Characterization of Winter Dysentery Bovine Coronavirus Isolated from Cattle in Israel

Abstract

Bovine coronavirus (BCoV) is the causative agent of winter dysentery (WD). In adult dairy cattle, WD is characterized by hemorrhagic diarrhea and a reduction in milk production. Therefore, WD leads to significant economic losses in dairy farms. In this study, we aimed to isolate and characterize local BCoV strains. BCoV positive samples, collected during 2017-2021, were used to amplify and sequence the S1 domain of S glycoprotein and the full hemagglutinin esterase gene. Based on our molecular analysis, local strains belong to different genetic variants circulating in dairy farms in Israel. Phylogenetic analysis revealed that all local strains clustered together and in proximity to other BCoV circulating in the area. Additionally, we found that local strains are genetically distant from the reference enteric strain Mebus. To our knowledge, this is the first report providing molecular data on BCoV circulating in Israel.

Keywords: S1; bovine coronavirus; hemagglutinin esterase; spike; winter dysentery.

Figure 1

Cytopathic effect and replication of Bovine coronavirus (BCoV) isolate ISR2182. (A) Uninfected HRT-18G cells show intact monolayer, while cells infected with local isolate ISR2182 (1:10) for 72 h show significant changes in monolayer (white arrows). (B) Replication of BCoV ISR2182 in HRT-18G, cells were infected with isolate ISR2182 and collected at different time points. BCoV replication was measured by RT-qPCR. (C) Immunofluorescence images showing replication of BCoV isolate ISR2182 in HRT-18G cells as detected by monoclonal antibody-targeting coronavirus nucleocapsid protein, with DAPI for nucleus staining.

Figure 2

Phylogenetic tree of the partial Spike gene of Bovine coronavirus (BCoV) from local isolates. (A) Phylogenetic tree of S1 region of BCoV spike glycoprotein. BCoV genomic RNA was isolated from feces of WD diseased cattle and used for molecular characterization of the S1 region. Local isolates (labeled in red) were aligned with selected reference strains. Phylogenetic tree generated based on S1 nucleotide sequences (nucleotides 1–2731) was generated via the neighbor-joining method with bootstrap analysis (1000 replicates, >70%). The scale bar shows the number of substitutions per site. We used HCoV OC43 as an outgroup strain. Spike partial sequences were retrieved from GenBank and embedded in the figure for each strain. (B) Deduced amino acid sequences of S1 from local isolates aligned to reference strain Mebus. Local isolate sequences were aligned using Geneious protein alignment tool to show only disagreements residues compared to the reference sequence of Mebus strain.

Figure 3

Phylogenetic tree of the Hemagglutinin Esterase (HE) gene of Bovine coronavirus (BCoV). Local isolates. (A) Local strains’ (labeled in red) alignment with selected reference BCoV HE sequences. Phylogenetic tree was generated based on full HE gene sequences. Phylogenetic trees were generated via the neighbor-joining method with bootstrap analysis (1000 replicates, >70%). The scale bar shows the number of substitutions per site. We used HCoV OC43 as an outgroup strain. HE sequences retrieved from GenBank and embedded in the figure for each strain. (B) Deduced amino acid sequences of local isolates aligned to reference strain Mebus. Amino acid alignment showing only disagreements residues compared to reference strain Mebus.