Detection of Astrovirus in Historical Cases of European Sporadic Bovine Encephalitis, Switzerland 1958-1976

Abstract

European sporadic bovine encephalitis is a frequent diagnosis in neurologically diseased cattle, but its etiology remained unresolved. Using in situ hybridization, we have detected a recently discovered neurotropic bovine astrovirus in historical tissues in a high proportion of brain samples of affected cattle. Our results suggest that astroviruses were already involved in the pathogenesis of the disease several decades ago, but have gone undetected.

Keywords: archive; astrovirus; cattle; encephalitis; historical; neurological disease.

Figure 1

Representative results of the in situ hybridization (ISH) for BoAstV-CH13/NeuroS1 RNA in cattle brain tissues. Two DIG labeled RNA probes [probe A: micrographs (A,C,E); probe B, micrographs (B,D,F)] were applied. Arrows indicate individual ISH-positive neurons. (A,B) Historical case of European sporadic bovine encephalitis (case 3628), which was diagnosed in 1959. Note that only probe A was reactive, but not probe B. (C,D) BoAstV-CH13/NeuroS1 positive control case, dating from 1995. Both probes show a clear labeling of neurons. (E,F) Negative control tissues with the absence of labeling by both probes.

Figure 2

Bovine astrovirus RNA and its correlation with histopathological lesions in brain tissues of historical cases of European sporadic bovine encephalitis. Astrovirus RNA was detected by in situ hybridization with probe A [ISH, purple labeling; (A,C,E,G)] and histopathological lesions were assessed after hematoxylin and eosin staining [HE; (B,D,F,H)]. (A,B) Case 11930, brainstem: good correlation of ISH labeling and histopathological lesions in perivascular cuffs (arrow). (C,D) Case 3628, brainstem: strong ISH labeling in neurons in vicinity of lesions (arrows), and weak ISH labeling of glia cells in a glial node (asterisk). (E,F) Case 3628, brainstem: strongly ISH labeled neurons and weakly labeled mononuclear cells in a perivascular cuff. (G,H) Case 11646, cerebellar cortex: lack of correlation between lesions and ISH labeling. While there is a moderate gliosis, cells remain ISH negative. Magnifications of microphotographs are indicated by scale bars.

Figure 3

Results of RT-PCR and sequencing of astrovirus RNA extracted from case 3466. A scheme of the BoAstV CH13 genome is presented. The viral genome is organized in three open-reading frames (ORF). The red bar indicates the target sequence for RT-PCR using primers MA2/MA4 within ORF 1b, which encodes for the RNA-dependent RNA polymerase. The blue and black bars show target sequences of in situ hybridization probes A and B, respectively, within ORF2 that encodes for structural capsid proteins. The nested RT-PCR protocol with primers bAV3/bAV4 (first round) and bAV1/bAV2 (second round) was designed to yield amplicons in the target region of ISH probe B (black bar). Sequence comparisons of the MA2/MA4 and of the bAV1/bAV2 amplicons of case 3466 with the BoAstV CH13 reference sequence (GenBank accession number: NC_024498) are shown. Alignments were generated with the Geneious R9 software, version 9.0.4 (Biomatters).