Minocycline neuroprotects, reduces microglial activation, inhibits caspase 3 induction, and viral replication following Japanese encephalitis

Minocycline is broadly protective in neurological disease models featuring inflammation and cell death and is being evaluated in clinical trials. Japanese encephalitis virus (JEV) is one of the most important causes of viral encephalitis worldwide. There is no specific treatment for Japanese encephalitis (JE) and no effective antiviral drugs have been discovered. Studies indicate that JE involves profound neuronal loss as well as secondary inflammation caused because of cell death. Minocycline is a semisynthetic second-generation tetracycline that exerts anti-inflammatory and antiapoptotic effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against experimental model of JE. Intravenous inoculation of GP78 strain of JEV in adult mice results in lethal encephalitis and caused primarily because of neuronal death and secondary inflammation caused because of cell death. Minocycline confers complete protection in mice following JEV infection ( p  < 0.0001). Neuronal apoptosis, microglial activation, active caspase activity, proinflammatory mediators, and viral titer were markedly decreased in minocycline-treated JEV infected mice on ninth day post-infection. Treatment with minocycline may act directly on brain cells, because neuronal cell line Neuro2a were also salvaged from JEV-induced death. Our data suggest that minocycline may be a candidate to consider in human clinical trials for JE patients.     

Structural abnormalities in neurons are sufficient to explain the clinical disease and fatal outcome of experimental rabies in yellow fluorescent protein-expressing transgenic mice

Under natural conditions and in some experimental models, rabies virus infection of the central nervous system causes relatively mild histopathological changes, without prominent evidence of neuronal death despite its lethality. In this study, the effects of rabies virus infection on the structure of neurons were investigated with experimentally infected transgenic mice expressing yellow fluorescent protein (YFP) in neuronal subpopulations. Six-week-old mice were inoculated in the hind-limb footpad with the CVS strain of fixed virus or were mock infected with vehicle (phosphate-buffered saline). Brain regions were subsequently examined by light, epifluorescent, and electron microscopy. In moribund CVS-infected mice, histopathological changes were minimal in paraffin-embedded tissue sections, although mild inflammatory changes were present. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase-3 immunostaining showed only a few apoptotic cells in the cerebral cortex and hippocampus. Silver staining demonstrated the preservation of cytoskeletal integrity in the cerebral cortex. However, fluorescence microscopy revealed marked beading and fragmentation of the dendrites and axons of layer V pyramidal neurons in the cerebral cortex, cerebellar mossy fibers, and axons in brainstem tracts. At an earlier time point, when mice displayed hind-limb paralysis, beading was observed in a few axons in the cerebellar commissure. Toluidine blue-stained resin-embedded sections from moribund YFP-expressing animals revealed vacuoles within the perikarya and proximal dendrites of pyramidal neurons in the cerebral cortex and hippocampus. These vacuoles corresponded with swollen mitochondria under electron microscopy. Vacuolation was also observed ultrastructurally in axons and in presynaptic nerve endings. We conclude that the observed structural changes are sufficient to explain the severe clinical disease with a fatal outcome in this experimental model of rabies.     

Extensive Attenuation of Rabies Virus by Simultaneously Modifying the Dynein Light Chain Binding Site in the P Protein and Replacing Arg333 in the G Protein

Rabies virus (RV) is a highly neurotropic virus that migrates from the portal of entry to the central nervous system (CNS). The cytoplasmic dynein light chain (LC8), which is involved in a variety of intracellular motile events, was shown to interact with RV phosphoprotein (P). In order to determine the functional significance of this interaction, P residues 143 to 149 or 139 to 149 encompassing a conserved LC8-interacting motif (K/RXTQT) were deleted from recombinant viruses SAD-L16 and SAD-D29. These viruses are identical except for a replacement of the arginine at position 333 (R333) of the RV glycoprotein by an aspartic acid in SAD-D29. SAD-L16 virus is fully pathogenic for mice, whereas SAD-D29 is nonpathogenic for adult mice but retained pathogenicity for suckling mice. The deletions introduced into the LC8 binding site abolished the P-LC8 interaction and blocked LC8 incorporation into virions. All the mutants propagated in cell culture as efficiently as the parent strains. The pathogenicity of the mutants was then compared with that of the parent viruses by inoculating suckling mice. SAD-L16 derivatives were as pathogenic as their parent virus after intramuscular inoculation, indicating that LC8 is dispensable for the spread of a pathogenic RV from a peripheral site to the CNS. In contrast, SAD-D29-derived deletion mutants were attenuated by as much as 30-fold after intramuscular inoculation but remained as pathogenic as the parent virus when inoculated directly into the brain. This remarkable attenuation after intramuscular but not after intracranial inoculation suggested that abolishing the P-LC8 interaction reduces the efficiency of peripheral spread of the more attenuated SAD-D29 strain. These results demonstrate that elimination of the LC8 ligand and simultaneous substitution of R333 considerably attenuate RV pathogenicity and may be helpful in designing and developing highly safe live-RV-based vaccines.     

