**Astrocyte Infection during Rabies Encephalitis Depends on the Virus Strain and Infection Route as Demonstrated by Novel Quantitative 3D Analysis of Cell Tropism**

**Madlin Potratz 1, Luca Zaeck 1, Michael Christen 1, Verena te Kamp 2, Antonia Klein 1, Tobias Nolden 3, Conrad M. Freuling 1, Thomas Müller 1 and Stefan Finke 1,\***


Received: 14 January 2020; Accepted: 5 February 2020; Published: 11 February 2020

**Abstract:** Although conventional immunohistochemistry for neurotropic rabies virus (RABV) usually shows high preference for neurons, non-neuronal cells are also potential targets, and abortive astrocyte infection is considered a main trigger of innate immunity in the CNS. While in vitro studies indicated di fferences between field and less virulent lab-adapted RABVs, a systematic, quantitative comparison of astrocyte tropism in vivo is lacking. Here, solvent-based tissue clearing was used to measure RABV cell tropism in infected brains. Immunofluorescence analysis of 1 mm-thick tissue slices enabled 3D-segmentation and quantification of astrocyte and neuron infection frequencies. Comparison of three highly virulent field virus clones from fox, dog, and raccoon with three lab-adapted strains revealed remarkable di fferences in the ability to infect astrocytes in vivo. While all viruses and infection routes led to neuron infection frequencies between 7–19%, striking di fferences appeared for astrocytes. Whereas astrocyte infection by field viruses was detected independent of the inoculation route (8–27%), only one lab-adapted strain infected astrocytes route-dependently [0% after intramuscular (i.m.) and 13% after intracerebral (i.c.) inoculation]. Two lab-adapted vaccine viruses lacked astrocyte infection altogether (0%, i.c. and i.m.). This suggests a model in which the ability to establish productive astrocyte infection in vivo functionally distinguishes field and attenuated lab RABV strains.

**Keywords:** rabies; uDISCO; 3D imaging; rabies pathogenicity; astrocyte infection
