Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus

Abstract

Bovine enteric caliciviruses (BEC) are associated with diarrhea in young calves. The BEC strains detected in Europe form a third genogroup within the genus "Norwalk-like viruses" (NLV) of the family Caliciviridae. In this report, we present sequence, clinical, and histological data characterizing a novel enteropathogenic BEC strain, NB, detected in fecal specimens from calves in the United States. The complete RNA genome of the NB virus is 7,453 bases long and is organized into two open reading frames (ORFs). ORF-1 is 2,210 amino acids long and encodes a large nonstructural polyprotein contiguous with the major capsid protein (VP1), similar to the lagoviruses and "Sapporo-like viruses" (SLV). The conserved calicivirus motifs were identified in the nonstructural proteins. ORF-2 is located at the 3' end of the genome and encodes a small basic protein (VP2) of 225 amino acids. The 5' and 3' untranslated regions are 74 and 67 bases long, respectively. Among caliciviruses, NB virus shows amino acid identities of 14.1 to 22.6% over the entire ORF-1 nonstructural-protein sequence with NLV, SLV, vesivirus, and lagovirus strains, while the overall sequence identity of the complete NB VP-1 with other caliciviruses is low, varying between 14.6 and 26.7%. Phylogenetic analysis of the complete VP1 protein, including strains from all four calicivirus genera, showed the closest grouping of NB virus to be with viruses in the genus Lagovirus, which cause liver infections and systemic hemorrhage in rabbits. In gnotobiotic calves, however, NB virus elicited only diarrhea and intestinal lesions that were most severe in the upper small intestine (duodenum and jejunum), similar to the NLV BEC strains. The tissues of major organs, including the lung, liver, kidney, and spleen, had no visible microscopic lesions.

FIG. 1.

Genome organization, 5′ UTR and subgenomic conserved sequences, and predicted ORF-1 NS proteins of the BEC-NB strain. (A) ORFs and UTR sequences in the complete 7,453-bp genome. (B) Locations and sequence conservation of the 5′ UTR (nucleotides 1 to 23) and subgenomic (nucleotides 5035 to 5057) sequence segments in BEC-NB. Only differences are indicated. (C) Predicted ORF-1 NS proteins of BEC-NB and the NLV BEC-Jena based on sequence comparison with maps of characterized cleavage products of a lagovirus, RHDV, and a human NLV, SOV. The calculated protein molecular weights are shown, and the COOH-terminal amino acid residue in the predicted virus ORF-1 sequence is shown above each box.

FIG. 2.

Unrooted consensus trees of 2C-like helicase/ATPase (A), 3C-like protease (B), 3D-like RdRp (C), and major capsid (VP-1) proteins (D) of BEC-NB. The lengths and ORF-1 locations of the amino acid sequences used to prepare the multiple alignments of the viruses used in the comparisons are shown in Table 1. The final trees depicted represent a consensus of trees for 100 bootstrapped data sets analyzed using the maximum-likelihood algorithm. The bootstrap values indicate the percentage of replicates supporting the branch points predicted in the consensus trees.