NorA efflux pump mediates Staphylococcus aureus response to Pseudomonas aeruginosa pyocyanin toxicity.

Endogenous transporters protect Staphylococcus aureus against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, NorC, and Tet38, on S. aureus survival following exposure to pyocyanin (PYO) of Pseudomonas aeruginosa, using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced S. aureus susceptibility to pyocyanin killing. The MICPYO of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a ΔnorA mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in norA expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using P. aeruginosa showed that ΔnorA mutant was less susceptible to killing by PYO-deficient mutants PA14phzM and PA14phzS than to killing by PA14. NorA overexpression led to reduced killing by all tested P. aeruginosa. We evaluated the NorA-PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both S. aureus (CF-SA) and P. aeruginosa (CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and norA transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low norA transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.

Staphylococcus aureus Tet38 Efflux Pump Structural Modeling and Roles of Essential Residues in Drug Efflux and Host Cell Internalization.

The Staphylococcus aureus Tet38 membrane protein has distinct functions, including drug efflux and host cell attachment and internalization mediated by interaction with host cell CD36. Using structural modeling and site-directed mutagenesis, we identified key amino acids involved in different functions. Tet38, a member of the major facilitator superfamily, is predicted to have 14 transmembrane segments (TMS), 6 cytoplasmic loops, and 7 external loops. Cysteine substitutions of arginine 106 situated at the junction of TMS 4 and external loop L2, and glycine 151 of motif C on TMS 5, resulted in complete or near-complete (8- to 16-fold) reductions in Tet38-mediated resistance to tetracycline, with minimal to no effect on A549 host cell internalization. In contrast, a three-amino-acid deletion, F411P412G413, in external loop L7 situated between TMS 13 and 14 led to a decrease of 4-fold in S. aureus internalization by A549 cells and a partial effect on tetracycline resistance (4-fold reduction). A three-amino-acid deletion, D38D39L40, in external loop L1 situated between TMS-1 and TMS-2, had a similar partial effect on tetracycline resistance but did not affect cell internalization. Using an Ni column retention assay, we showed further that the L7, but not the L1, deletion impaired binding to CD36. Thus, the L7 domain of Tet38 is key for interaction with CD36 and host cell internalization, and amino acids R106 and G151 (TMSs 4 and 5) are particularly important for tetracycline resistance without affecting internalization.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes.

We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes.

Role of the Tet38 Efflux Pump in Staphylococcus aureus Internalization and Survival in Epithelial Cells.

We previously identified the protein Tet38 as a chromosomally encoded efflux pump of Staphylococcus aureus that confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator of tet38, named tetracycline regulator 21 (TetR21), that bound specifically to the tet38 promoter and repressed pump expression. A ΔtetR21 mutant showed a 5-fold increase in tet38 transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to the tetR21 promoter and indirectly repressed the expression of tet38. To further assess the full role of Tet38 in S. aureus adaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We used S. aureus RN6390, its Δtet38, ΔtetR21, and ΔmgrA mutants, and a Δtet38 ΔtetR21 double mutant. After 2 h of contact, the Δtet38 mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ΔtetR21 mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ΔmgrA mutant. The growth patterns of RN6390 and the ΔmgrA and ΔtetR21 mutants within A549 cells were similar, while no growth was observed for the Δtet38 mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability of S. aureus to internalize and replicate within epithelial cells.

Oxidative stress and reduced glutamine synthetase activity in the absence of inflammation in the cortex of mice with experimental allergic encephalomyelitis.

Pathological changes occur in areas of CNS tissue remote from inflammatory lesions in multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE). To determine if oxidative stress is a significant contributor to this non-inflammatory pathology, cortex tissues from mice with clinical signs of EAE were examined for evidence of inflammation and oxidative stress. Histology and gene expression analysis showed little evidence of immune/inflammatory cell invasion but reductions in natural antioxidant levels and increased protein oxidation that paralleled disease severity. Two-dimensional oxyblots and mass-spectrometry-based protein fingerprinting identified glutamine synthetase (GS) as a particular target of oxidation. Oxidation of GS was associated with reductions in enzyme activity and increased glutamate/glutamine levels. The possibility that this may cause neurodegeneration through glutamate excitotoxicity is supported by evidence of increasing cortical Ca(2+) levels in cortex extracts from animals with greater disease severity. These findings indicate that oxidative stress occurs in brain areas that are not actively undergoing inflammation in EAE and that this can lead to a neurodegenerative process due to the susceptibility of GS to oxidative inactivation.

