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Title: Intravenous Inoculation of a Bat-Associated Rabies Virus Causes Lethal Encephalopathy in Mice through Invasion of the Brain via Neurosecretory Hypothalamic Fibers}
Article:
Abstract:
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.
Introduction:
Rabies is a fatal central nervous system( CNS) disease in mammals, caused by rabies virus( RV), a neurotropic lyssavirus from the family of the rhabdoviridae[1]. Generally, RV is transmitted by scratches or bites of rabid animals, which results in the dissemination of virions into skin and muscle tissue. After initial infection of cells at the infection site, RV enters axon terminals and migrates by retrograde axonal transport into the CNS[2]–[4], where it causes a lethal encephalopathy. The incubation period can vary from days to years[5],[6]; however, it is not known where the virus resides during this time. It is likely that a part of the virus that is introduced into damaged muscle or skin tissue after a bite is disseminated into the blood and transported via blood circuits to the CNS. Such an event could play a role in virus transmission by silver-haired bats where only few virus particles are minimally invasively introduced into small patches of skin, which have only few intraepidermal nerve fibers and therefore are not favorable for neuronal uptake[7],[8]. In contrast to natural RV infections, experimental RV infections are commonly done by intramuscular( i. m.), intranasal or intracerebral inoculation. Although injection into muscle probably imitates best natural infections, it causes much less local damage of skin, muscle tissue and microvasculature than an animal bite. Therefore, incidental hematogenous spread due to injury of vessels is less likely than in natural transmissions. Our study aimed to elucidate the pathogenetic relevance of hematogenous RV spread. In particular, we wanted to examine whether RV is able to directly invade the brain from the vasculature and where this invasion would preferentially take place. To accomplish this, we infected mice i. m. or intravenously( i. v.) with the dog-associated RV strain DOG4 or the silver-haired bat associated RV strain SB. Both of these RV strains are highly neuroinvasive[9], but differ greatly in their neurotropism. While DOG4 infects almost exclusively neuronal cells, SB can readily infect other cells in vitro such as endothelial cells and fibroblasts[10].
Discussion:
We show here that two street RV strains that are equally virulent in mice after i. m. infection differ completely in their pathogenicity when they are inoculated into the bloodstream. While i. v. inoculation of DOG4 did not cause any clinical signs of rabies, i. v. infection with SB resulted in a lethal encephalopathy. However, the early transient weight loss together with the detection of viral RNA in the CNS and the development of high VNA serum titers indicate that an infection of the brain did occur after i. v. inoculation of DOG4 but that it had apparently no or only little consequences on the health of the animals. The presence of RV in apparently healthy animals has been reported before[12],[13] It is not known, however, if these animals were only presymptomatic when they were examined, or if it the infection was under control. The failure to detect viral antigen immunohistochemically in CNS tissue of DOG4 i. v. infected mice in spite of the presence of viral RNA can be explained by the different sensitivities of RT-qPCR and the histochemical analysis. In addition, the attempt to isolate virus from the brains of two i. v. DOG4 infected mice 35 weeks pi likely failed because of the presence of VNA in the tissue homogenates[14]. Similar observations were previously made with the neurotropic Sindbis virus. This pathogen with a positive sense single-stranded RNA genome causes acute encephalitis in mice, but is usually cleared within eight days: virus isolation from brain failed as soon as 8 days pi[15], viral protein was not detectable by immunohistochemistry later than two weeks and RNA by in situ hybridization later than 20 days pi[16],[17] while demonstration of viral RNA by RT-PCR was still successful 24 weeks after infection[18]. Antibodies constitute the main effectors against RV[19],[20]. They are able to prevent rabies when given in the presymptomatic stage[21], or to clear apathogenic strains in experimental infections[22]. A conclusive explanation for the low and decreasing rate of virus production we observed in the CNS of DOG4 i. v. infected mice could be the inhibition of virus production by antibodies, a defense mechanism that is more beneficial for the host than the killing of virus-infected neurons[17],[19],[23]–[25]. A possible reactivation of the DOG4 virus in the CNS and development of a lethal encephalopathy when anti-RV antibody levels have decreased cannot be excluded, as shown for Sindbis virus[26]. Future experiments will have to further characterize the immune and infection status of DOG4 i. v. infected mice in order to clarify if the prolonged presence of DOG4 RNA in the CNS has any pathological relevance. The strain-specific differences in the distribution of viral antigen in the CNS suggest that the disparity in the survivorship of mice after i. v. inoculation of DOG4 or SB is likely due to differences in their neurotropism[27],[28]. However, they also indicate that the symptoms in the progressed stages of the disease, which were similar after i. m. infection with SB or