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.     

Retrograde neuronal tracing with a deletion-mutant rabies virus

We have constructed a deletion-mutant rabies virus encoding EGFP and find it to be an excellent tool for studying detailed morphology and physiology of neurons projecting to injection sites within the mammalian brain. The virus cannot spread beyond initially infected cells yet, unlike other viral vectors, replicates its core within them. The cells therefore fluoresce intensely, revealing fine dendritic and axonal structure with no background from partially or faintly labeled cells.     

Prevention of rabies virus infection in dogs by a recombinant canine adenovirus type-2 encoding the rabies virus glycoprotein

Safe and effective vaccination is important for rabies prevention in animals. Although several genetically engineered rabies vaccines have been developed, few have been licensed for use, principally due to biosafety concerns or due to poor efficacy in animal models. In this paper, we describe the construction and characterization of a replication-competent recombinant canine adenovirus type-2 expressing the rabies virus glycoprotein (SRV9 strain) by a different strategy from that reported previously, i.e., the recombinant genome carrying the glycoprotein cDNA was generated by a series of strictly gene cloning steps, infectious recombinant virus was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. This recombinant virus, CAV-E3delta-CGS, was subcutaneously injected into dogs. All vaccinated dogs produced effective neutralizing antibodies after one inoculation and a stronger anamnestic immune response was produced after booster injection. The immunized dogs could survive the challenge of 60,000 mouse LD50 CVS-24, which is lethal to all unimmunized dogs and is comparable to the conventional vaccines. The immunity lasts for months with a protective level of neutralizing antibody. This recombinant virus would be an alternative to the attenuated and the inactivated rabies vaccines and be prospective in immunizing dogs against rabies.     

Apoptosis and rabies virus neuroinvasion

Rabies virus (RV) causes a non-lytic infection of neurons leading to a fatal myeloencephalitis in mammals including humans. By comparing the infection of the nervous system of mice by a highly pathogenic neuroinvasive strain of RV (CVS) and by a strain of attenuated pathogenicity (PV) with restricted brain invasion, we showed that RV neuroinvasiveness results of three factors: not only neurotropic RV avoids induced neuron cell death but also "protective" T cells that migrate into the infected nervous system are killed by apoptosis and finally inflammation of the infected nervous system is limited. Our data suggest that the preservation of the neuronal network, the limitation of the inflammation and the destruction of T cells that invade the CNS in response to the infection are crucial events for RV neuroinvasion and for transmission of RV to another animal.     

Complete genome sequence of a street rabies virus from Mexico

A canine rabies virus (RABV) has been used as a street rabies virus in laboratory investigations. Its entire genome was sequenced and found to be closely related to that of canine RABV circulating in Mexico. Sequence comparison indicates that the virus is closely related to those in the "cosmopolitan" group, with high homology (89 to 93%) to clade I of rabies viruses. The virus is now termed dog rabies virus-Mexico (DRV-Mexico).     

The rabies virus glycoprotein receptor p75NTR is not essential for rabies virus infection

Rabies virus glycoprotein (RVG) is known to be the only factor that mediates rabies infection. The neurotrophin receptor (p75^NTR), through its cysteine-rich domain 1, is a specific receptor for RVG and neutralizes virus infectivity, but its role in virus infection has remained obscure. We used adult mouse dorsal root ganglion (DRG) neurons as a model to study the role of p75^NTR in RV infection of primary neurons. We show that RV infects around 20% of DRG neurons, of which more than 80% are p75^NTR positive, have large diameters, and are capsaicin insensitive. Surprisingly, RV binding and infection are absent in about half of the p75^NTR-expressing DRG neurons which have small diameters and are often capsaicin sensitive. This indicates that p75^NTR is not sufficient to mediate RV interaction in sensory neurons. The rate and specificity of neural infection are unchanged in RV-infected p75^{NTRExonIV−/−} mice that lack all extracellular receptor domains and in wild-type mice infected with two independent RV mutants that lack p75^NTR binding. Accordingly, the mortality rate is unchanged in the absence of RV-p75^NTR interaction. We conclude that although p75^NTR is a receptor for soluble RVG in transfected cells of heterologous expression systems, an RVG-p75^NTR interaction is not necessary for RV infection of primary neurons. This means that other receptors are required to mediate RV infection in vivo and in vitro.     

The efficacy of a cultured rabies vaccine studied in guinea pigs preliminarily infected with a fixed rabies virus]

In experiments of curative vaccination, carried out with the use of an experimental model similar to the current practice of treatment with antirabies preparations, the advantages of using tissue-culture rabies vaccine with immunogenic potency equal to 1.3 international units (I. U.) were shown. In these experiments the vaccine was introduced into guinea pigs infected with fixed rabies virus, the course of vaccination consisting of 14 daily injections. No correlation between the induction of virus-neutralizing antibodies and the immunogenic potency of tissue-culture rabies vaccine was established: the use of the vaccine with immunogenic potency equal to 0,3 and 1,3 I.U. had no essential influence on the level of antibody formation in the animals.     