IL-4(-/-) mice with lethal Mesocestoides corti infections--reduced Th2 cytokines and alternatively activated macrophages.

Protection against Mesocestoides corti, a cestode that invades vital organs, is dependent on the production of IL-4, as IL-4(-/-) mice were found to have higher parasite burdens when compared with wild-type mice. The goal of this study was to investigate the role of IL-4 in immunity to M. corti, focusing on the immunological profile and on potential mediators of pathology. IL-4(-/-) mice infected with M. corti showed 100% mortality by 32 days, whereas wild-type mice survived for approximately 1 year. Parasite burdens were significantly increased in the liver, peritoneal, and thoracic cavities of IL-4(-/-) mice, associated with impaired recruitment of inflammatory cells and a reduction in monocytes and macrophages. IL-5 production by splenocytes and expression in liver tissue was decreased in infected IL-4(-/-) mice compared with wild-type mice. In contrast, IL-4(-/-) mice produced increased amounts of IFNgamma and TNFalpha. Alternatively activated macrophages were a major feature of liver granulomas in wild-type mice evidenced by Arginase I expression, while livers from infected IL-4(-/-) mice showed impaired alternative macrophage activation without increased classical macrophage activation. Thus, lethality during M. corti infection of IL-4(-/-) mice is associated with decreased Th2 cytokines, increased Th1 cytokines and impairment of alternatively activated macrophages.

Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine.

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253-275) and nucleoprotein (N protein) (amino acids 404-418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.

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.

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.

Laser-induced shock waves enhance sterilization of infected vascular prosthetic grafts.

BACKGROUND AND OBJECTIVE: Bacteria that cause infection of vascular prosthetic grafts produce an exopolysaccharide matrix known as biofilm. Growth in biofilms protects the bacteria from leukocytes, antibodies and antimicrobial drugs. Laser-generated shock waves (SW) can disrupt biofilms and increase drug penetration. This study investigates the possibility of increasing antibiotic delivery and sterilization of vascular prosthetic graft. STUDY DESIGN/MATERIALS AND METHODS: Strains of Staphylococcus epidermidis and S. aureus were isolated from infected prosthetic grafts obtained directly from patients. Dacron grafts were inoculated with the isolated bacteria, which were allowed to form adherent bacterial colonies. The colonized grafts underwent the following treatments: (a) antibiotic (vancomycin) alone; (b) antibiotic and SW (c) saline only; and (d) saline and SW. Six hours after treatment, the grafts were sonicated, the effluent was cultured and the colony forming units (CFU) were counted. RESULTS: CFU recovered from control grafts colonized by S. epidermidis were comparable: saline, 3.05 x 10(8) and saline+SW 3.31 x 10(8). The number of S. epidermidis CFU diminished to 7.61 x 10(6) after antibiotic treatment but the combined antibiotic+SW treatment synergistically decreased CFU number to 1.27 x 10(4) (P<0.001). S. aureus showed a higher susceptibility to the antibiotic: 2.26 x 10(6) CFU; antibiotic +SW treatment also had an incremental effect: 8.27 x 10(4) CFU (P<0.001). CONCLUSIONS: This study demonstrates that laser-generated shock waves have no effects alone, but can enhance the effectiveness of antibiotics against bacteria associated with prosthetic vascular graft biofilms, suggesting that this treatment may be of value as adjunctive therapy for prosthetic graft infections.

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.

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.

Pathogenicity of different rabies virus variants inversely correlates with apoptosis and rabies virus glycoprotein expression in infected primary neuron cultures.

