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

Sequences recovered from BDV_LconsensusF and BDV_LconsensusR PCR products were aligned, and an additional set of ABV consensus primers biased towards the ABV sequences were identified: ABV_LconsensusF, 5'-CGCCTCGGAAGGTGGTCGG-3' (maps to positions aligning with residues 3724-3742 of BDV reference genome) and ABV_LconsensusR, 5'-GGCAYCAYCK-ACTCTTRAYYGTRTCAGC-3' (maps to positions aligning with residues 4233-4257 of BDV reference genome). Using identical PCR cycling conditions as described above for the microarray-based Bornaviridae PCR assay, these ABV consensus primers were found to be > 100X more sensitive for ABV detection compared to BDV_LconsensusF and BDV_LconsensusR primers, and were thus utilized to re-screen the initial set of PDD case and control samples provided for microarray analysis (no additional positives identified) and all subsequently provided samples. Two additional PCR primers in the N (ABV_NconsensusF: 5'-CCHCATGAGGCTATWGATT-GGATTAACG-3' and ABV_NconsensusR: 5'-GCMCGG-TAGCCNGCCATTGTDGG-3') and M (ABV_MconsensusF: 5'-GGRCAAGGTAATYGTYCCT-GGATGGCC-3' and ABV_PconsensusR: 5'-CCAACAC-CAATGTTCCGAAGMCG-3') that mapped to conserved sequences shared between the complete ABV genome sequence and the 14 other fully sequenced BDV genomes in the NCBI database were also employed for PCR screening of PDD cases and controls.