The acetylcholine receptor as a cellular receptor for rabies virus

Characterization of specific host cell receptors for enveloped viruses is a difficult problem because many enveloped viruses bind to a variety of substrates which are not obviously related to tissue tropisms in the intact host. Viruses with a limited cellular tropism in infected animals present useful models for studying the mechanisms by which virus attachment regulates the disease process. Rabies virus is a rhabdovirus which exhibits a marked neuronotropism in infected animals. Limited data suggest that spread occurs by transsynaptic transfer of virus. The results of recent experiments at Yale suggest that viral antigen is localized very soon after injection at neuromuscular junctions, the motor nerve endings on muscle tissue. On cultured muscle cells, similar co-localization with the acetylcholine receptor is seen both before and after virus multiplication. Pretreatment of these cells with some ligands of the acetylcholine receptor results in reduced viral infection. These findings suggest that a neurotransmitter receptor or a closely associated molecule may serve as a specific host cell receptor for rabies virus and thus may be responsible for the tissue tropism exhibited by this virus. In addition to clarifying aspects of rabies virus pathogenesis, these studies have broad implications regarding the mechanism by which other viruses or viral immunizations might mediate autoimmune diseases such as myasthenia gravis.     

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.     

Raccoon poxvirus recombinants expressing the rabies virus nucleoprotein protect mice against lethal rabies virus infection.

Raccoon poxvirus (RCN) recombinants expressing the rabies virus internal structural nucleoprotein (RCN-N) protected A/WySnJ mice against a lethal challenge with street rabies virus (SRV). Maximum survival was achieved following vaccination by tail scratch and footpad (FP) SRV challenge. RCN-N-vaccinated mice inoculated in the FP with SRV were resistant to infection for at least 54 weeks postvaccination. Protection was also elicited by RCN recombinants expressing the rabies virus glycoprotein (RCN-G). Vaccination with RCN-G evoked rabies virus neutralizing antibody. Rabies virus neutralizing antibody was not detected in RCN-N-vaccinated mice prior to or following SRV infection. Radioimmunoprecipitation assays showed that sera from RCN-N-vaccinated mice which survived SRV infection did not contain antibody to SRV structural protein G, M, or NS. The mechanism(s) of N-induced resistance appears to correlate with the failure of peripherally inoculated SRV to enter the central nervous system (CNS). Support for this correlation with resistance was documented by the observations that SRV-inoculated RCN-N-vaccinated mice did not develop clinical signs of CNS rabies virus infection, infectious SRV was not detected in the spinal cord or brain following FP challenge, and all RCN-N-vaccinated mice died following direct intracranial infection of the CNS with SRV. These results suggest that factors other than anti-G neutralizing antibody are important in resistance to rabies virus and that the N protein should be considered for incorporation with the G protein in recombinant vaccines.     

Intracerebral administration of recombinant rabies virus expressing GM-CSF prevents the development of rabies after infection with street virus

Recently it was found that prior immunization with recombinant rabies virus (RABV) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) (LBNSE-GM-CSF) resulted in high innate/adaptive immune responses and protection against challenge with virulent RABV (Wen et al., JVI, 2011). In this study, the ability of LBNSE-GM-CSF to prevent animals from developing rabies was investigated in mice after infection with lethal doses of street RABV. It was found that intracerebral administration of LBNSE-GM-CSF protected more mice from developing rabies than sham-treated mice as late as day 5 after infection with street RABV. Intracerebral administration of LBNSE-GM-CSF resulted in significantly higher levels of chemokine/cytokine expression and more infiltration of inflammatory and immune cells into the central nervous system (CNS) than sham-administration or administration with UV-inactivated LBNSE-GM-CSF. Enhancement of blood-brain barrier (BBB) permeability and increases in virus neutralizing antibodies (VNA) were also observed in mice treated with LBNSE-GM-CSF. On the other hand, intracerebral administration with UV-inactivated LBNSE-GM-CSF did not increase protection despite the fact that VNA were induced in the periphery. However, intracerebral administration with chemoattractant protein-1 (MCP-1, also termed CCL2) increased significantly the protective efficacy of UV-inactivated LBNSE-GM-CSF. Together these studies confirm that direct administration of LBNSE-GM-CSF can enhance the innate and adaptive immunity as well as the BBB permeability, thus allowing infiltration of inflammatory cells and other immune effectors enter into the CNS to clear the virus and prevent the development of rabies.     

Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles.

A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells.