Intranasal Borna Disease Virus (BoDV-1) Infection: Insights into Initial Steps and Potential Contagiosity

Abstract

Mammalian Bornavirus (BoDV-1) typically causes a fatal neurologic disorder in horses and sheep, and was recently shown to cause fatal encephalitis in humans with and without transplant reception. It has been suggested that BoDV-1 enters the central nervous system (CNS) via the olfactory pathway. However, (I) susceptible cell types that replicate the virus for successful spread, and (II) the role of olfactory ensheathing cells (OECs), remained unclear. To address this, we studied the intranasal infection of adult rats with BoDV-1 in vivo and in vitro, using olfactory mucosal (OM) cell cultures and the cultures of purified OECs. Strikingly, in vitro and in vivo, viral antigen and mRNA were present from four days post infection (dpi) onwards in the olfactory receptor neurons (ORNs), but also in all other cell types of the OM, and constantly in the OECs. In contrast, in vivo, BoDV-1 genomic RNA was only detectable in adult and juvenile ORNs, nerve fibers, and in OECs from 7 dpi on. In vitro, the rate of infection of OECs was significantly higher than that of the OM cells, pointing to a crucial role of OECs for infection via the olfactory pathway. Thus, this study provides important insights into the transmission of neurotropic viral infections with a zoonotic potential.

Keywords: OECs; borna disease virus; in vitro; in vivo; initial phase; olfactory ensheathing cells; olfactory epithelium.

Figure 1

Immunohistological detection of Mammalian Bornavirus nucleoprotein (BoDV-1-N) in the olfactory mucosa (OM). At (A) 4 days post infection (dpi); (B) 7 dpi; (C,D) 14 dpi; and (E,F) 21 dpi. AN—adult neurons; JN—juvenile neurons; GB—globose basal cells; HB—horizontal basal cells; NF—nerve fibers; OEC—olfactory ensheathing cells. Scale bar (A–E): 50 µm; (F) 500 µm. (G) Mean score of immunohistochemistry: arithmetic mean; error bars: min/max; hpi—hours post infection.

Figure 2

In situ hybridization for the detection of viral genomic RNA in the OM. At (A) 7 dpi; (B) OECs and nerve fibers located in the lamina propria at 7 dpi; (C) 14 dpi; (D) 21 dpi. AN—adult neurons; JN—juvenile neurons; NF—nerve fibers; OEC—olfactory ensheathing cells. Scale bar (A–D): 50 µm. (E) Mean score of in situ hybridization: arithmetic mean; error bars: min/max.

Figure 3

In situ hybridization for the detection of messenger RNA in the olfactory mucosa. At (A) 4 dpi; (B) 14 dpi; (C) nerve fibers located in the lamina propria at 21 dpi; (D) 21 dpi. AN—adult neurons; JN—juvenile neurons; NF—nerve fibers; OEC—olfactory ensheathing cells. Scale bar (A–D): 50 µm. (E) Mean score of in situ hybridization: arithmetic mean; error bars: min/max.

Figure 4

Immunofluorescence of the OM culture for the detection of olfactory receptor neurons (ORNs). (A) Olfactory neuron after two days in culture (ic). β III-Tubulin—Cy2; nuclear staining: 4’,6-diamidino-2-phenylindole (DAPI); ic—in culture. Scale bar: 20 µm. (B) Percentage of olfactory neurons in the primary culture of the OM over time. Arithmetic mean, error bars: standard deviation.

Figure 5

Percentage of BoDV-1-infected cells in the OM culture over time. (A) Olfactory neuron at seven dpi. Bo18—Cy3. Nuclear staining: DAPI. Scale bar: 20 µm. (B) Percentage of infected cells in the OM culture over time. Arithmetic mean, error bars: standard deviation.

Figure 6

Infection of the OM culture and OECs. (A) Rat OECs at five dpi; (B) Canine OECs at six dpi. (A,B) Immunofluorescence—Bo18: Cy3. Nuclear and cytoplasmatic staining: DAPI. Arrows: BoDV-1-N. Insert: magnification of a cell at six dpi. Scale bars: 50 µm. Arrows: intranuclear granular staining for BoDV-1. (C) Quantitative real time RT-PCR, normalized Mammalian Bornavirus 1 nucleoprotein BoDV-1-N copy numbers at four and seven dpi for viral genomic RNA (gRNA) and messenger RNA (mRNA) for the cultures of the olfactory mucosa (OM) and for rat olfactory ensheathing cells (OECs), respectively. Arithmetic mean, error bars: standard deviation. ** p = 0.0080, *** p = 0.0002.