Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: Identification of a candidate etiologic agent

Kistler, Amy L; Gancz, Ady; Clubb, Susan; Skewes-Cox, Peter; Fischer, Kael; Sorber, Katherine; Chiu, Charles Y; Lublin, Avishai; Mechani, Sara; Farnoushi, Yigal; Greninger, Alexander; Wen, Christopher C; Karlene, Scott B; Ganem, Don; DeRisi, Joseph L

Avian bornavirus (ABV) genome sequence recovery and comparative analysis to Borna disease virus (BDV) genomes. A. Bornaviridae genome schematic. Grey bar at base, non-segmented negative sense viral RNA (vRNA) of Bornaviridae genome; coordinates of major sequence landmarks highlighted below. Green bars and dashed lines, transcription initiation sites (TISs); red bars, transcription termination sites. Distinct ORF-encoding transcription products and the gene products they encode are diagrammed above: TIS1 transcripts encoding nucleocapsid (N) gene, pink; TIS2 transcripts encoding phosphoprotein (P) and X genes, green; TIS3 transcripts encoding the matrix (M), glycoprotein (G) and polymerase (large or 'L') gene, blue. Exons, thick solid black lines; introns, thin solid black lines; dashed black lines, 3'ends of transcripts generated transcription termination read-through; shaded boxes, location of ORFs in transcripts; reading frames for ORFs from multiple genes generated from TIS3 indicated at right. Array probes track, Bornaviridae oligonucleotide 70 mer probes from the Virochip array. PCR primers track, primers generated for PCR follow up and screening of specimens in this study for detection of Bornaviridae species with expected product diagrammed below. vRNA RT-PCR track, overlapping vRNA clones and RACE products recovered directly from RNA extracted from crop tissue of a histologically confirmed case of PDD. Solexa reads track shows distribution of 33 mer reads with at least 15 bp sequence identity to recovered ABV genome sequence. Sequence identity with BDV genomes track shows scanning average pairwise nucleotide sequence identity (window size of 100 nucleotides, advanced in single nucleotide steps) shared between ABV and all BDV genome sequences in NCBI. A dashed line on the graph indicates 50% identity threshold for reference. B. Phylogenetic analysis of ABV genome and the 4 representative BDV genome isolates. samples originating in the United States, consisted of crop biopsy specimens from 3 histologically confirmed PDD cases and 5 controls that were provided for nucleic acid extraction and follow-up Virus chip analysis. The samples from the second series (n = 8) originated in Israel, where total RNA and DNA from proventriculus, ventriculus and brain specimens were extracted from 5 PDD cases and 3 controls. For each series, total RNA was reverse-transcribed with random primers, PCR-amplified, and fluorescently labeled and hybridized to the Virus chip microarray as previously described [18] .