The mouse-adapted rabies virus strain CVS-24 has stable variants, CVS-B2c and CVS-N2c, which differ greatly in their pathogenicity for normal adult mice and in their ability to infect nonneuronal cells. The glycoprotein (G protein), which has previously been implicated in rabies virus pathogenicity, shows substantial structural differences between these variants. Although prior studies have identified antigenic site III of the G protein as the major pathogenicity determinant, CVS-B2c and CVS-N2c do not vary at this site. The possibility that pathogenicity is inversely related to G protein expression levels is suggested by the finding that CVS-B2c, the less pathogenic variant, expresses at least fourfold-higher levels of G protein than CVS-N2c in infected neurons. Although there is some difference between CVS-B2c- and CVS-N2c-infected neurons in G protein mRNA expression levels, the differential expression of G protein appears to be largely determined by posttranslational mechanisms that affect G protein stability. Pulse-chase experiments indicated that the G protein of CVS-B2c is degraded more slowly than that of CVS-N2c. The accumulation of G protein correlated with the induction of programmed cell death in CVS-B2c-infected neurons. The extent of apoptosis was considerably lower in CVS-N2c-infected neurons, where G protein expression was minimal. While nucleoprotein (N protein) expression levels were similar in neurons infected with either variant, the transport of N protein into neuronal processes was strongly inhibited in CVS-B2c-infected cells. Thus, downregulation of G protein expression in neuronal cells evidently contributes to rabies virus pathogenesis by preventing apoptosis and the apparently associated failure of the axonal transport of N protein.

Bacterial topoisomerases, anti-topoisomerases, and anti-topoisomerase resistance.

Topoisomerases are ubiquitous enzymes necessary for controlling the interlinking and twisting of DNA molecules. Among the four topoisomerases identified in eubacteria, two, DNA gyrase and topoisomerase IV have been exploited by nature and the pharmaceutical industry as antibacterial targets. Natural products that are inhibitors of one or both of these topoisomerases include the coumarin and cyclothialidine classes, which interfere with adenosine triphosphate hydrolysis, cinodine, flavones, and terpenoid derivatives. The plasmid-encoded bacterial peptides micron B17 and CcdB also inhibit DNA gyrase. The quinolones, a synthetic class of antibacterials that act on both DNA gyrase and topoisomerase IV have had the broadest clinical applications, however. Quinolone congeners differ in their relative potencies for DNA gyrase and topoisomerase IV Studies of an expanding set of resistant mutant enzymes and the crystal structure of the homologous enzyme in yeast have contributed to our understanding of interactions of these drugs with topoisomerase-DNA complexes and the ways in which mutations effect resistance.

Nocardia bacteremia. Report of 4 cases and review of the literature.

Bacteremic nocardiosis is reported rarely. We discuss 4 recent cases seen at our institution and 32 other cases described in the English literature. We found that patients with bacteremic nocardiosis were similar in presentation, risk factors, course, and therapeutic outcome to nonbacteremic patients with nocardiosis. The presence of endovascular foreign bodies appeared to be the only unique risk factor associated with bacteremic illness. Seeding of the central nervous system appeared to be relatively uncommon. Thirty percent of patients with nocardemia had concomitant bacteremia with other pathogens, mostly Gram-negative organisms. Nocardia grew in a variety of growth media, and the median incubation time to detection was 4 days. Fifty percent of patients with Nocardia bacteremia died. Positive blood cultures were a preterminal finding in the fatal, acute cases and occurred relatively early in the subacute, nonfatal cases. Poor outcome seemed to correlate with acute onset of nocardiosis (duration less than 1 month), late identification of nocardemia, involvement of more than 2 sites, and the lack of treatment with a sulfonamide-containing regimen.

Rabies virus quasispecies: implications for pathogenesis.

Passage of the mouse-adapted rabies virus strain CVS-24 (where CVS is challenge virus standard) in BHK cells results in the rapid selection of a dominant variant designated CVS-B2c that differs genotypically and phenotypically from the dominant variant CVS-N2c present in mouse-brain- or neuroblastoma-cell-passaged CVS-24. The glycoprotein of CVS-B2c has 10 amino acid substitutions compared with that of CVS-N2c. Because CVS-B2c can be reproducibly selected in BHK cells, it is likely to be a conserved minor subpopulation of CVS-24. CVS-N2c is more neurotropic in vitro and in vivo than CVS-B2c, which replicates more readily in nonneuronal cells in vitro and in vivo. These characteristics appear to be relevant to the pathogenicity of the two variants. CVS-N2c is more pathogenic for adult mice than CVS-B2c. In contrast, CVS-B2c is more pathogenic for neonatal mice. These differences in pathogenicity are reflected in the selection pattern when mixtures of CVS-N2c and CVS-B2c were used to infect neonatal and adult mice. Although CVS-N2c was highly selected in adult mice, no selection for either variant was seen in neonates, suggesting that certain aspects of development, such as maturation of the nervous and immune systems, may contribute to the selection process. We speculate that the existence of different variants within a rabies virus strain may facilitate the virus in overcoming barriers to its spread, both within the host and between species.

