The phenotype of the RABV glycoprotein determines cellular and global virus load in the brain and is decisive for the pace of the disease.

The Rabies lyssavirus glycoprotein (RABV-G) is largely responsible for the neuroinvasiveness of the virus and the induction of antiviral immune responses. To study the effects of RABV-G we compared the G of the attenuated RABV variant SPBN with that of the pathogenic DOG4 strain. Infection via the olfactory route caused 100% mortality in mice with both virus variants. Of note, with the attenuated SPBN, progression of the disease was accelerated, microglia response less pronounced and IL-6 expression higher than in the presence of RABV-G from the pathogenic DOG4. However, while virus spread was less extensive, viral gene expression in individual neurons was actually higher in SPBN-infected brains without causing apoptosis of infected neurons. These differences between the two variants were not observed in infected neuronal cultures indicating that the effects of RABV-G on virus spread and viral gene expression depend on factors only present in the intact brain.

Immunogenicity and safety of recombinant rabies viruses used for oral vaccination of stray dogs and wildlife.

Rabies is a zoonotic disease and stray dogs, wild carnivores and bats are the natural reservoirs of rabies. Oral immunization with live vaccines is the only practical approach to eradicate rabies in free ranging terrestrial animals. We have developed the double glycoprotein (G) rabies virus (RV) variant SPBNGAS-GAS that has great promise to be used as a live-attenuated vaccine. Oral immunization of rodents and several target animal species with this double G RV variant resulted in the induction of protective immunity, superior to that induced by a single RV G variant (SPBNGAS). The high oral efficacy of SPBNGAS-GAS is likely because of its increased ability to infect monocytes or immature dendritic cells (DCs), thereby inducing their conversion into mature DCs. Furthermore, infection of DCs with the double G variant resulted in a strong up-regulation of the expression of genes related to the NFjB signalling pathway including IFN-α and IFN-ß, which might underlie the protection conferred by this live RV vaccine. A potential problem associated with the use of live RVs for oral vaccination could rest in the possibility of reversion to the pathogenic phenotype because of the high mutation rate characteristic for all RNA viruses. In this respect, the presence of a second non-pathogenic G gene decreases considerably the risk of reversion to the pathogenic phenotype because a nonpathogenic G is dominant over a pathogenic G in determining the pathogenicity of the double G RV variant. Because of its excellent efficacy and safety, the SPBNGAS-GAS vaccine may provide a distinct advantage over other live RV vaccine in its ability to vaccinate a broad range of mammalian species.

Role of virus-induced neuropeptides in the brain in the pathogenesis of rabies.

Rabies virus (RABV) infection is characterized by the rapid neuronal spread of RABV into the CNS before a protective immune response is raised. Therefore, a typical feature of RABV infection is the paucity of inflammatory reactions in the brain. Here we examined whether the induction of immunosuppressive neuropeptides, in particular CGRP, may contribute to the ability of RABV to evade immune responses. RABV infection of mice caused a strong induction of calcitonin gene-related peptide (CGRP) in neurons and fibres in the neocortex as well as in the dentate gyrus and CA1 region of the hippocampus although RABV did not infect neurons in which CGRP expression was upregulated. Neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP) expressing neurons also were not infected by RABV. In contrast, somatostatin neurons were infected by RABV. There was evidence for an RABV-induced increase of VIP and somatostatin but not of NPY. To test how CGRP expression is related to TNFalpha-induced enhancement of CNS innate and adaptive immunity during RABV infection, we used recombinant RABVs that contained either an active (SPBN-TNFalpha(+)) or an inactive (SPBN-TNFalpha(-)) TNFalpha gene. As compared to SPBN-TNFalpha(-), infection with SPBN-TNFalpha(+) attenuated the induction of CGRP but simultaneously enhanced induction of the invariant chain of MHC II, microglial activation and T cell infiltration. In conclusion, distinct neuropeptidergic neurons in the brain are remarkably spared from RABV infection suggesting a pivotal role of neuropeptides during CNS virus infection. Given the inhibitory effect of CGRP on antigen presentation, we propose that the strong RABV-induced upregulation of CGRP in the brain may contribute to the mechanism by which RABV escapes immune detection. Targeting the expression of neuropeptides, in particular CGRP, that are induced during RABV infection may open a new avenue for therapeutic intervention in human rabies.

Pathogenesis of rabies.