Elemental Analysis in Murine Central Nervous System: Elevation of Rubidium Subsequent to Newcastle Disease Virus Encephalopathy

The Cg strain of Newcastle disease virus (NDV) produces neurologic signs and death in mice. This illness is unusual because of the lack of typical features of a viral encephalitis. Specifically, there is a paucity of infectious virus, detectable cellular inflammatory reaction, cytopathic effect, and viral antigen by immunofluorescence. We previously showed an elevation of alpha-aminoisobutyric acid in the CNS of moribund NDV-infected mice, indicating cellular membrane dysfunction. In an attempt to further our understanding of the pathogenesis of the illness, we evaluated CNS concentrations of sodium, potassium, iron, copper, zinc, magnesium, selenium, and rubidium. Elemental analysis revealed no difference between infected and control mice for all elements except for rubidium, which was significantly elevated in infected mice. Elevation in rubidium was detected in infected mice by X-ray fluorescence and atomic absorption spectrophotometry, whereas rubidium concentrations for control mice were similar by both methods. Neurologic symptoms correlated directly with rising rubidium concentrations. Our data suggest that abnormal trace element levels during viral infection may be one mechanism responsible for the clinical symptoms.     

Minocycline effects on the cerebrospinal fluid proteome of experimental autoimmune encephalomyelitis rats

To identify response biomarkers for pharmaceutical treatment of multiple sclerosis, we induced experimental autoimmune encephalomyelitis (EAE) in rats and treated symptomatic animals with minocycline. Cerebrospinal fluid (CSF) samples were collected 14 days after EAE induction at the peak of neurological symptoms, and proteomics analysis was performed using nano-LC-Orbitrap mass spectrometry. Additionally, the minocycline concentration in CSF was determined using quantitative matrix-assisted laser desorption/ionization-triple-quadrupole tandem mass spectrometry (MALDI-MS/MS) in the selected reaction monitoring (SRM) mode. Fifty percent of the minocycline-treated EAE animals did not show neurological symptoms on day 14 ("responders"), while the other half displayed neurological symptoms ("nonresponders"), indicating that minocycline delayed disease onset and attenuated disease severity in some, but not all, animals. Neither CSF nor plasma minocycline concentrations correlated with the onset of symptoms or disease severity. Analysis of the proteomics data resulted in a list of 20 differentially abundant proteins between the untreated animals and the responder group of animals. Two of these proteins, complement C3 and carboxypeptidase B2, were validated by quantitative LC-MS/MS in the SRM mode. Differences in the CSF proteome between untreated EAE animals and minocycline-treated responders were similar to the differences between minocycline-treated responders and nonresponders (70% overlap). Six proteins that remained unchanged in the minocycline-treated animals but were elevated in untreated EAE animals may be related to the mechanism of action of minocycline.     

Rabies virus infection of cultured rat sensory neurons.

