Shedding of Infectious Borna Disease Virus-1 in Living Bicolored White-Toothed Shrews

Abstract

Background: Many RNA viruses arise from animal reservoirs, namely bats, rodents and insectivores but mechanisms of virus maintenance and transmission still need to be addressed. The bicolored white-toothed shrew (Crocidura leucodon) has recently been identified as reservoir of the neurotropic Borna disease virus 1 (BoDV-1).

Principal findings: Six out of eleven wild living bicoloured white-toothed shrews were trapped and revealed to be naturally infected with BoDV-1. All shrews were monitored in captivity in a long-term study over a time period up to 600 days that differed between the individual shrews. Interestingly, all six animals showed an asymptomatic course of infection despite virus shedding via various routes indicating a highly adapted host-pathogen interaction. Infectious virus and viral RNA were demonstrated in saliva, urine, skin swabs, lacrimal fluid and faeces, both during the first 8 weeks of the investigation period and for long time shedding after more than 250 days in captivity.

Conclusions: The various ways of shedding ensure successful virus maintenance in the reservoir population but also transmission to accidental hosts such as horses and sheep. Naturally BoDV-1-infected living shrews serve as excellent tool to unravel host and pathogen factors responsible for persistent viral co-existence in reservoir species while maintaining their physiological integrity despite high viral load in many organ systems.

Fig 1. Relative body mass trend of non-infected and infected shrews.

Relative body mass trend of non-infected and infected shrews at 11 time points (weeks in husbandry 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40) with no differences in non-infected (demonstrated in green, Kruskal-Wallis-Test: H(10;42) = 4.3123; p = 0.9322) and infected animals (demonstrated in red, Kruskal-Wallis-Test: H(10;50) = 6.8237; p = 0.7420) and between groups (Mann-Whitney U-test, p > 0.06)

Fig 2. Detection of Borna disease virus-1 isolated from saliva (shrew #2) in rabbit embryonic brain cells (3^rd passage after isolation).

Immunofluorescence, polyclonal anti-BoDV-1 rat serum.

Fig 3. Phylogenetic analysis of BoDV-1 sequences obtained from isolated virus.

2150 nt long nucleic sequences comprising N, P, X genes of two isolated virus isolates (shrew #2, shrew #6), two sequences obtained from a shrew and a horse of the same region (KF275184, KF275185 [17]) and other representative BoDV-1 of endemic subclusters [25]. Cluster 1: Southwest Germany and Southern Rhine valley group with Cluster 1a: Baden-Wurttemberg and parts of Bavaria, Germany (L27077, AY374524) and Cluster 1b: Switzerland, The Principality of Liechtenstein and Austria (AY374550, AY374551), Cluster 2: South German Group (AY374521, AY374531), Cluster 3: Southern Saxony-Anhalt and Saxony (AY374519, AY374534), Cluster 4: Central German group (U04608, AY374522). Tree is rooted with BoDV-2 (AJ311524).

Fig 4. Demonstration of BoDV-1 nucleoprotein, messenger RNA and genomic RNA.

(A) Demonstration of BoDV-1 nucleoprotein by immunohistochemistry (IHC) in the brain of C. leucodon #5017; (B) Demonstration of BoDV-1 messenger RNA by in-situ hybridization (ISH) in the brain of C. leucodon #5017; (C) Demonstration of genomic BoDV-1 RNA by ISH in the brain of C. leucodon #5017; (D) Demonstration of BoDV-1 nucleoprotein by IHC in the trigeminal ganglion of C. leucodon #2001; (E) Demonstration of BoDV-1 messenger RNA by in-situ hybridization (ISH) in the trigeminal ganglion of C. leucodon #2001; (F) Demonstration of genomic BoDV-1 RNA by ISH in the trigeminal ganglion of C. leucodon #2001; (G) Demonstration of BoDV-1 nucleoprotein by IHC in the skin, mainly in the sebaceous glands of C. leucodon #2001; (H) Demonstration of BoDV-1 messenger RNA by in-situ hybridization (ISH) in the skin, mainly in the sebaceous glands of C. leucodon #2001; (I) Demonstration of genomic BoDV-1 RNA by ISH in the skin, mainly in the sebaceous glands of C. leucodon #2001;