Development of a nonhuman primate model for mammalian bornavirus infection

Abstract

Until recently, it was assumed that members of the family Bornaviridae could not induce severe disease in humans. Today, however, Borna disease virus 1 (BoDV-1), as well as the more recently emerged variegated squirrel bornavirus 1 (VSBV-1), are known as causative agents of lethal encephalitis in humans. In order to establish animal models reflecting the pathogenesis in humans and for countermeasure efficacy testing, we infected twelve rhesus macaques (Macaca mulatta) either with VSBV-1 or with BoDV-1. For each virus, three monkeys each were inoculated with 2 × 10⁴ focus forming units by the intracerebral route or by multiple peripheral routes (intranasal, conjunctival, intramuscular, and subcutaneous; same dose in total). All BoDV-1 and VSBV-1 intracerebrally infected monkeys developed severe neurological signs around 5 to 6 or 8 to 12 weeks postinfection, respectively. Focal myoclonus and tremors were the most prominent observations in BoDV-1 and VSBV-1-infected animals. VSBV-1-infected animals also showed behavioral changes. Only one BoDV-1 peripherally infected animal developed similar disease manifestations. All animals with severe clinical disease showed high viral loads in brain tissues and displayed perivascular mononuclear cuffs with a predominance of lymphocytes and similar meningeal inflammatory infiltrates. In summary, rhesus macaques intracerebrally infected with mammalian bornaviruses develop a human-like disease and may serve as surrogate models for human bornavirus infection.

Keywords: animal model; bornavirus; nonhuman primate.

Fig. 1.

Experimental schedule and time of viral RNA and antibody detection. Red dashed lines depict the time period of clinical signs for each individual animal. Viral antigen was found in brain samples from all seven diseased NHPs (brain graphic). Viral RNA was found in CSF (turquoise drop) and oral swab samples. Bornavirus reactive antibodies were found in five of the infected NHPs. Key: i.c. = intracerebral; non-i.c. = peripheral routes. Created with BioRender.com.

Fig. 2.

BoDV-1 (#7–12) and VSBV-1 (#1–6) genome load (A) and infectious titers (B) in selected organs derived from the individual NHPs. i.c. = intracerebral; non-i.c. = peripheral routes per mg tissue.

Fig. 3.

Histopathology and immunohistochemistry on brain tissues derived from NHPs infected with VSBV-1 and BoDV-1. (a)–(d): H&E 20X; bar = 200 µm, a/c: lymphocytic perivascular cuffing (box), and meningeal infiltrates (arrows), (b)/(d) = no histologic lesions. (e)–(h): H&E 100X; bar = 50 µm, e/g: prominent lymphocytic perivascular cuffing, and f/h: no perivascular cuffing. (i)–(l): IHC 100X; bar = 50 µm, (i)/(k): diffuse immunoreactive neurons, astrocytes, and microglia, (j)/(l): no immunoreactivity. I.C. = intracerebral; IN, IO, SC, and IM = peripheral routes.

Fig. 4.

Double staining with cross-reactive BoDV-1 P antibody and GFAP, IBA1, and NFP. 100X; bar = 20 µm. Bornavirus positive cells are purple. GFAP: yellow cells are astrocytes (arrowhead); IBA1: yellow cells are microglia (arrowhead); and NFP: yellow cells are nerve axons (arrowhead). Dual staining is colored orange/pink (arrows).