Dokument

Summary:
Rabies is a fatal disease in mammals which is transmitted by the neurotropic Rabies virus (RV). Most often, classical RV infections originate from muscle tissue after a bite through an infected canine and ascend to the central nervous system (CNS) via peripheral nerves. In contrast, transfer of non-classical RV by bat bites or scratches, the most common cause for human rabies in North America and also an emerging disease in Europe, most likely introduces RV in rather small amounts superficially into a new host. In both cases, classical and non-classical RV can have access to lymph and/or blood. The impact and effects of the hematogenously and lymphatically distributed share of the viral inoculum is unclear. Taking this into account combined with recent RV infections through unrecognized RV infected organ transplantations the questions arose whether RV is able to infect peripheral organs primarily via a vascular route or only by centrifugal spread via neuronal pathways from the CNS and if this postulated route is strain dependent. Subsequently it was thought to be elucidated, whether RV is able to replicate in organs and if its target cells for direct invasion of organs are different from those it reaches after centrifugal spread from the CNS. With regard to the transmission of RV by tissue transplants it was also investigated whether RV originating from organs is more likely to ascend into the CNS by neuronal pathways or on alternative routes. In order to answer these questions, mice were infected either with a dog-derived classical RV (DOG4) or a bat-derived non-classical RV (rSB) as representatives for the two RV strains with the largest impact in naturally occurring human rabies, and monitored for weight loss and disease symptoms. To maximize the hematogenous dissemination of the inoculum, mice were infected intravenously (i.v.) and compared to mice inoculated intramuscularly (i.m.). A TaqMan® probe based quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay was developed to quantify strain-specifically negativestranded as well as positive-stranded viral RNA in various tissues. For confirmation of replicating RV, virus was isolated from tissues and the nature of virus-positive cells in the periphery determined by immunohistochemistry. A kinetic study was undertaken to trace the pathways of RV into and within the CNS after i.v. and i.m. inoculation. I.m. inoculation with either DOG4 or rSB led to hind limb paralysis and death within twelve days. Viral RNA was detected in the CNS and all analyzed organs (lungs, heart, liver, kidneys) from morbid animals. rSB killed mice in a dose-dependent way also when injected i.v., however without causing typical symptoms of rabies. Surprisingly, i.v. inoculation of DOG4 rendered the infection completely harmless. The mice recovered from a short period of mild weight loss and survived for longer than eight months, showing no signs of viral replication in organs, but low virus load in blood cells and CNS. This and persistent high virus neutralizing antibody (VNA) titers suggest an ongoing immune-controlled latent RV infection after DOG4 i.v. inoculation. After rSB i.m. inoculation, the spread of RV to the periphery was only detected after viral progression throughout the CNS. Importantly, viral RNA was detected at early time points in organs after i.v. inoculation and infectious RV was isolated from the heart before it was isolated from the brain. After i.m. as well as after i.v. inoculation with rSB only neuronal cells were found to be positive for viral antigen. This data reveal for the first time the possibility of a primary infection of peripheral ganglionic cells in organs by rSB via a nonneuronal route. Immunohistochemical kinetic studies of CNS tissue after rSB i.m. inoculation confirmed the motor pathway from the muscle to the brain as the main route for viral invasion whereby the sensory system was affected only secondarily through its connections to the motor system. In contrast, the forebrains of i.v. inoculated mice were infected independently from the presence of viral antigen in spinal cord and brain stem. Our immunohistochemical findings suggest for the first time a direct invasion of the CNS by rSB from the vascular system, most preferentially through hypothalamic neurosecretory axons in the neurohypophysis and the median eminence, whereas retrograde neuronal transport of RV from peripheral organs to the CNS proved to be unlikely.

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