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

vRNA genome sequence recovery To ensure recovery of accurate sequence across the ABV genome, especially at splice junctions and transcription initiation and termination sites, we utilized the sequence from ABV hybrid assembly to design primers for recovery of 3 overlapping products by RT-PCR directed against the vRNA present in the specimen. Aliquots of 500 ng of DNAse-treated total RNA extracted from the clinical specimen were annealed with 3 primers complementary to the predicted vRNA sequences: ABV1r, 5'-ATGACCAGGAC-GAGGAGATG-3' (maps to residues 8831-8812 of vRNA), ABV2r, 5'-CCTGTGAATGTCTCGTTTCTG-3' (maps to residues 5754-5733 of vRNA), and ABV3r 5-TTCTTTCAG-CAACCACTGACG-3' (maps to residues 2563-2543 of vRNA). Reverse transcription was carried out at 50°C for 1 hr with SuperScriptIII (Invitrogen, Carlsbad CA) according to manufacturer's instructions. Following RNase H treatment, PCR was performed on the resulting cDNA with Phusion polymerase (NEB, Ipswich, MA) with the primers used for reverse transcription and the following primers: ABV1f: 5'-GGATCATTCCTTGATGATGTATTAGC-3', (maps to residues 5567-5589) ABV2f: 5'-CAAATGGA-GAGCCTGATTGG-3' (maps to residues 2378-2397) ABV3f: 5'-AATCGGTAAGTCCAGAGTCAAGG-3' (maps to residues 155-177). All products were amplified for 35 cycles under the following conditions: 98°C, 3 minutes; 98°C, 10 seconds, 50°C, 30 seconds, 72°C 3 minutes. Resulting products were gel purified, and subcloned into the TOPO T/A cloning vector pCR2.1 after incubation with Taq polymerase and dATP for 10 minutes at 72°C. For each product, 4 independent transformants were prepared for standard dideoxy sequencing on an ABI3730 sequencer (ElimBio, Hayward CA). Forward and reverse reads spanning each clone were generated using M13F and M13R and additional overlapping primers spaced at 600-800 bp intervals across the each of the clones. 5' and 3' RACE to sequence at vRNA termini vRNA RT-PCR products containing uncapped vRNA termini were captured using the First Choice RLM RACE kit (Ambion, Austin TX) with the following modifications to the standard protocol: 1) tobacco acid phosphotase treatment was omitted, 2) a phosphorylated RNA, RNAligate, 5'-p-GUUAUCACUUUCACCC-3' (gift of J. Shock, DeRisi lab) was substituted for the 3' RNA ligation-mediated RACE primer provided in the kit and ligated to 3' ends as per manufacterer's 5' RACE protocol, and 3) in the 3' RACE reverse transcription reactions, two reverse transcription reactions were performed and carried forward in parallel: one with random decamers and one with a DNA oligo complementary to oJSmer utilized in the RNA ligation step (ligateRC, 5'-p-GGGTGAAAGTGATAAC-3'). For 5' RACE, a single round of PCR was sufficient to generate a product using the vRNA specific primer ABV5RaceOuter, 5'-CAGTCGGTTCTTGGACTTGAAGTATCTAGG-3' (maps to residues 346-317 of vRNA) and manufacturer provided outer PCR primer. For 3' RACE, nested PCR was required to recover detectable PCR product of expected size using outer PCR primers oJSmerRC and the gene specific primer ABV3RaceOuter, 5'-CCCGTCTACTGTTCTTTCGCCG-3' (maps to residues 8479-8497 of vRNA), followed by inner PCR using Tailed_RNAligateRC, 5'-AAGCAGTGGTAACAACGCAGAGTACGGGTGAAAGT-GATAAC-3' and the gene specific primer, ABV3RaceInner, 5'-GCAATCCAGGAATAAGCAAGCACAAA-3' (maps to residues 8595-8620 of vRNA). Both of the RACE PCR reactions were carried out with Platinum Taq polymerase (Invitrogen, Carlsbad, CA) in 35 cycles of gradient PCR (with varying