Rabies is a central nervous system (CNS) disease that is almost invariably fatal. The causative agent is rabies virus (RV), a negative-stranded RNA virus of the rhabdovirus family. RV pathogenesis, like that of other viruses, is a multigenic trait. Recent findings indicate that in addition to the RV G protein viral elements that regulate gene expression, especially expression of the L gene, are also likely to play a role in RV pathogenesis. In vivo, RV infects almost exclusively neurons, and neuroinvasiveness is the major defining characteristic of a classical RV infection. A key factor in the neuroinvasion of RV is transsynaptic neuronal spread. While the ability of RV to spread from the post-synaptic site to the pre-synaptic site is mediated by the RV G protein, the RV P protein might be an important determinant of retrograde transport of the virus within axons. Although the mechanism(s) by which an RV infection cause(s) a lethal neurological disease are still not well understood, the most significant factor underlying the lethal outcome of an RV infection appears to be the neuronal dysfunction due to drastically inhibited synthesis of proteins required in maintaining neuronal functions.

Second-generation rabies virus-based vaccine vectors expressing human immunodeficiency virus type 1 gag have greatly reduced pathogenicity but are highly immunogenic.

Rabies virus (RV) vaccine strain-based vectors show great promise as vaccines against other viral diseases such as human immunodeficiency virus type 1 (HIV-1) infection and hepatitis C, but a low residual pathogenicity remains a concern for their use. Here we describe several highly attenuated second-generation RV-based vaccine vehicles expressing HIV-1 Gag. For this approach, we modified the previously described RV vaccine vector SPBN by replacing the arginine at position 333 (R333) within the RV glycoprotein (G) with glutamic acid (E333), deleting 43 amino acids of the RV G cytoplasmic domain (CD), or combining the R333 exchange and the CD deletion. In addition, we constructed a new RV vector that expresses HIV-1 Gag from an RV transcription unit upstream of the RV phosphoprotein gene (BNSP-Gag) instead of upstream of the G gene. As expected and as demonstrated for SPBN-Gag, all vaccine vehicles were apathogenic after peripheral administration. However, the new, second-generation vaccine vectors containing modifications in the RV G were also apathogenic after intracranial infection with 10(5) infectious particles, and BNSP-Gag produced a 50%-reduced mortality in mice. Of note, the observed attenuation of pathogenicity did not result in either the attenuation of the humoral response against the RV G or the previously observed robust cellular response against HIV-1 Gag. These findings demonstrate that very safe and highly effective RV-based vaccines can be constructed and further emphasize their potential utility as efficacious antiviral vaccines.

Silver-haired bat rabies virus variant does not induce apoptosis in the brain of experimentally infected mice.

To examine whether induction of apoptosis plays a role in the pathogenesis of street rabies, we compared the distribution of viral antigens, histopathology, and the induction of apoptosis in the brain of mice infected with a street rabies virus (silver-haired bat rabies virus, SHBRV) and with a mouse-adapted laboratory rabies virus strain (challenge virus standard, CVS-24). Inflammation was identified in the meninges, but not in the parenchyma of the brain of mice infected with either CVS-24 or SHBRV. Necrosis was present in numerous cortical, hippocampal, and Purkinje neurons in CVS-24-infected mice, but only minimal necrosis was identified in mice infected with SHBRV. Likewise, extensive terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) staining was observed in the brain of mice infected with CVS-24 but little or none in the brain of mice infected with SHBRV. Rabies virus antigens were distributed similarly in the CNS infected with either virus. However, the expression of the glycoprotein (G) is more widespread and the staining of G is generally stronger in CVS- than SHBRV-infected mice, whereas the expression of rabies virus nucleoprotein (N) is similar in mice infected with either CVS or SHBRV. The positive TUNEL staining thus correlates with the high level of G expression in CVS-infected mouse brain. Northern blot hybridization revealed that the ratio between the N and G transcripts is similar in brains infected with either virus, indicating that the reduced expression of G protein is not caused by reduced transcription in SHBRV-infected animals. Taken together, these observations suggest that apoptosis is not an essential pathogenic mechanism for the outcome of a street rabies virus infection and that other pathologic processes may contribute to the profound neuronal dysfunction characteristic of street rabies.

Overexpression of cytochrome C by a recombinant rabies virus attenuates pathogenicity and enhances antiviral immunity.

