L 15 growth medium

All 11 virus strains analyzed in this study were derived from the WHO Collaborating Center (http://apps.who.int/whocc/Detail.aspx?cc_ref=SEN-5&cc_code=sen) for arboviruses and viral hemorrhagic viruses in Senegal at Institut Pasteur de Dakar (Dakar, Senegal) (Table 2). Viral stocks were prepared by inoculating viral strains into Aedes albopictus (C6/36) cell line in Leibovitz 15 (L-15) growth medium (GibcoBRL, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) (GibcoBRL, Grand Island, NY, USA), 10% Tryptose Phosphate and antibiotics (Sigma, Gmbh, Germany). BAGV infection was confirmed after 4 days of propagation by immunofluorescence assay (IFA) using specific hyper-immune mouse ascitic fluid, as previously described [15 (link)]. Cultures supernatants were collected for virus RNA isolation. Extraction of viral RNA from supernatants was performed with the QIAamp viral RNA mini kit (Qiagen, Heiden, Germany) according to manufacturer’s instructions. Extracted RNA was frozen at −80 °C prior to downstream applications.

Xenopus laevis were obtained from the Korean Xenopus Resource Center for Research (Seoul, Korea). The embryos were injected following in vitro fertilization of eggs [31 (link)], the frogs having been induced by injection of 500 units of human chorionic gonadotropin (Sigma, St. Louis, MI, USA). RNAs were injected into the embryo animal pole at the one- or two-cell stage and then cultured in 30% Marc’s Modified Ringer’s (MMR) solution. RNAs of β-galactosidase (1 ng/embryo) or eGFP (1 ng/embryo) were injected as control to check for gastrulation defects at stage 11 [32 (link)]. Developmental stages were designated according to the Nieuwkoop and Faber Normal Table of Xenopus laevis (Daudin) [33 ]. Animal caps (ACs) were then dissected from the injected and noninjected embryos at stage 8.0–8.5 and incubated in 1× L-15 growth medium (Gibco/Thermo Fisher, Waltham, MA, USA) until stages 11 and 24 in preparation for RT-PCR.