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

When possible, a sample of brain as well as a combined proventricular/ventricular sample was prepared for RNA extraction for each bird. If not available, other tissues and/ or gastrointestinal content were used (see Additional file 5: Summary of clinical and molecular data for specimens provided in this study). Frozen samples were allowed to thaw for 1-2 hours at room temperature prior to handling. Then, under a laminar flow biohazard hood and using aseptic technique, approximately 1 cm 3 of each tissue was macerated by two passages through a 2.5 ml sterile syringe and transferred into sterile test tubes containing 4 ml nuclease-free PBS. The content of the tubes was mixed by vortex for 30 sec, and the tubes were placed overnight at 4°C. RNA extraction was performed on the following day, using either the QIAamp ® viral RNA kit (Qiagen, Valencia, CA; batch1&2, specimens 1-8) or the TRI Reagent ® kit (Molecular Research Center, Cincinnati, OH; all other specimens), following the manufacturers' instructions. The end product was either provided lyophilized (batches 1 and 2, samples 1-9) as a dry pellet, or re-suspended in 40 ul nuclease-free water.