Complete genome sequence of an astrovirus identified in a domestic rabbit (Oryctolagus cuniculus) with gastroenteritis

Abstract

A colony of domestic rabbits in Tennessee, USA, experienced a high-mortality (~90%) outbreak of enterocolitis. The clinical characteristics were one to six days of lethargy, bloating, and diarrhea, followed by death. Heavy intestinal coccidial load was a consistent finding as was mucoid enteropathy with cecal impaction. Preliminary analysis by electron microscopy revealed the presence of virus-like particles in the stool of one of the affected rabbits. Analysis using the Virochip, a viral detection microarray, suggested the presence of an astrovirus, and follow-up PCR and sequence determination revealed a previously uncharacterized member of that family. Metagenomic sequencing enabled the recovery of the complete viral genome, which contains the characteristic attributes of astrovirus genomes. Attempts to propagate the virus in tissue culture have yet to succeed. Although astroviruses cause gastroenteric disease in other mammals, the pathogenicity of this virus and the relationship to this outbreak remains to be determined. This study therefore defines a viral species and a potential rabbit pathogen.

Figure 1

Electron micrograph of virus like particles in the stool of one animal (Table1). Scale bar indicates 50 nm.

Figure 2

The rabbit astrovirus genome. (A) A cartoon showing the organization of the viral genome. Scale bar indicates nucleotide position. The location of a putative ribosomal frameshift motif is indicated by an arrowhead. The position of the two overlapping Virochip probes that were originally positive for an astrovirus are indicated. (B) Location of overlapping PCR, 5^′, and 3^′ RACE amplicons used to determine and confirm the sequence of the viral genome. (C) A histogram showing the number of Ion Torrent reads (x1000 reads) aligning at each nucleotide position in the genome. (D) The average pairwise percent identity between the predicted rabbit astrovirus protein sequences and the sequences of the proteins from the astroviruses shown in Figure 3. The histogram is binned into 20 amino acid windows.

Figure 3

Maximum likelihood phylogenies created from multiple sequence alignments of the predicted rabbit astrovirus proteins and the related proteins of all astroviruses in Genbank with complete genome sequences. (A) Phylogeny based on ORF1A sequences. (B) Phylogeny based on ORF1B sequences. (C) Phylogeny based on the first ~420 amino acids of the indicated ORF2 (capsid) sequences. See Methods for accession numbers of sequences used. The bootstrap percentages of selected nodes are indicated. Scalebars indicate 0.2 amino acid substitutions per site.