Frequency and Pathological Phenotype of Bovine Astrovirus CH13/NeuroS1 Infection in Neurologically-Diseased Cattle: Towards Assessment of Causality

Abstract

Next-generation sequencing (NGS) has opened up the possibility of detecting new viruses in unresolved diseases. Recently, astrovirus brain infections have been identified in neurologically diseased humans and animals by NGS, among them bovine astrovirus (BoAstV) CH13/NeuroS1, which has been found in brain tissues of cattle with non-suppurative encephalitis. Only a few studies are available on neurotropic astroviruses and a causal relationship between BoAstV CH13/NeuroS1 infections and neurological disease has been postulated, but remains unproven. Aiming at making a step forward towards assessing the causality, we collected brain samples of 97 cases of cattle diagnosed with unresolved non-suppurative encephalitis, and analyzed them by in situ hybridization and immunohistochemistry, to determine the frequency and neuropathological distribution of the BoAstV CH13/NeuroS1 and its topographical correlation to the pathology. We detected BoAstV CH13/NeuroS1 RNA or proteins in neurons throughout all parts of the central nervous system (CNS) in 34% of all cases, but none were detected in cattle of the control group. In general, brain lesions had a high correlation with the presence of the virus. These findings show that a substantial proportion of cattle with non-suppurative encephalitis are infected with BoAstV CH13/NeuroS1 and further substantiate the causal relationship between neurological disease and astrovirus infections.

Keywords: astrovirus; bovine; cattle; encephalitis; infection; neurotropic.

Figure 1

Assessment of causation of bovine astrovirus (BoAstV) CH13/NeuroS1 infection in non-suppurative bovine encephalitis (Figure adapted from: Lipkin and Anthony [18]). The first reports of non-suppurative encephalitis in cattle date back to 1961, but all studies aiming at identifying a possible agent have remained inconclusive [12,13,14]. After discovery of BoAstV CH13/NeuroS1 in such cases, the criteria for a “possible causal relationship” were met but not yet for a probable causal relationship due to limitations in the study design [6,7]. To confirm that the BoAstV CH13/NeuroS1 is a probable cause for bovine non-suppurative encephalitis, we investigated the statistical association of the virus and the disease, and compared the topography of virus infection to that of the histopathological brain lesions.

Figure 2

Age distribution (A) and seasonal distribution (B) of BoAstV CH13/NeuroS1 positive cases.

Figure 3

Comparison of the detection of the viral RNA (vRNA) by in situ hybridization (ISH) and of the viral proteins by immunohistochemistry (IHC). The vast majority of analyzed sections show a good correlation of ISH and IHC, with a comparable staining in neurons, as seen in the cerebrum of case 50898 (a, b; examples are indicated by arrows). Only in three sections can a lack of correlation be observed, with either a negative ISH-result (c), or a red, granular staining in the IHC (d, arrow) as seen in the brainstem of case 50898, or vice versa, in case 25018: a deep purple ISH staining in neurons of the brainstem (e, arrows) is observed, while the IHC remains negative (f).

Figure 4

Correlation of pathological lesions and presence of the virus. Brainstem sections of two BoAstV CH13/NeuroS1-positive animals show glial nodes (*), neuronal necrosis (**), and perivascular cuffing (***) typical for non-suppurative bovine encephalitis in HE-stained sections. Severe lesions in the brainstem of case 50898 (a) show a good correlation to the results from the viral detection by IHC in most parts of the brainstem (b, arrow). In the brainstem of case 26875 perivascular cuffs are observed (c), correlating well to the BoAstV CH13/NeuroS1 positive staining of neurons in ISH in the same area (d, arrow).