The pathogenicity of individual rabies virus strains appears to correlate inversely with the extent of apoptotic cell death they induce and with the expression of rabies virus glycoprotein, a major inducer of an antiviral immune response. To determine whether the induction of apoptosis by rabies virus contributes to a decreased pathogenicity by stimulating antiviral immunity, we have analyzed these parameters in tissue cultures and in mice infected with a recombinant rabies virus construct that expresses the proapoptotic protein cytochrome c. The extent of apoptosis was strongly increased in primary neuron cultures infected with the recombinant virus carrying the active cytochrome c gene [SPBN-Cyto c(+)], compared with cells infected with the recombinant virus containing the inactive cytochrome c gene [SPBN-Cyto c(-)]. Mortality in mice infected intranasally with SPBN-Cyto c(+) was substantially lower than in SPBN-Cyto c(-)-infected mice. Furthermore, virus-neutralizing antibody (VNA) titers were significantly higher in mice immunized with SPBN-Cyto c(+) at the same dose. The VNA titers induced by these recombinant viruses paralleled their protective activities against a lethal rabies virus challenge infection, with SPBN-Cyto c(+) revealing an effective dose 20 times lower than that of SPBN-Cyto c(-). The strong increase in immunogenicity, coupled with the marked reduction in pathogenicity, identifies the SPBN-Cyto c(+) construct as a candidate for a live rabies virus vaccine.

The central nervous system inflammatory response to neurotropic virus infection is peroxynitrite dependent.

We have recently demonstrated that increased blood-CNS barrier permeability and CNS inflammation in a conventional mouse model of experimental allergic encephalomyelitis are dependent upon the production of peroxynitrite (ONOO(-)), a product of the free radicals NO* and superoxide (O2*(-)). To determine whether this is a reflection of the physiological contribution of ONOO(-) to an immune response against a neurotropic pathogen, we have assessed the effects on adult rats acutely infected with Borna disease virus (BDV) of administration of uric acid (UA), an inhibitor of select chemical reactions associated with ONOO(-). The pathogenesis of acute Borna disease in immunocompetent adult rats results from the immune response to the neurotropic BDV, rather than the direct effects of BDV infection of neurons. An important stage in the BDV-specific neuroimmune response is the invasion of inflammatory cells into the CNS. UA treatment inhibited the onset of clinical disease, and prevented the elevated blood-brain barrier permeability as well as CNS inflammation seen in control-treated BDV-infected rats. The replication and spread of BDV in the CNS were unchanged by the administration of UA, and only minimal effects on the immune response to BDV Ags were observed. These results indicate that the CNS inflammatory response to neurotropic virus infection is likely to be dependent upon the activity of ONOO(-) or its products on the blood-brain barrier.

High level expression of a human rabies virus-neutralizing monoclonal antibody by a rhabdovirus-based vector.

Humans exposed to rabies virus must be promptly treated by passive immunization with anti-rabies antibody and active immunization with rabies vaccine. Currently, antibody prepared from pooled human serum or from immunized horses is utilized. However, neither of these reagents are readily available, entirely safe, or consistent in their biological activity. An ideal reagent would consist of a panel of human monoclonal antibodies. Such antibodies are now available, their only drawback being the cost of production. Using recombinant technology, we constructed a rabies virus-based vector which expresses high levels (approximately 60 pg/cell) of rabies virus-neutralizing human monoclonal antibody. The vector is a modified vaccine strain of rabies virus in which the rabies virus glycoprotein has been replaced with a chimeric vesicular stomatitis virus glycoprotein, and both heavy and light chain genes encoding a human monoclonal antibody have been inserted. This recombinant virus can infect a variety of mammalian cell lines and is non-cytolytic, allowing the use of cell culture technology routinely employed to produce rabies vaccines.

Genetic engineering of live rabies vaccines.

Rabies virus is not a single entity but consists of a wide array of variants that are each associated with different host species. These viruses differ greatly in the antigenic makeup of their G proteins, the primary determinant of pathogenicity and major inducer of protective immunity. Due to this diversity, existing rabies vaccines have largely been targeted to individual animal species. In this report, a novel approach to the development of rabies vaccines using genetically modified, reverse-engineered live attenuated rabies viruses is described. This approach entails the engineering of vaccine rabies virus containing G proteins from virulent strains and modification of the G protein to further reduce pathogenicity. Strategies employed included exchange of the arginine at position 333 for glutamine and modification of the cytoplasmic domain. The recombinant viruses obtained were non-neuroinvasive when administered via a peripheral route. The ability to confer protective immunity depended largely upon conservation of the G protein antigenic structure between the vaccine and challenge virus, as well as on the route of immunization.

Effect of rabies virus infection on gene expression in mouse brain.

