The glycoprotein and the matrix protein of rabies virus affect pathogenicity by regulating viral replication and facilitating cell-to-cell spread

While the glycoprotein (G) of rabies virus (RV) is known to play a predominant role in the pathogenesis of rabies, the function of the RV matrix protein (M) in RV pathogenicity is not completely clear. To further investigate the roles of these proteins in viral pathogenicity, we constructed chimeric recombinant viruses by exchanging the G and M genes of the attenuated SN strain with those of the highly pathogenic SB strain. Infection of mice with these chimeric viruses revealed a significant increase in the pathogenicity of the SN strain bearing the RV G from the pathogenic SB strain. Moreover, the pathogenicity was further increased when both G and M from SB were introduced into SN. Interestingly, the replacement of the G or M gene or both in SN by the corresponding genes of SB was associated with a significant decrease in the rate of viral replication and viral RNA synthesis. In addition, a chimeric SN virus bearing both the M and G genes from SB exhibited more efficient cell-to-cell spread than a chimeric SN virus in which only the G gene was replaced. Together, these data indicate that both G and M play an important role in RV pathogenesis by regulating virus replication and facilitating cell-to-cell spread.     

Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence

An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen.     

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.     

Rescue of rabies virus from cloned cDNA and identification of the pathogenicity-related gene: glycoprotein gene is associated with virulence for adult mice

In order to identify the viral gene related to the pathogenicity of rabies virus, we tried to establish a reverse genetics system of the attenuated RC-HL strain, which causes nonlethal infection in adult mice after intracerebral inoculation. A full-length genome plasmid encoding the complete antigenomic cDNA of the RC-HL strain and helper plasmids containing cDNAs of the complete open reading frame of the N, P, and L genes, respectively, were constructed. After transfection of these plasmids into BHK-21 cells infected with the T7 RNA polymerase-expressing vaccinia virus, infectious rabies virus with almost the same biological properties as those of the wild-type RC-HL strain was rescued. Using this reverse genetics system of the RC-HL strain, we generated a chimeric virus with the open reading frame of the glycoprotein gene from the parent Nishigahara strain, which kills adult mice after intracerebral inoculation, in the background of the RC-HL genome. Since the chimeric virus killed adult mice following intracerebral inoculation, it became evident that the open reading frame of the glycoprotein gene is related to the pathogenicity of the Nishigahara strain for adult mice.     

糖蛋白G 基因在基因组P-M 位的插入重组表达对狂犬病毒Flury LEP 致病力的影响

【目的】研究狂犬病病毒Flury鸡胚低代毒株(Flury LEP)在基因组P-M位增加糖蛋白基因(G基因)的重组表达对病毒致病力的影响。【方法】利用反向遗传操作技术,构建了P、M基因之间额外插入G基因的重组狂犬病病毒Flury LEP株(rLEP-PGM),并对重组病毒的生物学特性及对小鼠的致病性进行了初步研究。【结果】亲本株和重组病毒具有相似的生长特性,LEP和rLEP-PGM在BHK-21细胞的生长滴度分别为4×106 FFU/mL和2.5×106 FFU/mL,在小鼠神经母细胞(NA)的生长滴度分别为4×107 FFU/mL和2.5×107 FFU/mL;嗜神经指数均为1;Western blot显示,rLEP-PGM在感染细胞的G蛋白表达量比LEP显著提高;小鼠感染试验显示,rLEP-PGM与LEP脑内注射小鼠的LD50分别为3 FFU和1 FFU,肌肉注射途径的LD50分别为4×104 FFU和3.2×105 FFU。【结论】P、M基因之间插入一个额外的G基因能够提高G蛋白的表达水平,同时增强重组病毒外周侵入中枢神经系统的能力。     

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.     