DOG4 but different after i. v. infection with SB, are not determined by the infection of particular regions of the brain. The finding that SB was able to directly invade neurosecretory fibers from the vascular system, while i. v. injected DOG4 failed to establish a lethal infection of the brain by using this way of entry, suggests that both viruses may use different cell surface molecules that facilitate their uptake. However, since dog- as well as bat-associated RV strains can vary substantially in their neurotropism[9], it is possible that different bat RV strains might actually utilize different cellular attachment molecules like the neural cell adhesion molecule, the neurotrophin receptor p75TNR, distinct subunits of the nicotinic acetylcholine receptor or other yet undefined molecules, which could vary with the specific glycoproteins of these viruses. Therefore, future experiments must reveal whether our findings can be generalized for other canine- and bat-associates RV strains. The migration of SB can be precisely traced after i. m. inoculation. Immunohistochemical analysis shows that motoneurons in the spinal cord are the exclusive source for virus progression into higher-order CNS structures; this is in agreement with a previous study made in skunks[29]. The fact that early infection can only be detected in the ventral horn neurons excludes the possibility that SB uses sensory fibers in addition to motor axons for CNS invasion from the i. m. inoculation site as it has been proposed for other RV strains[30]–[32]. The observed symptoms of mice after i. v. inoculation strongly suggest that SB directly infects the brain[11]. Our conclusion that SB invades the CNS at the neurovascular interface of the hypothalamus-hypophysis system, is somewhat in conflict with an earlier report[33] that the transit of virus into neuronal tissue occurs via infection of ependymal cells that line the ventricles which contain the cerebral liquor transsudated from vessels of the choroid plexus. In addition, the possibility of passing the blood-brain-barrier via nicotinic acetylcholine receptor-mediated endocytosis into endothelial cells of brain capillaries[34], as it has been suggested in a recent study[35], can be excluded by our findings. A third possibility is the invasion of SB via CVO, which are highly vascularized sites that facilitate direct communication of neurons with blood and liquor through fenestrated endothelium. CVO either consist of neuronal cell bodies and sense various circulating substances( sensory CVO), or they are formed by neurosecretory axons and glial cells( secretory CVO). Their special composition exposes them also as targets for invasion of pathogens, such as trypanosoma[36]. The retrograde invasion of SB from vessels into the CNS through neurosecretory fibers of the CVO ME and NPH is strongly indicated, because mice sacrificed four days after i. v. inoculation showed an almost exclusive involvement of the hypothalamic nuclei PVN, SO and ARC. PVN and SO project oxytocin- and antidiuretic hormon-secreting axons to the NPH, and ARC is considered to form a functional complex together with the ME[37], where hormones for the regulation of the adenohypophysis are released. In summary, our study revealed a route of brain invasion alternative to the migration in peripheral nerves, which might be advantageous for certain RV strains. The transit from the vascular system to the brain via neurosecretory fibers of the ME and NPH might also present a possible explanation for the RV infection of immunosuppressed recipients of RV infected organs that was reported in 2004 in the U. S. A. and in 2005 in Germany[38],[39]. Transplanted tissue is deprived of direct nerval input for many months[40]; thus the classical retrograde pathway of CNS invasion by RV via visceromotor fibers of the autonomic nervous system would have still been unavailable at the time of the actual incubation for the reported transplantation incidents. Instead, virions present in the transferred organs might have been spilled into the recipients' bloodstream and reached the CNS on this newly described route.
Rabies virus ( RV ) infects mammalian neurons and cycles in regionally distinct animal populations such as the red fox in Europe , domestic canines in Asia , or raccoons , skunks and bats in Northern America . Although human rabies can be prevented by pre- and post-exposure prophylaxis , more than 50 , 000 people die annually from the severe encephalopathy caused by RV . Recently , two cases of RV transmission by organ transplantation were reported . In our study , using intravenous inoculation of mice , we evaluated the pathogenetic relevance of virions that reach the bloodstream . Mice inoculated intravenously with a canine-derived RV survived the infection in contrast to intramuscularly injected mice , while mice infected with a silver-haired bat-related RV succumbed to the disease regardless of the route of inoculation . We found that the silver-haired bat-related RV was able to transit from the blood to the brain by invading neurosecretory fibers of the hypothalamus , which form neurohemal synapses lacking a blood-brain-barrier . This newly described route of brain invasion might reflect how RV reached the central nervous system from transplanted organs , since it takes longer to establish the neural connections between host and grafted tissue necessary for classical RV migration than the time until the infection became symptomatic in the two reported cases .