Mmessage in vitro transcription kit

The voltage clamp recording of Xenopus oocytes was performed as previously described^{38 (link),39 (link)} with slight modifications. Adult Xenopus laevis were acquired from Xenopus Aquaculture Materials. The oocytes were extracted from anesthetized Xenopus laevis. cRNA of AVR-14, GLR-1, SOL-1, and STG-1 was synthesized using the mMessage in vitro transcription kit (Ambion). Approximately 50 nL of the cRNA solution (containing 100 ng/µl cRNA) was injected into each oocyte. After 3–4 days of incubation at 17 °C, voltagec clamp measurements were performed using oc-725c (Warner Instruments). The two electrodes were inserted into oocytes, and oocytes were voltage-clamped to −50 to −60 mV in the extracellular solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, pH 7.2). Glutamate was applied for 20 s at each concentration (0.1, 0.2, 0.5, 1, 2, 5, and 10 mM, respectively) followed by at least 3 min of wash periods using a perista pump. Dataset was analyzed using Clampfit (10.3.2.1).

Synthetic RNA fragments of the viruses were prepared for LASV, RVFV, and YFV, MARV and CCHFV [24 (link)]. The target regions of LASV, RVFV and YFV viruses to be detected were amplified by RT-PCR, purified using QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA), and cloned into the expression vector pGEM T Easy (Promega, Madison, WI) containing the T7 promoter region. The plasmids were purified, and the presence of complete inserts was confirmed by sequencing the inserts using vector-specific primers. Target regions for MAR and CCHF were synthetically constructed and inserted into the pIDT Blue vector (Integrated DNA Technologies Coralville, IA). The complete inserts of the target regions thus generated were linearized and in vitro transcribed using the mMessage in vitro transcription kit (Ambion, Austin, TX). Following DNAse treatment, the synthetic RNA was purified using an RNeasy column (Qiagen, Inc., Valencia, CA). The quantity of RNA generated for each transcript was determined using the Ribogreen® RNA Quantitation Kit (Molecular Probes, Eugene, OR) following the manufacturer’s instructions.

Xenopus laevis oocytes were defolliculated with collagenase A (Roche Applied Science), and mature stage VI oocytes were isolated for microinjection. Mouse ZIP8 cDNA was cloned into pGHJ [47 (link)] for oocyte expression, which was linearized and used to synthesize capped RNA (cRNA) with the mMessage in-vitro transcription kit (Ambion); the cRNA was then microinjected into oocytes as described [48 (link)]. Oocytes were incubated at 16°C in ND96 complete buffer for 3-4 days until the transport assay. ND96 buffer contains 5 mM Hepes, 1 mM MgCl₂, 1.8 mM CaCl₂, 96 mM NaCl, and 2 mM KCl; a careful balance of this cation/anion ratio was always maintained (i.e. when adding the HCO₃⁻ anion, Cl⁻ anion was removed to maintain equal molality). Oocytes were transferred to well plates containing ND96 buffer without magnesium or calcium and containing freshly-added 3.5 mM HCO₃⁻ for transport assays. HSeO₃⁻ and/or Zn²⁺ was added for 30 min and then washed in ice-cold ND96 buffer; individual oocytes were then digested in nitric acid for Se quantification by ICP-MS.