Abstract
The highly neurotropic rabies virus (RABV) enters peripheral neurons at axon termini and requires long distance axonal transport and trans-synaptic spread between neurons for the infection of the central nervous system (CNS). Recent 3D imaging of field RABV-infected brains revealed a remarkably high proportion of infected astroglia, indicating that highly virulent field viruses are able to suppress astrocyte-mediated innate immune responses and virus elimination pathways. While fundamental for CNS invasion, in vivo field RABV spread and tropism in peripheral tissues is understudied. Here, we used three-dimensional light sheet and confocal laser scanning microscopy to investigate the in vivo distribution patterns of a field RABV clone in cleared high-volume tissue samples after infection via a natural (intramuscular; hind leg) and an artificial (intracranial) inoculation route. Immunostaining of virus and host markers provided a comprehensive overview of RABV infection in the CNS and peripheral nerves after centripetal and centrifugal virus spread. Importantly, we identified non-neuronal, axon-ensheathing neuroglia (Schwann cells, SCs) in peripheral nerves of the hind leg and facial regions as a target cell population of field RABV. This suggests that virus release from axons and infected SCs is part of the RABV in vivo cycle and may affect RABV-related demyelination of peripheral neurons and local innate immune responses. Detection of RABV in axon-surrounding myelinating SCs after i.c. infection further provided evidence for anterograde spread of RABV, highlighting that RABV axonal transport and spread of infectious virus in peripheral nerves is not exclusively retrograde. Our data support a new model in which, comparable to CNS neuroglia, SC infection in peripheral nerves suppresses glia-mediated innate immunity and delays antiviral host responses required for successful transport from the peripheral infection sites to the brain.
Highlights
Rabies is estimated to cause 59,000 human deaths annually in over 150 countries, with 95% of cases occurring in Africa and Asia
Comparable to field rabies virus (RABV) isolates, even at an extremely low infection dose (3 T CID50/ mouse), the complete in vivo cycle had been undergone by animals that succumbed to infection, as shown by detection of viral RNA in both salivary glands and in oral swabs using RT-qPCR (Table 1; Additional file 1: Fig. S1)
RABV infects peripheral neuron‐associated neuroglia in the hind leg after i.m. inoculation Immunostaining of solvent-cleared hind leg slices of diseased animals for RABV phosphoprotein (P), neurofilament M (NEFM), and cell nuclei (TO-PROTM-3) was performed for light sheet and confocal laser scanning microscopy analysis (Fig. 1)
Summary
Rabies is estimated to cause 59,000 human deaths annually in over 150 countries, with 95% of cases occurring in Africa and Asia (reviewed in [21, 26]). Prolonged replication of pathogenic RABV in the brain eventually leads to the onset of a progressive rabies encephalitis, invariably resulting in fatal disease progression. In animals suffering from RABV encephalitis, the virus can be detected in multiple nervous or innervated tissues including hind leg, spinal cord, brain, face, and salivary glands [5, 15]. For the transmission of infectious virus to new hosts via virus-containing saliva, centrifugal spread of RABV in the peripheral nervous system (PNS) is required [18]. The secretion of RABV-containing saliva by salivary glands is commonly accepted as the main source of virus shedding and virus transmission to animals via bites (reviewed in [16, 21])
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