣ further decreased RABV pathogenicity by inducing a transient innate immune response. In contrast, overexpression of RANTES or IP-10 increased RABV pathogenicity by causing neurological diseases, which is due to persistent and high-level expression of chemokines, excessive infiltration and accumulation of inflammatory cells in the central nervous system, and severe enhancement of blood-brain barrier permeability. These studies indicate that overexpression of chemokines, although important in controlling virus infection, may not always be beneficial to the host.Rabies virus (RABV) is a negative-strand RNA virus belonging to the Rhabidoviridae family, genus Lyssavirus, which causes rabies (fatal encephalomyelitis) in many species of mammals (5). More than 55,000 humans die of rabies each year worldwide (26). Once clinical signs develop, rabies is always fatal (12, 53). Despite the lethality of rabies, only mild inflammation and little neuronal destruction were observed in the central nervous system (CNS) of rabies patients (31, 32). Adaptation of wild-type (wt) RABV in laboratory animals and/or cell culture leads to attenuation in phenotype, and laboratory-adapted RABVs have been used for vaccine development (1, 10). To delineate the mechanism(s) of RABV attenuation, previous studies compared the host responses to infection with either laboratory-attenuated or wt RABV (52). It was found that laboratory-attenuated RABV induced extensive inflammation, apoptosis, and neuronal degeneration, as well as induction of expression of innate immune genes in the CNS; however, wt RABV caused little or no neuronal damage and avoided the activation of expression of innate molecule genes. Other investigators also reported the induction of innate immunity in mice or neuronal cells infected with laboratory-attenuated viruses (20,33,37). The mostly upregulated genes in the innate immune responses after infection with attenuated RABV include genes encoding for inflammatory chemokines and type I interferon (IFN) as well as 33, 378). Further studies have shown that the expression of chemokines (mRNA and proteins), particularly macrophage inflammatory protein 1␣ (MIP-

). In the present study, the immunogenicity of recombinant RABV expressing MIP-1␣ (rHEP-MIP1␣) was determined. It was found that intramuscular immunization of BALB/c mice with rHEP-MIP1␣ resulted in a higher level of expression of MIP-1␣ at the site of inoculation, increased recruitment of dendritic cells (DCs) and mature B cells into the draining lymph nodes and the peripheral blood, and higher virus-neutralizing antibody titers than immunization with the parent rHEP and recombinant RABVs expressing RANTES (CCL5) or IP-10 (CXCL10). Our data thus demonstrate that expression of MIP-1␣ not only reduces viral pathogenicity but also enhances immunogenicity by recruiting DCs and B cells to the site of immunization, the lymph nodes, and the blood.Rabies continues to present public health problems worldwide and causes more than 55,000 human deaths each year, most of which occur in the developing nations of Asia and Africa, where dog rabies remains the main source of human exposure (8, 31). Current rabies vaccines are made with inactivated rabies virus (RABV) grown in cultured cells. Although these vaccines are safe and efficacious, multiple doses (at least 4) must be administered over an extended period of time (14 days) to stimulate optimal immune responses (24). Furthermore, the high cost of cell culture-based vaccines makes it difficult to utilize them effectively in developing countries where they are needed most (28). A live attenuated RABV vaccine (SAG-2) and a recombinant vaccinia virus expressing RABV glycoprotein (VRG) have been licensed particularly for use in the oral immunization of wild animals (10, 29). These vaccines are effective; however, VRG may cause intense skin inflammation and systemic vaccinia infection (3, 25), and SAG-2 induces a low level of virus-neutralizing antibody (VNA) responses in wild animals (11).Recent studies demonstrated that the activation of innate immune responses is one of the mechanisms by which RABV is attenuated (16, 30). Induced innate-response genes include inflammatory chemokines and cytokines, interferons (IFNs) and IFN-related genes, and Toll-like receptors (14,(22)(23). Furthermore, it was found that overexpression of the chemokine MIP-1␣ (CCL3) in mouse brain further decreased RABV pathogenicity, while overexpression of RANTES (CCL5) or IP-10 (CXCL10) increased RABV pathogenicity (35). In this study, the immunogenicity of the recombinant high egg passage (rHEP) Flury strain of RABV that contains the MIP-1␣ gene (rHEP-MIP1␣) was investigated.To ensure that the decreased pathogenicity of rHEP-MIP1␣, as shown previously (35), is due to the overexpression of MIP-1␣, another rHEP virus was constructed with a MIP-1␣ gene cloned into the rHEP genome that does not express MIP-1␣ protein because two stop codons were introduced near the N terminus of the MIP-1␣ gene, one stop codon (TAG) replacing TCA (residues 68 to 70) and the other replacing the codon ATG (residues 78 to 80). The recombinant RABV was rescued, using the procedures described by Inoue et al. (13), and was de...

We investigated the antigenic maturation of rabies virus N protein, for which we used some conformational epitope-specific monoclonal antibodies (MAbs) and an MAb (5-2-26) against a phosphorylation-dependent linear epitope. Infected cells were lysed with a deoxycholate-free lysis buffer and separated by ultracentrifugation into the soluble top and the nucleocapsid fractions. None of the study MAbs recognized N proteins in the top fraction, whereas nucleocapsid-associated N proteins were recognized by all of the MAbs. Immunoprecipitation with polyclonal anti-N antibodies coprecipitated the P proteins from the top fraction, indicating that soluble N proteins are mostly associated with the P protein. The N proteins dissociated from both the N-P complex and nucleocapsids were recognized by none of the study MAbs, whereas the MAb 5-2-6 recognized the SDS-denatured N proteins of the nucleocapsid but not of the top fraction. In addition, the phosphorylation-deficient mutant N proteins were shown to be similarly accumulated as the wild-type N proteins into the viral inclusion bodies, defined as the virus-specific structures composed of viral nucleocapsids, that are produced in the cytoplasm of the infected cells. Based on these results, we believe that newly synthesized N proteins are not immediately phosphorylated at serine-389 (a common phosphorylation site) but are first associated with the P protein. After being used for encapsidation of the viral RNA, the N proteins undergo conformational changes, whereby epitopes for the conformation-specific MAbs are formed and become phosphorylated at serine-389.

We investigated the relationship between the two forms of rabies virus P protein, a non‐catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37‐ and 40‐kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [3H]leucine and [32P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37‐kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37‐ and 40‐kDa products in P cDNA‐transfected animal cells, while only a 37‐kDa product was produced in Escherichia coli. Incubation of 37‐kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40‐kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37‐kDa polypeptide, which depends on certain heparin‐sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus‐induced factor(s).