狂犬病病毒SRV_9突变株的构建与拯救

为构建人工修饰的狂犬病病毒,首先用人细胞色素C基因替换狂犬病病毒SRV9株基因间隔区中的非必需区域Ψ区并缺失基因组全长cDNA的糖蛋白CD编码区,得到突变型SRV9cDNA质粒。然后,该质粒与表达野生型SRV9四种结构蛋白N、P、G和L的质粒共转染BHK-21细胞。免疫荧光试验显示转染细胞中有大量特异性荧光,电子显微镜观察可见大量典型的狂犬病病毒粒子。上述结果表明已成功地拯救出了人工修饰的狂犬病病毒。狂犬病病毒SRV9突变株的成功构建与拯救,为新型狂犬病减毒活疫苗的研究提供了重要的实验工具。     

狂犬病毒糖蛋白DNA疫苗的研制及其免疫效果的观察

构建了含有狂犬病毒（RV）CVS株糖蛋白（GP）基因的重组质粒pCMVCVSRG,将其转染至鼠NIH3T3细胞中,用间接免疫荧光法和APAAP法均证实RVGP能在真核细胞中表达。分别将合RV不同毒株的GP基因的质粒（DNA疫苗）及空白载体质粒（对照组）免疫小鼠,仅DNA疫苗免疫的小鼠产生了中和抗体。以RV攻击后,DNA疫苗免疫组小鼠的存活率与对照组相比,差异有极显著性意义（P＜0．01）;不同的启动子（CMV或SV40）与不同GP基因（来源于CVS株或ERA株）对DNA疫苗的免疫效果无明显影响。在注射120d后．用PCR方法仍可检测出RVGP基因。结果表明：狂犬病DNA疫苗能够诱生低水平的中和抗体和记忆性B淋巴细胞,并能保护小鼠抵抗RV的攻击。该疫苗能在体内稳定存在。狂犬病DNA疫苗的研制为狂犬病免疫开辟了一条新途径,并可为防治其他疾病的DNA疫苗的研制奠定基础。     

用lac基因筛选表达狂犬病毒糖蛋白的重组痘苗病毒

为了研制基因工程狂犬病疫苗,我国于1991年首次报道了在痘苗病毒天坛株中表达狂犬病毒糖蛋白,但报道中重组病毒的选择是先经人骨髓瘤细胞(TK-143)在诱变剂5-溴脱氧尿苷(BrudR)作用下通过标记拯救技术筛选出携带有同源基因的重组病毒,然后再利用重组病毒中携带的Lac基因为选择标记,通过噬斑纯化获得重组病毒,用这种选择方式获得的重组病毒,经过了TK-143细胞和BrudR,因此不宜发展成疫苗,本研究探索不经过TK-143细胞和BrudR,仅利用Lac基因为选择标记,直接在鸡胚细胞上通过噬斑纯化获得重组病毒,现将研究结果报道如下。     

A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity

Several rabies virus (RV) vaccine strains containing an aspartic acid (Asp) or glutamic acid (Glu) instead of an arginine (Arg) at position 333 of the RV glycoprotein (G) are apathogenic for immunocompetent mice even after intracranial inoculation. However, we previously showed that the nonpathogenic phenotype of the highly attenuated RV strain SPBNGA, which contains a Glu at position 333 of G, is unstable when this virus is passaged in newborn mice. While the Glu(333) remained unchanged after five mouse passages, an Asn(194)-->Lys(194) mutation occurred in RV G. This mutation was associated with increased pathogenicity for adult mice. Using site-directed mutagenesis to exchange Asn(194) with Lys(194) in the G protein of SPBNGA, resulting in SPBNGA-K, we show here that this mutation is solely responsible for the increase in pathogenicity and that the Asn(194)-->Lys(194) mutation does not arise when Asn(194) is exchanged with Ser(194) (SPBNGA-S). Our data presented indicate that the increased pathogenicity of SPBNGA-K is due to increased viral spread in vivo and in vitro, faster internalization of the pathogenic virus into cells, and a shift in the pH threshold for membrane fusion. These results are consistent with the notion that the RV G protein is a major contributor to RV pathogenesis and that the more pathogenic RVs escape the host responses by a faster spread than that of less pathogenic RVs.     

Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity

The genetic diversity of representative members of the Lyssavirus genus (rabies and rabies-related viruses) was evaluated using the gene encoding the transmembrane glycoprotein involved in the virus-host interaction, immunogenicity, and pathogenicity. Phylogenetic analysis distinguished seven genotypes, which could be divided into two major phylogroups having the highest bootstrap values. Phylogroup I comprises the worldwide genotype 1 (classic Rabies virus), the European bat lyssavirus (EBL) genotypes 5 (EBL1) and 6 (EBL2), the African genotype 4 (Duvenhage virus), and the Australian bat lyssavirus genotype 7. Phylogroup II comprises the divergent African genotypes 2 (Lagos bat virus) and 3 (Mokola virus). We studied immunogenic and pathogenic properties to investigate the biological significance of this phylogenetic grouping. Viruses from phylogroup I (Rabies virus and EBL1) were found to be pathogenic for mice when injected by the intracerebral or the intramuscular route, whereas viruses from phylogroup II (Mokola and Lagos bat viruses) were only pathogenic by the intracerebral route. We showed that the glycoprotein R333 residue essential for virulence was naturally replaced by a D333 in the phylogroup II viruses, likely resulting in their attenuated pathogenicity. Moreover, cross-neutralization distinguished the same phylogroups. Within each phylogroup, the amino acid sequence of the glycoprotein ectodomain was at least 74% identical, and antiglycoprotein virus-neutralizing antibodies displayed cross-neutralization. Between phylogroups, the identity was less than 64.5% and the cross-neutralization was absent, explaining why the classical rabies vaccines (phylogroup I) cannot protect against lyssaviruses from phylogroup II. Our tree-axial analysis divided lyssaviruses into two phylogroups that more closely reflect their biological characteristics than previous serotypes and genotypes.     

Dominance of a nonpathogenic glycoprotein gene over a pathogenic glycoprotein gene in rabies virus

The nonpathogenic phenotype of the live rabies virus (RV) vaccine SPBNGAN is determined by an Arg-->Glu exchange at position 333 in the glycoprotein, designated GAN. We recently showed that after several passages of SPBNGAN in mice, an Asn-->Lys mutation arose at position 194 of GAN, resulting in GAK, which was associated with a reversion to the pathogenic phenotype. Because an RV vaccine candidate containing two GAN genes (SPBNGAN-GAN) exhibits increased immunogenicity in vivo compared to the single-GAN construct, we tested whether the presence of two GAN genes might also enhance the probability of reversion to pathogenicity. Comparison of SPBNGAN-GAN with RVs constructed to contain either both GAN and GAK genes (SPBNGAN-GAK and SPBNGAK-GAN) or two GAK genes (SPBNGAK-GAK) showed that while SPBNGAK-GAK was pathogenic, SPBNGAN-GAN and SPBNGAN-GAK were completely nonpathogenic and SPBNGAK-GAN showed strongly reduced pathogenicity. Analysis of genomic RV RNA in mouse brain tissue revealed significantly lower virus loads in SPBNGAN-GAK- and SPBNGAK-GAN-infected brains than those detected in SPBNGAK-GAK-infected brains, indicating the dominance of the nonpathogenic phenotype determined by GAN over the GAK-associated pathogenic phenotype. Virus production and viral RNA synthesis were markedly higher in SPBNGAN-, SPBNGAK-GAN-, and SPBNGAN-GAK-infected neuroblastoma cells than in the SPBNGAK- and SPBNGAK-GAK-infected counterparts, suggesting control of GAN dominance at the level of viral RNA synthesis. These data point to the lower risk of reversion to pathogenicity of a recombinant RV carrying two identical GAN genes compared to that of an RV carrying only a single GAN gene.     

Rabies Virus Glycoprotein with a Consensus Amino Acid Sequence and a Lysosome Targeting Signal Causes Effective Production of Antibodies in DNA-Immunized Mice

Safe and effective anti-rabies vaccines are intensely sought worldwide. DNA vaccines have already shown their efficacy and safety and have occupied a special place in the field. Two prototype anti-rabies DNA vaccines were compared for the potential to induce virus-specific antibody production. One vector contained a codon-optimized gene with a territory-adapted consensus sequence of the rabies virus glycoprotein. The other one expressed the same glycoprotein in fusion with a c-CD63 lysosome targeting motif at the C terminus. ELISA of serum samples from immunized mice showed that the c-CD63 variant induced more efficient antibody production and shifted the IgG2a/IgG1 ratio towards the Th2-type immune response. The results gave grounds to believe that the approach successfully applied to the rabies glycoprotein may help to develop new-generation anti-rabies vaccines.     

