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.

No abstract

Coronaviruses are enveloped, positive-stranded RNA viruses responsible for enteric, hepatitis, neurological and respiratory infections in humans and animals. Porcine haemagglutinating encephalomyelitis virus (HEV) is a member of the antigenic subgroup of haemagglutinating coronaviruses, including human respiratory coronavirus (HCoV-OC43), bovine coronavirus (BCoV) and some North American isolates of

Flaviviral NS2B/NS3 heterocomplex serine proteases are a primary target for anti-flavivirus drug discovery. To gain insights into the enzymatic properties and molecular determinants of flaviviral NS2B/NS3 protease substrate specificity in host cells, we developed and applied a novel series of membrane-anchored red-shifted fluorescent protein substrates to detect West Nile virus (WNV) NS2B/NS3 endoproteolytic activity in human cells. The substrate consists of a fluorescent reporter group (DsRed) tethered to the endoplasmic reticulum membrane by a membrane-anchoring domain. Between the two domains is a specific peptide linker that corresponds to the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 protein junctions within the WNV polyprotein precursor. When the protease cleaves the peptide linker, the DsRed reporter group is released, changing its localization in the cell from membrane-bound punctate perinuclear to diffuse cytoplasmic. This change in protein location can be monitored by fluorescent microscopy, and cleavage products can be quantified by Western blotting. Our data demonstrate the robustness of our trans-cleavage fluorescence assay to capture single-cell imaging of membrane-associated WNV NS2B/NS3 endoproteolytic activity and to perform in-cell selectivity profiling of the NS2B/NS3 protease. Our study is the first to provide cellular insights into the biological and enzymatic properties of a prime target for inhibitors of WNV replication.