A variety of molecular genetic approaches were used to study the effect of rabies virus (RV) infection on host gene expression in mouse brain. The down-regulation of gene expression was found to be a major effect of RV infection by using subtraction hybridization. However, a combination of techniques identified approximately 39 genes activated by infection. These included genes involved in regulation of cell metabolism, protein synthesis, synaptic activity, and cell growth and differentiation. Northern blot analysis to monitor temporal activation of several of these genes following infection revealed essentially two patterns of activation: (i) an early response with up-regulation beginning within 3 days after infection and correlating with transcription of RV nuclear protein; and (ii) a late response with enhanced expression occurring at days 6-7 after infection and associated with peak RV replication. The gene activation patterns and the known functions of their products suggest that a number of host genes may be involved in the replication and spread of RV in the brain.

A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection.

To investigate the importance of the rabies virus (RV) glycoprotein (G) in protection against rabies, we constructed a recombinant RV (rRV) in which the RV G ecto- and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein (rRV-VSV-G). We were able to recover rRV-VSV-G and found that particle production was equal to rRV. However, the budding of the chimeric virus was delayed and infectious titers were reduced 10-fold compared with the parental rRV strain containing RV G. Biochemical analysis showed equal replication rates of both viruses, and similar amounts of wild-type and chimeric G were present in the respective viral particles. Additional studies were performed to determine whether the immune response against rRV-VSV-G was sufficient to protect against rabies. Mice were primed with rRV or rRV-VSV-G and challenged with a pathogenic strain of RV 12 days later. Similar immune responses against the internal viral proteins of both viruses indicated successful infection. All mice receiving the rRV vaccine survived the challenge, whereas immunization with rRV-VSV-G did not induce protection. The results confirm the crucial role of RV G in an RV vaccine.

Reinvestigation of the role of the rabies virus glycoprotein in viral pathogenesis using a reverse genetics approach.

The rabies virus glycoprotein (G) gene of the highly neuroinvasive and neurotropic strains SHBRV-18, CVS-N2c, and CVS-B2c was introduced into the non-neuroinvasive and less neurotropic SN-10 strain to provide further insight into the role of G in the pathogenesis of rabies. Phenotypic analyses of the recombinant viruses revealed, as expected, that the neurotropism of a particular rabies virus strain was a function of its G. Nevertheless, the pathogenicity of the recombinant viruses was, in every case, markedly lower than that of the wild-type viruses suggesting that while the G dictates neurotropism, other viral attributes are also important in pathogenesis. The low pathogenicity of the recombinant viruses is at least in part due to a strong increase in transcription activity. On the other hand, the production of infectious virus by the R-SHB18 recombinant virus-infected cells was significantly delayed by comparison with SHBRV-18 wild-type virus infected-cells. Replacement of the R-SHB18 G cytoplasmic domain, transmembrane domain, and stem region with its SN-10 G counterparts neither results in a significant increase in budding efficiency nor an increase in pathogenicity. These results suggest that an optimal match of the cytoplasmic domain of G with the matrix protein may not be sufficient for maximal virus budding efficiency, which is evidently a major factor of virus pathogenicity. Our studies indicate that to maintain pathogenicity, the interactions between various structural elements of rabies virus must be highly conserved and the expression of viral proteins, in particular the G protein, must be strictly controlled.

Genotypic and phenotypic diversity of rabies virus variants involved in human rabies: implications for postexposure prophylaxis.

OBJECTIVES: Rabies virus variants associated with silver-haired bats (SHBRV) are responsible for most recent human rabies cases in the United States, which are not associated with a history of exposure. We compared their genotype and phenotype with those of dog rabies virus (DRV) variants, the classic cause of rabies in humans, to determine whether differences in these strains might have ramifications for therapeutic intervention, particularly vaccination. METHODS: Eleven silver-haired bat and 8 dog rabies virus isolates were characterized by sequencing the glycoprotein gene, by assessing their ability to replicate in neuronal versus nonneuronal cultures at optimal and suboptimal temperatures, by assessing their pathogenicity in mice, and by determining the resistance of these viruses to therapeutic immunization with commercial vaccines. RESULTS: SHBRV isolates were less genetically diverse, less neuronal cell specific, more temperature sensitive, but as pathogenic, on average, as DRV isolates. Immune protection was equivalent for SHBRV and DRV strains of similar pathogenicity. CONCLUSIONS: SHBRV strains have unique characteristics that may explain their exceptional association with human rabies but have little bearing on their lethality in mice. The pathogenicity of a particular virus, rather than its antigenic makeup, determines the outcome of immunization.

Recombinant rabies virus as potential live-viral vaccines for HIV-1.

