VIRUS ISOLATION AND CHARACTERIZATION
Eleven Chinese firebelly newt (Cynops orientalis) carcasses that were part of a larger shipment of live newts imported to the United States from Hong Kong, China in August 2015 were subjected to virus culture to screen for ranavirus, a major pathogen of amphibia.9 Pools of sterilely collected organs from dead salamanders, including liver, kidney and spleen, were homogenized in Dulbecco's minimal essential medium in a 1:10 (weight to volume) ratio and inoculated into either epithelioma papulosum cyprini (EPC10) or zebrafish (ZF411) epithelial cell monolayers. A different set of dissection instruments was used for each animal. Cultures were incubated at 10 °C–16 °C and examined for cytopathic effects (CPE) daily for 7–10 days. Cultures that did not exhibit CPE were blind passed at least once before being classified as negative. Cultures that exhibited CPE were frozen at −80 °C and thawed three times, and cellular debris was removed by centrifugation at 1000 g for 30 min at 4 °C. For electron microscopic analysis, aliquots of the supernatant were re-centrifuged at 100 000 g for 10 min in a Beckman Airfuge (lndianapolis, CA, USA). The pelleted viral particles were negatively stained with 2% phosphotungstic acid and examined in a Hitachi H-7600 transmission electron microscope (Schaumnberg, lL, USA).
For reverse transcription-PCR (RT-PCR) analysis, aliquots of the initial low-speed supernatant were extracted using the following kits according to the manufacturer's instructions: Qiagen DNeasy Blood and Tissue Kit (Valencia, CA, USA) (for DNA) or Ambion MagMAX Viral RNA Isolation Kit (Waltham, MA, USA) (for RNA). Primer SVCV F1 (5′-TCT TGG AGC CAA ATA GCT CAR RTC-3′) and SVCV R2 (5′-AGA TGG TAT GGA CCC CAA TAC ATH ACN CAY-3′) were used in a 25-μL one-step RT-PCR reaction based on Stone et al.12 using Qiagen One-Step reagents with 5 μL of RNA according to the manufacturer's instructions. Cycle conditions were as follows: 50 °C for 20 min and 95 °C for 10 min; 45 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. Reaction products (5 μL) were examined using a 1% agarose gel, samples with the expected amplicon size (714 bp) were sequenced with the PCR primers and the resulting sequence was characterized by BLAST analysis. A real-time RT-PCR (rRT-PCR) assay targeting the G gene was used to determine individual infection status of animals whose tissues had originally been pooled for virus isolation.13