Glycoprotein of nonpathogenic rabies viruses is a key determinant of human cell apoptosis

We showed that, unlike pathogenic rabies virus (RV) strain CVS, attenuated RV strain ERA triggers the caspase-dependent apoptosis of human cells. Furthermore, we observed that the induction of apoptosis is correlated with a particular virus antigen distribution: the overexpression of the viral G protein on the cell surface, with continuous localization on the cytoplasmic membrane, and large cytoplasmic inclusions of the N protein. To determine whether one of these two major RV proteins (G and N proteins) triggers apoptosis, we constructed transgenic Jurkat T-cell lines that drive tetracycline-inducible gene expression to produce the G and N proteins of ERA and CVS individually. The induction of ERA G protein (G-ERA) expression but not of ERA N protein expression resulted in apoptosis, and G-ERA was more efficient at triggering apoptosis than was CVS G protein. To test whether other viral proteins participated in the induction of apoptosis, human cells were infected with recombinant RV in which the G protein gene from the attenuated strain had been replaced by its virulent strain counterpart (CVS). Only RV containing the G protein from the nonpathogenic RV strain was able to trigger the apoptosis of human cells. Thus, the ability of RV strains to induce apoptosis is largely determined by the viral G protein.     

Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence.

Twelve monoclonal antibodies neutralizing the CVS strain of rabies virus were used to characterize antigenic site II of the viral glycoprotein. Nineteen antigenic mutants resistant to neutralization by some of these antibodies were selected; some continued to normally or partially bind the antibody, whereas others did not. Mutations conferring resistance to neutralization by site II-specific monoclonal antibodies were localized into two clusters, the first between amino acids 34 and 42 (seven groups of mutants) and the second at amino acids 198 and 200 (three groups of mutants). Two intermediate mutations were identified at positions 147 and 184. Four mutations resulted in reduced pathogenicity after intramuscular inoculation of the virus in adult mice. One of the mutants, M23, was 300 times and the others were 10 to 30 times less pathogenic than CVS. In three cases the attenuated phenotype was related to an important modification of antigenic site II, whereas the other known antigenic sites were unchanged.     

Era vaccine-derived cases of rabies in wildlife and domestic animals in Ontario, Canada, 1989-2004

A vaccination program for the control of terrestrial rabies in the province of Ontario, Canada, began in 1989. During the period between 1989 and 2004, over 13 million baits containing the live, attenuated rabies virus ERA-BHK21 were distributed across the province, with the aim of immunizing foxes by the oral route. Animals recovered from bait distribution areas were assayed by fluorescent antibody test for rabies virus infection. Immunoreactivity with a panel of monoclonal antibodies that discriminate between ERA and rabies virus variants known to circulate in Ontario, and molecular genetic analyses were used to identify animals infected with ERA. Nine cases of ERA variant rabies were identified over the 16-yr period of study; these did not appear to be stratified by species, year of discovery, or location of capture. The ERA-positive animals were found across the province in eight counties, all of which had been baited in the year of case discovery. The nine ERA-positive cases included four red foxes (Vulpes vulpes), two raccoons (Procyon lotor), two striped skunks (Mephitis mephitis), and one bovine calf (Bos taurus). Molecular phylogenetic analyses of the partial N gene sequences generated from these isolates indicated that these nine cases were due to infection with the ERA variant. The glycoprotein sequences were predicted from G gene sequencing of all nine field isolates and two laboratory stock ERA viruses. This revealed some heterogeneity at residue 120 (either arginine or histidine) in both field and laboratory stocks as well as a few other mutations in field isolates. The significance of this heterogeneity remains unclear. Our data demonstrate that the ERA vaccine distributed in Ontario carried residual pathogenicity; however, there does not appear to be any evidence of ERA establishment in wildlife populations over the 16-yr period. These results are consistent with previous reports of the rare detection of ERA vaccine-induced rabies and with laboratory studies of ERA pathogenicity.     

Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein

Using four neutralizing monoclonal antibodies which presumably bind to the same antigenic site on the CVS glycoprotein (antigenic site III as defined by cross-neutralization tests), we isolated 58 mutants of the CVS strain of rabies virus. These mutants were highly resistant to the selecting antibodies and grew efficiently in cell cultures. We classified them into five groups on the basis of the pattern of resistance to the four antibodies. We determined pathogenicities of the mutants for adult mice by intracerebral inoculation. Group 2 mutants were nonpathogenic or had attenuated pathogenicity. On the contrary, mutants from the other groups were pathogenic, causing paralysis and death as does CVS. We determined the nucleotide alterations of representative mutants from each group by using the dideoxy method of RNA sequencing. In the glycoproteins of eight nonpathogenic or attenuated mutants, we identified an amino acid substitution at position 333. Arginine 333 was replaced by either glutamine or glycine. In the glycoprotein of eight pathogenic mutants, we identified an amino acid substitution at lysine 330, asparagine 336, or isoleucine 338. Thus, although all substitutions affected neutralization and were located close to each other in the glycoprotein sequence, only substitutions at position 333 affected pathogenicity.     

Detection and genetic characterization of rabies virus from human patients

Saliva and blood were collected from two patients who had not received post exposure prophylaxis in the cities of Wenzhou and Xinning respectively. Both patients were confirmed as positive for rabies by detection of rabies virus specific nucleoprotein antibodies in the sera by Western Blot. However, rabies virus specific RNA was only identified in the saliva collected from the patient in Wenzhou. Furthermore, the isolate Zhejiang Wz0 (H) was obtained by inoculating one-day-old suckling mice. Both nucleoprotein (N) and glycoprotein (G) genes from the isolate were amplified by RT-PCR and sequenced. Phylogenetic analysis indicated that the isolate belonged to classic rabies virus, and shared a higher homology with the street viruses from dogs in the main endemic areas in China and the street virus from dogs in Indonesia than with other known strains. Further comparison of the deduced amino acid sequences between the isolate and the vaccine strains used in China showed that the virus had a higher level of homology with the vaccine strain CTN than with the other vaccine strains (3aG, PV, PM and ERA). In particular, amino acid residues substitutions located in antigenic site III in the G protein, which could react with the neutralizing antibodies, were observed. These results suggested that the virus belonged to the classic rabies virus, and both N and G genes diverged from the current vaccine strains used in China at either the nucleotide or the amino acid level. Foundation items: The project was supported by the Grants (2003BA712A08-02 and 2004BA718B03) from the Chinese Ministry of Science and Technology.     

Neutralization-resistant variants of infectious hematopoietic necrosis virus have altered virulence and tissue tropism.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes an acute disease in salmon and trout. In this study, a correlation between changes in tissue tropism and specific changes in the virus genome appeared to be made by examining four IHNV neutralization-resistant variants (RB-1, RB-2, RB-3, and RB-4) that had been selected with the glycoprotein (G)-specific monoclonal antibody RB/B5. These variants were compared with the parental strain (RB-76) for their virulence and pathogenicity in rainbow trout after waterborne challenge. Variants RB-2, RB-3, and RB-4 were only slightly attenuated and showed distributions of viral antigen in the livers and hematopoietic tissues of infected fish similar to those of the parental strain. Variant RB-1, however, was highly attenuated and the tissue distribution of viral antigen in RB-1-infected fish was markedly different, with more viral antigen in brain tissue. The sequences of the G genes of all four variants and RB-76 were determined. No significant changes were found for the slightly attenuated variants, but RB-1 G had two changes at amino acids 78 and 218 that dramatically altered its predicted secondary structure. These changes are thought to be responsible for the altered tissue tropism of the virus. Thus, IHNV G, like that of rabies virus and vesicular stomatitis virus, plays an integral part in the pathogenesis of viral infection.     

Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein.

Dogs were vaccinated intradermally with vaccinia virus recombinants expressing the rabies virus glycoprotein (G protein) or nucleoprotein (N protein) or a combination of both proteins. The dogs vaccinated with either the G or G plus N proteins developed virus-neutralizing antibody titers, whereas those vaccinated with only the N protein did not. All dogs were then challenged with a lethal dose of a street rabies virus, which killed all control dogs. Dogs vaccinated with the G or G plus N proteins were protected. Five (71%) of seven dogs vaccinated with the N protein sickened, with incubation periods 3 to 7 days shorter than that of the control dogs; however, three (60%) of the five rabid dogs recovered without supportive treatment. Thus, five (71%) of seven vaccinated with the rabies N protein were protected against a street rabies challenge. Our data indicate that rabies virus N protein may be involved in reducing the incubation period in dogs primed with rabies virus N protein and then challenged with a street rabies virus and, of more importance, in subsequent sickness and recovery.