Recombinant, replication-competent rabies virus (RV) vaccine strain-based vectors were developed expressing HIV type I (HIV-1) envelope glycoprotein (gp160) from both a laboratory-adapted (CXCR4-tropic) and a primary (dual-tropic) HIV-1 isolate. An additional transcription stop/start unit within the RV genome was used to express HIV-1 gp160 in addition to the other RV proteins. The HIV-1 gp160 protein was stably and functionally expressed, as indicated by fusion of human T cell lines after infection with the recombinant RVs. Inoculation of mice with the recombinant RVs expressing HIV-1 gp160 induced a strong humoral response directed against the HIV-1 envelope protein after a single boost with recombinant HIV-1 gp120 protein. Moreover, high neutralization titers up to 1:800 against HIV-1 could be detected in the mouse sera. These data indicate that a live recombinant RV, a rhabdovirus, expressing HIV-1 gp160 may serve as an effective vector for an HIV-1 vaccine.

The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure.

To provide a more defined and safer replacement for the human rabies immune globulin (HRIG) from pooled serum which is currently used for treatment of exposure to rabies virus we have developed a series of human rabies virus-specific monoclonal antibodies. Mouse-human heterohybrid myeloma cells producing rabies virus-specific human monoclonal antibodies were prepared using B cells obtained from volunteers recently-immunized with a commercial rabies virus vaccine (HDCV). Cell lines producing antibody which neutralized the Evelyn-Rokitnicki-Abelseth (ERA) rabies virus strain in vitro were cloned and the resulting monoclonal antibodies characterized for isotype, specificity against a variety of rabies virus isolates, and neutralization capacity. The ability of the monoclonal antibodies to neutralize a variety of rabies virus strains in vitro correlated with their binding specificity for these viruses in an enzyme-linked immunoadsorbant assay (ELISA). A number of these antibodies have proven suitable for the formulation of a prophylactic human monoclonal antibody-based reagent which would provide significant advantages to the HRIG in having defined, reproducible specificity, lessened possibility of contamination with viral pathogens, and consistent availability.

Neonatal Borna disease virus infection in the rat causes a loss of Purkinje cells in the cerebellum.

Viral insults that occur during early postnatal periods, can affect neuronal systems which exhibit significant postnatal development, such as the cerebral cortex and cerebellum. Borna disease virus (BDV) is a single-strand RNA virus which replicates in the nervous system of many species after experimental inoculation and causes acute neurological disease. Neonatal rats infected with BDV do not mount an aggressive response to the virus like their adult counterparts, but instead develop a persistent BDV infection with less overt clinical sequelae. Recently, the cerebellum, a neural structure associated with regulation of motor behavior, and perhaps with higher cognitive functions, has been demonstrated to be a target of neonatal BDV infections in rats (Bautista et al, 1995). In the present study neonatal rats were infected with BDV and their cerebella were analyzed histologically and immunohistochemically at 7 months of age. The cerebella of infected animals were reduced in size but normal foliation and laminar organization was present. However, as visualized with immunohistochemistry for the Purkinje cell-specific antigen calbindin, there were numerous gaps within the Purkinje cell layer and in the molecular layer which contains the Purkinje cell dendritic trees. We estimated the number of Purkinje cells and found there was an approximately 75% loss of PC in adult rats neonatally infected with BDV. These results suggest that neonatal BDV infection may either (1) target the PC and cause the death of these cells directly or (2) acts indirectly by triggering an immune response which is then responsible for the loss of these cells.

Upregulation of COX-2 and CGRP expression in resident cells of the Borna disease virus-infected brain is dependent upon inflammation.

Infection of immunocompetent adult rats with Borna disease virus (BDV) causes severe encephalitis and neural dysfunction. The expression of COX-2 and CGRP, genes previously shown to be implicated in CNS disease and peripheral inflammation, was dramatically upregulated in the cortical neurons of acutely BDV-infected rats. Neuronal COX-2 and CGRP upregulation was predominantly seen in brain areas where ED1-positive macrophages/microglia accumulated. In addition, COX-2 expression was strongly induced in brain endothelial cells and the number of COX-2 immunoreactive microglial cells was increased. In contrast, despite increased expression of viral antigens, neither COX-2 nor CGRP expression was altered in the CNS of BDV-infected rats treated with dexamethasone, or tolerant to BDV. Thus, increased CGRP and COX-2 expression in the BDV-infected brain is the result of the inflammatory response and likely to be involved in the pathogenesis of virus-induced encephalitis.