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.

Clinical molecular diagnostics. Portions of fresh frozen lung specimens were selected from several of the above cases for identification of nucleic acid sequences for several viruses implicated as causative agents of respiratory tract disease of snakes. RNA was extracted from lung of BP1 and tested by using previously described consensus PCR methods for the genus Ferlavirus in the Paramyxoviridae, the Rhabdoviridae, and the genus Orthoreovirus in the Reoviridae (71) (72) (73) . Additionally, RNA was extracted from portions of lungs of BP6 and -7 and tested for gene sequences for members of the family Rhabdoviridae and Filoviridae. For molecular diagnostic detection of filoviruses (Marburg viruses and Ebola viruses), RNA was sent to the Viral Special Pathogens Branch, Centers for Disease Control and Prevention. Two independent panfilovirus RT-PCR assays were utilized as previously described (74, 75) using SuperScript III reverse transcriptase (Life Technologies) according to the manufacturer's instructions. Briefly, RNA was reverse transcribed for 30 min at either 45°C or 50°C, respectively, and then denatured for 2 min at 94 to 95°C. For RNAs amplified according to the protocol described elsewhere (74) , reaction mixtures were thermocycled 40 times at 94°C for 15 s, followed by successive incubations at 45°C and 68°C for 30 s each. For RNAs amplified according to the previously described protocol (75) , reaction mixtures were treated identically except that the annealing and extension temperatures were 53°C and 72°C degrees, respectively. Positive-and negativecontrol reactions were carried out in parallel to validate the performance of the testing.