Immunization against rabies with plant-derived antigen.

We previously demonstrated that recombinant plant virus particles containing a chimeric peptide representing two rabies virus epitopes stimulate virus neutralizing antibody synthesis in immunized mice. We show here that mice immunized intraperitoneally or orally (by gastric intubation or by feeding on virus-infected spinach leaves) with engineered plant virus particles containing rabies antigen mount a local and systemic immune response. After the third dose of antigen, given intraperitoneally, 40% of the mice were protected against challenge infection with a lethal dose of rabies virus. Oral administration of the antigen stimulated serum IgG and IgA synthesis and ameliorated the clinical signs caused by intranasal infection with an attenuated rabies virus strain.

Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1.

The coat protein (CP) of alfalfa mosaic virus was used as a carrier molecule to express antigenic peptides from rabies virus and HIV. The antigens were separately cloned into the reading frame of alfalfa mosaic virus CP and placed under the control of the subgenomic promoter of tobacco mosaic virus CP in the 30BRz vector. The in vitro transcripts of recombinant virus with sequences encoding the antigenic peptides were synthesized from DNA constructs and used to inoculate tobacco plants. The plant-produced protein (virus particles) was purified and used for immunization of mice. Both antigens elicited specific virus-neutralizing antibodies in immunized mice.

Intrinsic responses to Borna disease virus infection of the central nervous system.

Immune cells invading the central nervous system (CNS) in response to Borna disease virus (BDV) antigens are central to the pathogenesis of Borna disease (BD). We speculate that the response of the resident cells of the brain to infection may be involved in the sensitization and recruitment of these inflammatory cells. To separate the responses of resident cells from those of cells infiltrating from the periphery, we used dexamethasone to inhibit inflammatory reactions in BD. Treatment with dexamethasone prevented the development of clinical signs of BD, and the brains of treated animals showed no neuropathological lesions and a virtual absence of markers of inflammation, cell infiltration, or activation normally seen in the CNS of BDV-infected rats. In contrast, treatment with dexamethasone exacerbated the expression of BDV RNA, which was paralleled by a similarly elevated expression of mRNAs for egr-1, c-fos, and c-jun. Furthermore, dexamethasone failed to inhibit the increase in expression of mRNAs for tumor necrosis factor alpha, macrophage inflammatory protein 1 beta, interleukin 6, and mob-1, which occurs in the CNS of animals infected with BDV. Our findings suggest that these genes, encoding transcription factors, chemokines, and proinflammatory cytokines, might be directly activated in CNS resident cells by BDV. This result supports the hypothesis that the initial phase of the inflammatory response to BDV infection in the brain may be dependent upon virus-induced activation of CNS resident cells.

Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America.

The silver-haired bat variant of rabies virus (SHBRV) has been identified as the etiological agent of a number of recent human rabies cases in the United States that are unusual in not having been associated with any known history of conventional exposure. Comparison of the different biological and biochemical properties of isolates of this virus with those of a coyote street rabies virus (COSRV) revealed that there are unique features associated with SHBRV. In vitro studies showed that, while the susceptibility of neuroblastoma cells to infection by both viruses was similar, the infectivity of SHBRV was much higher than that of COSRV in fibroblasts (BHK-21) and epithelial cells (MA-104), particularly when these cells were kept at 34 degrees C. At this temperature, low pH-dependent fusion and cell-to-cell spread of virus is seen in BHK-21 cells infected with SHBRV but not with COSRV. It appears that SHBRV may possess an unique cellular tropism and the ability to replicate at lower temperature, allowing a more effective local replication in the dermis. This hypothesis is supported by in vivo results which showed that while SHBRV is less neurovirulent than COSRV when administered via the intramuscular or intranasal routes, both viruses are equally neuroinvasive if injected intracranially or intradermally. Consistent with the above findings, the amino acid sequences of the glycoproteins of SHBRV and COSRV were found to have substantial differences, particularly in the region that contains the putative toxic loop, which are reflected in marked differences in their antigenic composition. Nevertheless, an experimental rabies vaccine based on the Pittman Moore vaccine strain protected mice equally well from lethal doses of SHBRV and COSRV, suggesting that currently used vaccines should be effective in the postexposure prophylaxis of rabies due to SHBRV.