The axonal transport of rabies virus (challenge virus strain of fixed virus) was studied in differentiated rat embryonic dorsal root ganglion cells. In addition, we observed the attachment of rabies virus to neuronal extensions and virus production by infected neurons. A compartmentalized cell culture system was used, allowing infection and manipulation of neuronal extensions without exposing the neural soma to the virus. The cultures consisted of 60% large neuronal cells whose extensions exhibited neurofilament structures. Rabies virus demonstrated high binding affinity to unmyelinated neurites, as suggested by assays of virus adsorption and immunofluorescence studies. The rate of axoplasmic transport of virus was 12 to 24 mm/day, including the time required for internalization of the virus into neurites. The virus transport could be blocked by cytochalasin B, vinblastine, and colchicine, none of which negatively affected the production of virus in cells once the infection was established. It was concluded that, for the retrograde transfer of rabies virus by neurites from the periphery to the neuronal soma, the integrity of tubulin- and actin-containing structures is essential. The rat sensory neurons were characterized as permissive, moderately susceptible, but low producers of rabies virus. These neurons were capable of harboring rabies virus for long periods of time and able to release virus into the culture medium without showing any morphological alterations. The involvement of sensory neurons in rabies virus pathogenesis, both in viral transport and as a site for persistent viral infection, is discussed.     

Gamma interferon is critical for neuronal viral clearance and protection in a susceptible mouse strain following early intracranial Theiler's murine encephalomyelitis virus infection

We evaluated the role of gamma interferon (IFN-gamma) in protecting neurons from virus-induced injury following central nervous system infection. IFN-gamma(-/-) and IFN-gamma(+/+) mice of the resistant major histocompatibility complex (MHC) H-2(b) haplotype and intracerebrally infected with Theiler's murine encephalomyelitis virus (TMEV) cleared virus infection from anterior horn cell neurons. IFN-gamma(+/+) H-2(b) mice also cleared virus from the spinal cord white matter, whereas IFN-gamma(-/-) H-2(b) mice developed viral persistence in glial cells of the white matter and exhibited associated spinal cord demyelination. In contrast, infection of IFN-gamma(-/-) mice of the susceptible H-2(q) haplotype resulted in frequent deaths and severe neurologic deficits within 16 days of infection compared to the results obtained for controls. Morphologic analysis demonstrated severe injury to spinal cord neurons in IFN-gamma(-/-) H-2(q) mice during early infection. More virus RNA was detected in the brain and spinal cord of IFN-gamma(-/-) H-2(q) mice than in those of control mice at 14 and 21 days after TMEV infection. Virus antigen was localized predominantly to anterior horn cells in infected IFN-gamma(-/-) H-2(q) mice. IFN-gamma deletion did not affect the humoral response directed against the virus. However, the level of expression of CD4, CD8, class I MHC, or class II MHC in the central nervous system of IFN-gamma(-/-) H-2(q) mice was lower than those in IFN-gamma(+/+) H-2(q) mice. Finally, in vitro analysis of virus-induced death in NSC34 cells and spinal motor neurons showed that IFN-gamma exerted a neuroprotective effect in the absence of other aspects of the immune response. These data support the hypothesis that IFN-gamma plays a critical role in protecting spinal cord neurons from persistent infection and death.     

Effect of rabies virus infection on the expression of parvalbumin, calbindin and calretinin in mouse cerebral cortex

Some clinical features of rabies and experimental evidence from cell culture and laboratory animals suggest impairment of gabaergic neurotransmission. Several types of gabaergic neurons occur in the cerebral cortex. They can be identified by three neuronal markers: the calcium binding proteins (CaBPs) parvalbumin (PV), calbindin (CB) and calretinin (CR). Rabies virus spreads throughout the cerebral cortex; however, rabies cytopathic effects on gabaergic neurons are unknown. The expression of calcium-binding proteins (CaBPs) parvalbumin (PV), calbindin (CB) and calretinin (CR) was studied in the frontal cortex of mice. The effect of gabaergic neurons was evaluated immunohistochemically. The distribution patterns of CaBPs in normal mice and in mice infected with 'fixed' or 'street' rabies virus were compared. PV was found in multipolar neurons located in all cortical layers except layer I, and in pericellular clusters of terminal knobs surrounding the soma of pyramidal neurons. CB-immunoreactivity was distributed in two cortical bands. One was composed of round neurons enclosed by a heavily labeled neuropil; this band corresponds to supragranular layers II and III. The other was a weakly stained band of neuropil which contained scattered multipolar CB-ir neurons; this corresponds to infragranular layers V and VI. The CR-ir neurons were bipolar fusiform cells located in all layers of cortex, but concentrated in layers II and III. A feature common to samples infected with both types of viruses was a more intense immunoreactivity to PV in contrast to normal samples. The infection with 'street' virus did not cause additional changes in the expression of CaBPs. However, the infection with 'fixed' virus produced a remarkable reduction of CB-immunoreactivity demonstrated by the loss of CB-ir neurons and low neuropil stain in the frontal cortex. In addition, the size of CR-ir neurons in the cingulate cortex was decreased.     

