Identification, Characterization, and In Vitro Culture of Highly Divergent Arenaviruses from Boa Constrictors and Annulated Tree Boas: Candidate Etiological Agents for Snake Inclusion Body Disease

Stenglein, Mark D.; Sanders, Chris; Kistler, Amy L.; Ruby, J. Graham; Franco, Jessica Y.; Reavill, Drury R.; Dunker, Freeland; DeRisi, Joseph L.

Sample collection and processing. At necropsy, portions of tissues were fixed in formalin or frozen at Ϫ80°C until further processing. For histopathological analysis, tissue samples were preserved in 10% neutral buff- For RNA extraction, 100-mg tissue pieces were added to 1 ml of Trizol reagent (Invitrogen) and a ball bearing in a centrifuge tube. Tissue was disrupted using the TissueLyzer (Qiagen) for 2 min at 30 Hz. Samples were clarified by centrifugation at 10,000 ϫ g for 2 min, and then 200 l chloroform was added. Samples were mixed, incubated for 2 min at room temperature, and then centrifuged for 15 min at 12,000 ϫ g at 4°C. The RNA in the aqueous phase was further purified using an RNA Clean and Concentrator column (Zymo Research) according to the manufacturer's protocol, including the optional on-column DNase digestion to remove residual DNA. RNA quantity and quality were determined by spectroscopy.