A chimeric bovine enteric calicivirus: evidence for genomic recombination in genogroup III of the Norovirus genus of the Caliciviridae

Abstract

The Norovirus genus of the Caliciviridae encompasses viruses that cause outbreaks of gastroenteritis in human and viruses that have been associated with diarrhea in cattle. The two bovine noroviruses, Bo/Newbury2/76/UK and Bo/Jena/80/DE, represent two distinct genetic clusters in the newly described genogroup III. In the present study, Jena-like polymerase sequences were identified for the first time in the UK, but one of these, Bo/Thirsk10/00/UK, was a chimeric virus. Bo/Thirsk10/00/UK had a Jena-like polymerase gene but Newbury2-like capsid and ORF3 genes by comparison of their genome organization, nucleotide, and amino acid identities and phylogenetic analyses. The present study is one of few studies to clearly demonstrate the existence of chimeric genomes in the Norovirus genus and the first, to our knowledge, to identify a chimeric genome in genogroup III. It provides additional support that genomic recombination is part of the natural evolution of noroviruses and is relevant to the diagnosis and immunological control of norovirus diarrhea outbreaks.

Fig. 1

HMA with the 316 bp M13-forward/M13-reverse amplicons from plasmids containing the 114 bp NI/E3 amplicons from the RdRp of BoCVs Bo/Newbury2/76/UK, Bo/Aberystwyth65/00/UK, and Bo/Thirsk10/00/UK. Lane A—Bo/Newbury2/76/UK, B—Bo/Thirsk10/00/UK, C—Bo/Aberystwyth65/00/UK, D—Bo/Newbury2/76/UK with Bo/Aberystwyth65/00/UK, E—Bo/Aberystwyth65/00/UK with Bo/Thirsk10/00/UK, F—Bo/Newbury2/76/UK with Bo/Thirsk10/00/UK. The dotted box outlines homoduplexes and the solid box outlines heteroduplexes. The figures in italics show nucleotide identities determined by sequence analysis between the 76-nucleotide fragments of the RdRp of the BoCVs in the relevant lane.

Fig. 2

Multipie alignment of the BoCV ORF3 proteins starting at the methionine of the Bo/Newbury2/76/UK initiation codon (boxed). The methionine for the initiation codons of Bo/Thirk10/00/UK and Bo/Jena/80/DE is highlighted with a black background. Asterisks (highlighted with a gray background) represent noncoding residues caused by premature termination codons in the Bo/Thirsk10/00/UK and Bo/Jena/80/DE ORF3 genes. Dots represent identical amino acids to Bo/Newbury2/76/UK. Dashes represent gaps in the aligned ORF3 proteins. Numbers represent amino acid position in the Bo/Jena/80/DE ORF3 protein.

Fig. 3

Phylogenetic analyses generated using the maximum likelihood method of TreePuzzle for the translated amino acid sequences of (A) the partial RdRp sequence (193 amino acids), (B) the complete capsid, and (C) 113 amino acids from the 5′ end of the ORF3 gene (starting from the Bo/Jena/80/DE ORF3 initiation codon) of the UK BoCVs, the German Bo/Jena/80/DE, and representatives of the human genogroups I and II noroviruses. The Sapovirus Hu/Manchester/93/UK was used as the out-group. The UK and German reference viruses that represent the two genetic clusters of genogroup III are highlighted in bold italicized text. The numbers at the nodes (quartet puzzling values) indicate the frequencies of occurrence for 1000 replicate trees.

Fig. 4

Nucleotide identity plot of the 3′ end (2818 nucleotides) of the Bo/Thirsk10/00/UK genome compared with the UK (Bo/Newbury2/76/UK) and German (Bo/Jena/80/DE) reference BoCVs. The bars below the plot represent the three ORFs. The capsid gene (ORF2) is divided into the S (dotted line), P1 (solid line), and P2 (dashed line) domains of the Hu/Norwalk/68/US as determined by X-ray crystallography (Prasad et al., 1999). The arrow indicates the point after the recombination site (nucleotide 586) identified by LARD analysis. The Bo/Jena/80/DE and Bo/Thirsk10/00/UK ORF3 initiation codon was used.

Fig. 5

The genome recombination site of Bo/Thirsk10/00/UK identified at the ORF1 (solid line)–ORF2 (dotted line) overlap. The scale indicates the nucleotide position in the 2818 nucleotides from the 3′ end of the Bo/Thirsk10/00/UK genome. The point of recombination as determined by LARD analysis is indicated by the arrow. The ORF1 termination codon is in bold text and the ORF2 initiation codon is shown in italic underlined text. The arrow indicates the point after the recombination site (nucleotide 586) identified by LARD analysis.