Rabies virus ocular disease: T-cell-dependent protection is under the control of signaling by the p55 tumor necrosis factor alpha receptor, p55TNFR

Following brain infection, the Challenge Virus Standard strain of rabies virus infects the retina. Rabies virus ocular infection induces the infiltration of neutrophils and predominantly T cells into the eye. The role of tumor necrosis factor alpha (TNF-alpha)-lymphotoxin signaling in the control of rabies virus ocular infection and inflammatory cell infiltration was assessed using mice lacking the p55 TNF-alpha receptor (p55TNFR(-/-) mice). The incidence of ocular disease and the intensity of retinal infection were greater in p55TNFR(-/-) mice than in C57BL/6 mice: the aggravation correlated with less neutrophil and T-cell infiltration. This indicates that cellular infiltration is under the control of the p55 TNF-alpha receptor and suggests that inflammatory cells may protect the eye against rabies virus ocular infection. The role of T cells following rabies virus ocular disease was assessed by comparison of rabies virus infection in nude mice with their normal counterparts. Indeed, the incidence and severity of the rabies virus ocular disease were higher in athymic nude mice than in BALB/c mice, indicating that T lymphocytes are protective during rabies virus ocular infection. Moreover, few T cells and neutrophils underwent apoptosis in rabies virus-infected retina. Altogether, these data suggest that T lymphocytes and neutrophils are able to enter the eye, escape the immune privilege status, and limit rabies virus ocular disease. In conclusion, rabies virus-mediated eye disease provides a new model for studying mechanisms regulating immune privilege during viral infection.     

Differential effects of rabies and borna disease viruses on immediate-early- and late-response gene expression in brain tissues.

In situ hybridization and Northern blot analysis were used to examine expression of the immediate-early-response genes (IEGs) egr-1, junB, and c-fos, and the late response gene encoding enkephalin in the brains of rats infected intranasally with Borna disease virus (BDV) or rabies virus. In both Borna disease and rabies virus infections, a dramatic and specific induction of IEGs was detected in particular regions of the hippocampus and the cortex. Increased IEG mRNA expression overlapped with the characteristic expression patterns of BDV RNA and rabies virus RNA, although relative expression levels of viral RNA and IEG mRNA differed, particularly in the hippocampal formation. Furthermore, the temporal relationship between viral RNA synthesis and activation of IEG mRNA expression in BDV infection differed markedly from that in rabies virus infection, suggesting that IEG expression is upregulated by different mechanisms. Expression of proenkephalin (pENK) mRNA was also significantly increased in BDV infection, whereas in rabies virus infection, pENK mRNA levels and also the levels of glyceraldehyde-3-phosphate dehydrogenase mRNA were reduced at terminal stages of the disease, probably reflecting a generalized suppression of cellular protein synthesis due to massive production of rabies virus mRNA. The correlation between activated IEG mRNA expression and the strong increase in viral RNA raises the possibility that IEG products induce some phenotypic changes in neurons that render them more susceptible to viral replication.     

Validation of the inactivant binary ethylenimine for inactivating rabies virus for veterinary rabies vaccine production.

The rabies vaccine is produced by inactivation of rabies virus propagated on BHK21 cells. In the rabies inactivation process, BEI is added at a final concentration of 1.6 mM to the viral harvest at 37 degrees C, followed by a second dose of BEI at 24 h post-inactivation. Inactivation was confirmed by the mice innocuity test and tissue culture amplification test as per B.P (Vet) 2004. Validation of test procedure is essential as per cGMP requirement. The dose of BEI was validated by using lower and higher concentrations of BEI in inactivation process. The study indicated that BEI at a lower concentration (0.4 mM) was able to inactivate the rabies virus within 30 h and the routine concentration (1.6 mM) of BEI is effective in inactivating rabies virus within 18 h. The amplification test used for confirming the inactivation of the live virus was validated by spiking the sample with different dilutions of pretitrated live rabies virus. The test revealed that the amplification method is sensitive to detect live rabies virus if present in the inactivated sample. The validation of BEI as an inactivant and the amplification test are discussed.     

