Ball Python Nidovirus: a Candidate Etiologic Agent for Severe Respiratory Disease in Python regius

Stenglein, Mark D.; Jacobson, Elliott R.; Wozniak, Edward J.; Wellehan, James F. X.; Kincaid, Anne; Gordon, Marcus; Porter, Brian F.; Baumgartner, Wes; Stahl, Scott; Kelley, Karen; Towner, Jonathan S.; DeRisi, Joseph L.

Tissue culture and virus isolation. Cells were cultured as described previously (59) . To prepare inocula, frozen tissues were thawed on ice, and 1-g portions were minced with scalpels and disrupted by Dounce homogenization in ice-cold minimum essential medium (MEM) (Gibco) supplemented with 25 mM HEPES buffer, pH 7.4. Homogenate was clarified by centrifugation at 10,000 ϫ g for 1 min and filtered through a 0.45-m filter. Filtrate was diluted 1:10 in MEM plus HEPES and added to cultures of 80% confluent cells. Culture medium was harvested and replaced after 20 h and every 2 to 3 days thereafter for an additional 14 days and stored at Ϫ80°C until further processing. RNA was isolated from clarified culture supernatant using the ZR viral RNA kit (Zymo Research), according to the manufacturer's protocol. RNA was reverse transcribed and analyzed by qRT-PCR as described above.