Bovine coronaviruses associated with enteric and respiratory diseases in Canadian dairy cattle display different reactivities to anti-HE monoclonal antibodies and distinct amino acid changes in their HE, S and ns4.9 protein

Bovine coronavirus isolates associated with recent outbreaks of respiratory disease in Ontario and Quebec dairy farms were compared to reference strains known to be responsible for neonatal calf diarrhea (NCD) or winter dysentery (WD) of adult cattle. In respect to their hemagglutinating properties and their higher RDE activities with rat erythrocytes, WDBCoV strains differed from NCDBCoV strains and respiratory bovine coronaviruses RBCoV strains. Serologically, three MAbs directed to the HE glycoprotein of the WDBCoV strain BCQ.2590 recognized two serogroups amongst NCDBCoV strains by hemagglutination inhibition, whereas only one of the MAbs failed to react toward three of the four RBCoV isolates tested. Sequencing analysis of the S (S1 portion), HE, ORF4 and ORF5 genes of BCoV isolates associated with different clinical syndromes indicated that neither insertions or deletions could explain their distinct tropism. For the HE glycoprotein, a total of 15 amino acids (aa) substitutions were identified by comparing field isolates to the prototype Mebus strain. Two specific proline substitutions were identified for virulent strains being located in the signal peptides (aa 5) and aa position 367; one specific aa change was revealed at position 66 for RBCoV field isolates. Analysis of the S1 portion of the S glycoprotein revealed a total of eight aa changes specific to enteropathogenic (EBCoV) strains and eight aa changes specific to RBCoV strains. For all BCoV isolates studied, the region located between the S and M genes (ORF4) apparently encodes for two non-structural (ns) proteins of 4.9 and 4.8 kDa. A specific non-sense mutation was identified for the nucleotide at position 88 of the putative 4.9 kDa protein gene of RBCoV isolates resulting in 29 rather that 43 aa residues. The ORF5, which encodes a 12.7 ns protein and the 9.5 kDa E protein, was highly conserved amongst the BCoV field isolates.