狂犬病毒鼠脑复壮后的典型形态恢复及感染细胞内的形态发生学

[目的]观察比较鼠脑复壮前后狂犬病毒的形态变化,并观察病毒感染BHK-21细胞后不同时间的形态发生情况.[方法]以保存时间较长的SRV9毒株为原始材料,经乳鼠脑传代复壮后接种BHK-21细胞,浓缩、纯化后观察.[结果](1)未经复壮的病毒中DI粒子占较高比例,典型粒子只占少数,而复壮后典型粒子所占比例升高到病毒粒子总数的90%.(2)感染24h后在细胞浆内可以观察到典型病毒粒子,其数量随着培养时间的延长而增加.带毒传代之后的培养过程中细胞内病毒数量增加不明显.(3)病毒可以在细胞内的空泡膜表面以多种方式成堆出芽.[结论](1)鼠脑复壮可恢复狂犬病毒中典型粒子所占比例.(2)带毒传代1～2次时为狂犬病毒收获的最佳时机.(3)本研究为狂犬病毒的装配机制补充了数据.     

A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r=0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.     

Intracerebral vaccination suppresses the spread of rabies virus in the mouse brain

To investigate the efficacy of intracerebral (IC) immunization in preventing viral spread in the brain, we immunized mice with inactivated rabies virus via the subcutaneous (SC) or IC route, followed by administration of a lethal dose of rabies virus (challenge virus standard strain), directly into the brains of immunized mice. Progressive paralytic neurological signs were observed in control and 75% of SC immunized mice, whereas only 20% of IC immunized mice exhibited symptoms. Neutralizing antibody titers in blood plasma were significantly elevated in SC and IC immunized mice, with the highest levels seen in IC immunized mice. Analysis of whole brain lysates revealed a strong induction of immunoglobulin in the brains of IC immunized mice that had virus neutralizing activity. Histopathological examination of brain tissue revealed mild encephalitis and disseminated viral antigen in control and SC immunized mice, but rare in IC immunized mice. These results suggest that IC immunization induces a preventive humoral immune response against intracerebrally inoculated rabies virus. Induction of neutralizing antibody in cerebrospinal fluid represents a putative therapeutic measure for the treatment of rabid animals and humans.     

Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro.

We previously reported that A/WySnJ mice vaccinated via a tail scratch with a recombinant raccoon poxvirus (RCN) expressing the rabies virus internal structural nucleoprotein (N) (RCN-N) were protected against a street rabies virus (D. L. Lodmell, J. W. Sumner, J.J. Esposito, W.J. Bellini, and L. C. Ewalt, J. Virol. 65:3400-3405, 1991). To improve our understanding of the mechanism(s) of this protection, we investigated whether sera of A/WySnJ mice that had been vaccinated with RCN-N but not challenged with street rabies virus had anti-rabies virus activity. In vivo studies illustrated that mice inoculated in the footpad with preincubated mixtures of anti-N sera and virus were protected. In addition, anti-N sera inoculated into the site of virus challenge protected mice. The antiviral activity of anti-N sera was also demonstrated in vitro. Infectious virus was not detected in cultures 24 h following infection with virus that had been preincubated with anti-N sera. At later time points, infectious virus was detected, but inhibition of viral production was consistently > or = 99% compared with control cultures. The protective and antiviral inhibitory activity of the anti-N sera was identified as anti-N antibody by several methods. First, absorption of anti-N sera with goat anti-mouse immunoglobulin serum, but not normal goat serum, removed the activity. Second, radioimmuno-precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sucrose density gradient-fractionated anti-N sera showed that antiviral activity was present only in the fraction containing anti-N antibody. Finally, absorption of anti-N sera with insect cells infected with a baculovirus expressing the N protein removed the protective activity. These data indicate that anti-N antibody is a component of the resistance to rabies virus infections.     

