Intravenous inoculation of a bat-associated rabies virus causes lethal encephalopathy in mice through invasion of the brain via neurosecretory hypothalamic fibers.

The majority of rabies virus (RV) infections are caused by bites or scratches from rabid carnivores or bats. Usually, RV utilizes the retrograde transport within the neuronal network to spread from the infection site to the central nervous system (CNS) where it replicates in neuronal somata and infects other neurons via trans-synaptic spread. We speculate that in addition to the neuronal transport of the virus, hematogenous spread from the site of infection directly to the brain after accidental spill over into the vascular system might represent an alternative way for RV to invade the CNS. So far, it is unknown whether hematogenous spread has any relevance in RV pathogenesis. To determine whether certain RV variants might have the capacity to invade the CNS from the periphery via hematogenous spread, we infected mice either intramuscularly (i.m.) or intravenously (i.v.) with the dog-associated RV DOG4 or the silver-haired bat-associated RV SB. In addition to monitoring the progression of clinical signs of rabies we used immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to follow the spread of the virus from the infection site to the brain. In contrast to i.m. infection where both variants caused a lethal encephalopathy, only i.v. infection with SB resulted in the development of a lethal infection. While qRT-PCR did not reveal major differences in virus loads in spinal cord or brain at different times after i.m. or i.v. infection of SB, immunohistochemical analysis showed that only i.v. administered SB directly infected the forebrain. The earliest affected regions were those hypothalamic nuclei, which are connected by neurosecretory fibers to the circumventricular organs neurohypophysis and median eminence. Our data suggest that hematogenous spread of SB can lead to a fatal encephalopathy through direct retrograde invasion of the CNS at the neurovascular interface of the hypothalamus-hypophysis system. This alternative mode of virus spread has implications for the post exposure prophylaxis of rabies, particularly with silver-haired bat-associated RV.

The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription.

Recent studies indicate that the interaction between rabies virus (RV) phosphoprotein and the dynein light chain 8 (LC8) is essential for RV pathogenesis. Through its association with the dynein motor complex, LC8 has been suggested as a molecular factor that links the viral ribonucleoprotein to the host cell transport system. Recent structural investigations, however, dispute this model. To understand the role of LC8 in RV pathogenesis, we generated recombinant RVs with or without the LC8 binding domain (LC8-BD) deleted from the RV phosphoprotein. Peripheral infection of adult mice showed that removal of the LC8-BD did not inhibit entry into the CNS, although it prevented onset of RV-induced CNS disease. However, deletion of the LC8-BD significantly attenuated viral transcription and replication in the CNS. Studies in RAG2 knockout (KO) mice infected with the same recombinant RVs confirmed this finding and indicated that the adaptive immune system is not a factor in the attenuation of viral replication early in the infection. In cell culture, the deletion of the LC8-BD greatly attenuated growth on neuronal cells whereas the growth pattern on nonneuronal cells remained unchanged. However, deletion of the LC8-BD did not affect production of RV virions. We provide evidence that removal of the LC8-BD decreases primary transcription. In this study, we propose that LC8 does not play a role in the retrograde axonal transport of RV and that the deletion of the LC8-BD impairs the infectivity of the virions by reducing early transcription and replication in neurons.

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.

Overexpression of tumor necrosis factor alpha by a recombinant rabies virus attenuates replication in neurons and prevents lethal infection in mice.

The effect of tumor necrosis factor alpha (TNF-alpha) on rabies virus (RV) infection of the mouse central nervous system (CNS) was studied, using recombinant RV engineered to express either soluble TNF-alpha [SPBN-TNF-alpha+] or insoluble membrane-bound TNF-alpha [SPBN-TNF-alpha(MEM)]. Growth curves derived from infections of mouse neuroblastoma NA cells revealed significantly less spread and production of SPBN-TNF-alpha+ than of SPBN-TNF-alpha(MEM) or SPBN-TNF-alpha-, which carries an inactivated TNF-alpha gene. The expression of soluble or membrane-bound TNF-alpha was not associated with increased cell death or induction of alpha/beta interferons. Brains of mice infected intranasally with SPBN-TNF-alpha+ showed significantly less virus spread than did mouse brains after SPBN-TNF-alpha- infection, and none of the SPBN-TNF-alpha+-infected mice succumbed to RV infection, whereas 80% of SPBN-TNF-alpha- -infected mice died. Reduced virus spread in SPBN-TNF-alpha+-infected mouse brains was paralleled by enhanced CNS inflammation, including T-cell infiltration and microglial activation. These data suggest that TNF-alpha exerts its protective activity in the brain directly through an as yet unknown antiviral mechanism and indirectly through the induction of inflammatory processes in the CNS.