Identification of a Novel Astrovirus Associated with Bovine Respiratory Disease

Abstract

Astroviruses (AstVs) cause gastrointestinal disease in mammals and avians. Emerging evidence suggests that some AstVs have extraintestinal tissue tropism, with AstVs detected in the liver, kidney, central nervous system, and the respiratory tract variably associated with disease. In cattle, AstV infection has been linked to gastroenteric or neurologic disease. Here, metagenomic sequencing of a lung from a bovine with respiratory disease identified a novel AstV with a predicted capsid-encoding ORF2 amino acid sequence with 66% identity to caprine astrovirus (CAstV G2.1). A quantitative reverse transcription PCR (qRT-PCR) targeting ORF2 found four out of 49 (8%) lungs and one out of 48 (2%) enteric samples obtained from bovine diagnostic submissions positive for the novel bovine astrovirus (BAstV). In two strongly qRT-PCR-positive lung samples, intense novel BAstV nucleic acid signals were mainly localized in the cytoplasm of alveolar macrophages and mononuclear cells using RNAscope® in situ hybridization (ISH). Genetic analysis of two novel BAstV genomes determined from qRT-PCR positive samples found high similarity for ORF1ab nucleotide sequence (92.1% and 93.9%) to BAstV strain BSRI-1, while ORF2 nucleotide sequence was most similar to CAstV G2.1 (74.6% and 77.6%). Phylogenetic analysis of the novel BAstV sequences found a close genetic relationship to the single BAstV (BSRI-1) previously identified from a bovine respiratory sample as well as bovine and caprine AstVs identified from various tissues. Further research is needed to determine the clinical significance of BAstV in respiratory diseases.

Figure 1

Phylogenetic analysis of astrovirus. Phylogenetic trees were constructed by maximum likelihood analysis with 500 bootstrap replicates using MEGA11 software. Bootstrap values ≥70 are shown above the branches. GenBank accession numbers are indicated along with AstV host. The scale bar indicates 0.5 substitutions per site for Figure 1(a) and 1.0 substitution per site for Figure 1(b). ORF1ab amino acid sequence clades are arbitrarily labeled A-I. Clade B contains BAstV amino acid sequences identified in this study and highlighted in yellow (a) ORF2 amino acid sequence clades are arbitrarily labeled A-H. Clade F contains BAstV amino acid sequences identified in this study highlighted in yellow (b).

Figure 2

Detection of the novel bovine astrovirus nucleic acid in lungs from cattle with respiratory disease by in situ hybridization (ISH). Signal for the novel BAstV ranged from patches in 21-24401, indicated by arrows (a), to intensely diffuse in 20-25551 (b), 100x. Pinpoint signals from 21-24401 were detected in the cytoplasm of cells that corresponded to large alveolar macrophages, as well as mononuclear cells indicated by arrows (c), 400x. Diffuse, intense novel BAstV ISH signals obscured entire cells in sample 20-25551 indicated by arrows (d), 400x. No signal was detected in the bronchial epithelium, indicated by arrow, (e) or neutrophils, indicated by arrow, (f), in 20-25551 and 21-24401, respectively 400x. A negative control is inserted in (e).

Figure 3

Hematoxylin and eosin staining of serial lung sections used for ISH analysis. Arrows indicate areas where the novel BAstV ISH signal was identified in patches of 21-24401 (a), 100x. Serial H&E stained section with an intense diffuse ISH novel BAstV signal of 20-25551 (b), 100x. Large alveolar macrophages and mononuclear cells are indicated by the arrows (c, d), 400x. Bronchiolar epithelial cells, indicated by arrows, (e) and neutrophils, indicated by arrow, (f), 400x. A negative control is inserted in (e).