Plasmodium berghei: Development of resistance to clindamycin and minocycline in mice

Strains of Plasmodium berghei resistant to clindamycin or minocycline were selected by a procedure in which groups of infected mice were treated with increasing doses of drug during each of a series of subpassages. Groups of five mice, each infected by intravenous inoculation with 10 million parasitized erythrocytes, were treated orally with different doses of drug for four consecutive days beginning on the day of infection. Subpassages were routinely made by Day 7, using donor mice from the group that had been treated with the highest dose of drug that allowed for some development of parasitemia during the preceding passage. Drug doses were increased in each passage as dictated by the development of parasitemia during the previous treated passage.The rate of development of resistance to clindamycin or minocycline was much slower than to conventional antimalarials such as chloroquine, quinine, or pyrimethamine. P. berghei developed total resistance to the latter compounds in nine to 12 treated passages in mice over a period of 60 to 85 days. In contrast, development of total resistance to clindamycin required 42 treated passages over a period of 300 days. Total resistance to minocycline was not attained during 86 successive minocycline-treated passages in mice over a period of 600 days, but a sixfold increase in resistance to minocycline was observed.The clindamycin-resistant strain was normally sensitive to minocycline, chloroquine, quinine, and pyrimethamine. The strain partially resistant to minocycline was normally sensitive to clindamycin, chloroquine, quinine, and pyrimethamine. Resistance to clindamycin was stable during 51 drug-free passages in mice over a period of 1 year. Resistance to minocycline was unstable. During 16 drug-free passages in mice the strain reverted towards normal sensitivity to minocycline. Strains resistant to clindamycin or minocycline showed no difference in rate of development in mice as compared to the parent strain. Likewise, only minor morphological modifications were seen in Giemsa-stained blood smears between the two resistant strains and the parent strain.These results suggest that other species of malaria may develop resistance to clindamycin or minocycline. Should resistance to one of these compounds appear, however, it should not invalidate the use of the other in the treatment of malaria.     

Isolation and replication of rabies virus in C6 rat glioma cells (clone CCL-107).

The susceptibility of the C6 rat glioma cell line (ATCC; CCL-107) to rabies virus was characterized. The kinetics of infection performed with a fixed and a wild strain (from an infected cow) of rabies virus was monitored by direct immunofluorescence. Fluorescent cytoplasmic bodies were readily observed by UV microscopy from 24 hours post-infection (hpi) onwards. The ability of C6 to produce rabies infective virion particles was confirmed by determining the viral titres present in the supernatants of infected cultures, by both BHK-21 cell infection and mice inoculation. C6 cells produced similar viral titres to those produced by BHK-21 for both strains used. In addition, the yield of rabies glycoprotein was assessed by ELISA. In general, BHK-21 and C6 cells infected either by PV or with the wild rabies strain produced similar amounts of rabies glycoprotein. At 96 hpi, however, when the glycoprotein production peaked, BHK-21 infected with the wild strain produced significantly higher amounts of glycoprotein than C6. Subsequently, the optimal conditions for isolation of wild rabies virus strains from C6 cells were established and these proved to be as sensitive as NA cells in detecting 10 wild rabies samples. Due to the high sensitivity exhibited, C6 rat glioma cells present a new and useful system for rabies virus investigation.     

The induction of protective immune response in mice vaccinated by recombinant avian adenovirus CELO expressing glycoprotein G of the rabies virus

The recombinant avian adenovirus CELO-gpRb expressing glycoprotein G of rabies virus (strain TS-80, ARRIW&M, Pokrov, Russia) was used for mice vaccination against rabies. Double intramuscular immunization by recombinant CELO-gpRb adenovirus in a dose 10(9) pfu per mouse caused the induction of virus neutralizing antibodies (VNA) synthesis in 78% of mice, while twice repeated intradermal injections of the recombinant adenovirus failed to induce the VNA production. The protection level in groups of vaccinated mice after intracerebral injection of CVS rabies virus in a dose of 100 MLD50 was equal to 45% at single intramuscular immunization and to 91% after twice repeated intramuscular immunization. The recombinant adenoviral vaccine against rabies, based on CELO viral genome, has a good perspective for domestic and wild animal vaccination, not only due to rather high protection level, but also because the production of adenoviral CELO vaccine in chicken embryos is of high technology and inexpensive.