Material and Methods
Three red-necked wallabies were clinically examined after noticed to
show neurological symptoms (Table 1). Therapeutic attempts failed and
the animals were euthanized. A complete necropsy and histopathology were
performed. Samples from different tissues, including lungs, liver,
kidney, heart, stomach, small and large intestine, as well as brain,
were routinely fixed in 4% formalin, paraffin embedded (FFPE) and cut
into 3 µm sections followed by hematoxylin and eosin staining (H&E) for
light microscopic evaluation. Consecutive sections were standardly
stained with Luxol fast blue/cresyl violet (LFBKV) to evaluate myelin
sheaths and Nissl substance, with von Kossa to demonstrate dystrophic
mineralization and with Prussian blue reaction to detect hemosiderin as
previously described (Bennett et al., 2020b). Immunohistochemistry for
CD3 as a T cell marker, ionized calcium-binding adaptor molecule 1
(iba-1) as a marker for microglia and macrophages, and glial fibrillary
acidic protein (GFAP) for astrocyte detection was performed as
previously described (Bennett et al., 2020b) using
3-amino-9-ethylcarbazole as a substrate (AEC, Dako Carpinteria, CA, USA)
for chromogen labeling. Mayer’s hematoxylin was used for
counterstaining. In addition to formalin fixation, tissue samples of the
brain, lung, spleen, liver and small intestine were stored at -80°C for
subsequent molecular analysis.
Total RNA extraction and RT-qPCR was performed as previously described
(Pfaff et al., 2022). For all three animals, partial E1 protein-encoding
sequences of 715 nucleotides (nt) length were determined via
conventional RT-PCR with subsequent Sanger sequencing of the amplicons
by Microsynth Seqlab (Balgach, Switzerland). A Juke-Cantor
Neighbor-Joining tree was calculated from the three new sequences
together with all RusV sequences available in public databases using
Geneious Prime 2021.0.1 (Biomatters Ltd., Auckland, New Zealand). In
addition, the RNAScope 2-5 HD Reagent Kit-Red (Advanced Cell
Diagnostics, USA) was employed according to manufacturer’s instructions
for RNA in situ hybridization (ISH) using a custom-designed probe
targeting the RusV non-structural protein (p200, NSP) open reading
frame. A probe against the dihydrodipicolinate reductase (DapB )
gene was used as a technical negative control. Brain tissue from a
red-necked wallaby diagnosed with lumpy jaw disease without signs of
meningoencephalitis served as a negative control for detection of RusV
in ISH.
To exclude other known neuropathogens, brain samples were tested
via PCR for rabies virus (Fischer
et al., 2014), mammalian
bornaviruses (Schlottau et al., 2018), West Nile virus (Eiden et al.,
2010), Usutu virus (Jöst et al., 2011), tick-borne encephalitis
virus (Klaus et al., 2010), herpesviruses (Ehlers et al., 1999) andToxoplasma gondii (Talabani et al., 2009).