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

Mapping these 1400 reads onto their corresponding positions on a consensus sequence for the 14 publicly available BDV genome sequences revealed spikes of high read coverage distributed discontinuously across the entire span of the BDV genome consensus. Reads containing blastn scores ≥ 90% identity to known BDV sequences were used as source sequences for primer design for PCR and sequence recovery of additional bornavirus sequence from both mRNA and vRNA templates present in the PDD specimen. Sequences recovered in this manner facilitated subsequent primer design for recovery of complete genome sequence via RT-PCR of 3 large overlapping fragments of the genome and 5'-and 3'-RACE ( Figure 2A , vRNA RT-PCR track) directly from negative stranded vRNA present in the total RNA extracted from this clinical specimen.