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).