Author: Lelli, Davide; Lavazza, Antonio; Prosperi, Alice; Sozzi, Enrica; Faccin, Francesca; Baioni, Laura; Trogu, Tiziana; Cavallari, Gian Luca; Mauri, Matteo; Gibellini, Anna Maria; Chiapponi, Chiara; Moreno, Ana
Title: Hypsugopoxvirus: A Novel Poxvirus Isolated from Hypsugo savii in Italy Document date: 2019_6_19
ID: 1axsebya_9
Snippet: After necropsy, organ samples (lungs, heart, kidney, brain, and intestines) were mechanically homogenized in minimal essential medium (1 g/10 mL), which contained antibiotics. They were then centrifuged at 3000 g for 15 min. Samples were inoculated in confluent monolayers of VERO and MARC 145 cells (African green monkey), incubated at 37 • C with 5% CO 2 and observed daily for seven days to assess their cytopathic effects (CPEs). In the absence.....
Document: After necropsy, organ samples (lungs, heart, kidney, brain, and intestines) were mechanically homogenized in minimal essential medium (1 g/10 mL), which contained antibiotics. They were then centrifuged at 3000 g for 15 min. Samples were inoculated in confluent monolayers of VERO and MARC 145 cells (African green monkey), incubated at 37 • C with 5% CO 2 and observed daily for seven days to assess their cytopathic effects (CPEs). In the absence of CPEs, the cryolysates were sub-cultured twice onto fresh monolayers. Cell culture supernatants showing CPE were partially purified by ultracentrifugation at 35,000 rpm for 2 h (rotor TST41 Kontron) through a 25% (w/w) sucrose cushion, and the pellet was re-suspended in PBS. This antigen was kept at −70 • C and then submitted for viral identification with the NGS approach and negative-staining electron microscopy (nsEM) by using the Airfuge (Beckman Instruments, Palo Alto, CA, USA) method [17] .
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