Apoptosis plays an important role in experimental rabies virus infection.

Cultured rat prostatic adenocarcinoma (AT3) cells infected with the challenge virus standard (CVS) strain of fixed rabies virus showed characteristic morphologic features of apoptosis, evidence of oligonucleosomal DNA fragmentation, and expression of the Bax protein. CVS-infected Bcl-2-transfected AT3 cells did not demonstrate these features. Adult ICR mice inoculated intracerebrally with CVS showed morphologic changes of apoptosis, DNA fragmentation, and increased Bax expression in neurons, with changes most marked in the hippocampus and cerebral cortex. Ultrastructurally, some neurons demonstrated morphologic features more typical of necrosis. These studies provide evidence that apoptosis plays an important role in the pathogenesis of rabies virus infection.     

Two modes of pseudorabies virus neuroinvasion and lethality in mice

We describe two distinct modes of neuroinvasion and lethality after murine flank inoculation with virulent and attenuated strains of pseudorabies virus (PRV). Mice infected with virulent (e.g., PRV-Becker, PRV-Kaplan, or PRV-NIA3) strains self-mutilate their flank skin in response to virally induced pruritus, die rapidly with no identifiable symptoms of central nervous system (CNS) infection such as behavioral abnormalities, and have little infectious virus or viral antigen in the brain. In distinct contrast, animals infected with an attenuated PRV vaccine strain (PRV-Bartha) survive approximately three times longer than wild-type PRV-infected animals, exhibit severe CNS abnormalities, and have an abundance of infectious virus in the brain at the time of death. Interestingly, these animals have no skin lesions and do not appear pruritic at any time during infection. The severe pruritus and relatively earlier time until death induced by wild-type PRV infection may reflect the peripheral nervous system (PNS) and immune responses to infection rather than a fatal, virally induced CNS pathology. Based on previously characterized afferent (sensory) and efferent (motor) neuronal pathways that innervate the skin, we deduced that wild-type virulent strains transit through the PNS via both afferent and efferent routes, whereas PRV-Bartha travels by only efferent routes in the PNS en route to the brain.     

Influence of pseudorabies virus proteins on neuroinvasion and neurovirulence in mice

Neurotropism is a distinctive feature of members of the Alphaherpesvirinae. However, its molecular basis remains enigmatic. In the past, research has been focused mainly on the role of viral envelope proteins in modulating herpesvirus neuroinvasion and neurovirulence (T. C. Mettenleiter, Virus Res. 92:192-206, 2003). To further analyze the molecular requirements for neuroinvasion of the alphaherpesvirus pseudorabies virus (PrV), adult mice were infected intranasally with a set of single- or multiple-deletion mutants lacking the UL3, UL4, UL7, UL11, UL13, UL16, UL17, UL21, UL31, UL34, UL37, UL41, UL43, UL46, UL47, UL48, UL51, US3, US9, glycoprotein E (gE), gM, UL11/US9, UL11/UL16, UL16/UL21, UL11/UL16/UL21, UL11/gE, UL11/gM, UL43/gK, UL43/gM, or UL43/gK/gM genes. Neurovirulence was evaluated by measuring mean survival times compared to that after wild-type virus infection. Furthermore, by immunohistochemical detection of infected neurons, the kinetics of viral spread in the murine central nervous system was investigated.     

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.     

Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion

Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.     

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.     

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.     

Homology between the glycoproteins of vesicular stomatitis virus and rabies virus

We compared the predicted amino acid sequences of the vesicular stomatitis virus and rabies virus glycoproteins by using a computer program which provides an optimal alignment and a statistical significance for the match. Highly significant homology between these two proteins was detected, including identical positioning of one glycosylation site. A significant homology between the predicted amino acid sequences of vesicular stomatitis virus and influenza virus matrix proteins was also found.     

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.     

Double-labeled rabies virus: live tracking of enveloped virus transport

Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.     

Density gradient centrifugation studies on rabies virus

Neurath, A. Robert (The Wistar Institute, Philadelphia, Pa.), Tadeusz J. Wiktor, and Hilary Koprowski. Density gradient centrifugation studies on rabies virus. J. Bacteriol. 92:102-106. 1966.-Cesium chloride density gradient centrifugation of rabies virus revealed a heterogeneous population of infectious virus particles, the majority of which showed a density of 1.20 g/ml. From results obtained by rate zonal centrifugation in preformed sucrose gradients, it was possible to calculate a sedimentation coefficient of about 600S for rabies virus. Sedimentation coefficients of about 23S and 10S were calculated for two soluble rabies antigens present in infected tissue-culture fluids, and they showed a density of 1.26 g/ml in cesium chloride solutions.