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

To determine if the sequence fragments we detected among specimens derived from PDD cases corresponded to the presence of a full-length bornavirus, we performed unbiased deep sequencing on a PCR-confirmed bornavirus positive PDD case that contained the highest concentration of RNA. To recover both mRNA and vRNA present in the sample, RNA from this specimen was linearly amplified with both oligo(dT) and random hexamer primers, and then PCR-amplified using a modified random amplification strategy compatible with the Solexa sequencing platform (Materials and Methods). An initial set of 1.4 million 33 mer reads was obtained from this template material. Filtering on read quality, insert presence, and sequence complexity reduced this data set to 600,000 unique reads. Additional ELAND and iterative BLAST analyses ( [19] Materials and Methods) of these reads against all avian sequences in NCBI (including ESTs, n = 918,511) identified reads in the dataset with at least 22 nucleotides of sequence identity likely derived from host transcripts randomly amplified during sequencing sample preparation. The 322,790 reads that passed this host filter were next screened for the presence of bornavirus sequence through similar ELAND and iterative BLAST analyses (Materials and Methods) using a database generated from all Borna Disease virus (BDV) sequences present in NCBI (n = 207) and the sequences we had recovered from PCR follow-up of the PDD samples that tested positive for bornavirus by Virus chip microarray (n = 5). These analyses provided us with 1400 reads with at least a match of 15 or more nucleotides (blastn) or 7 or more predicted amino acids (tblastx) to known BDV sequences.