Sephadex g 25 dna grade

Nucleotide exchange was performed according to previously established protocols60 (link)62 (link). Briefly, for nucleotide removal, Mg²⁺ was removed by the addition of a 5-fold excess of EDTA and reacted for 1 h at room temperature. The protein solution was desalted using a PD-10 desalting column Sephadex G-25 DNA Grade (GE Healthcare) with elution buffer consisting of 20 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP. After removal of Mg²⁺, the protein was diluted to 80–100 μM before the addition of ZnCl₂ (500 μM) and (NH₄)₂SO₄ (200 mM). After the addition of alkaline phosphatase (5 U mg⁻¹Rab protein), the mixture was incubated for 16 h at 4 °C. For nucleotide exchange, the mixture contained a 5-fold excess of GppNHp during alkaline phosphatase incubation. Afterwards, the mixture was desalted using a PD-10 desalting column Sephadex G-25 DNA Grade (GE Healthcare) with elution buffer consisting of 25 mM HEPES (pH 7.5) 150 mM NaCl, 1 mM TCEP, 1 mM MgCl₂ and 1 μM GppNHp.

Viral strains and isolates of TBEV can be classified into three subtypes: the European, the Siberian and the Far Eastern [31] . In this study we used the European TBEV strain Ljubljana 1 [32] (link). TBEV was grown 7 days on Vero E6 cells. Supernatant was collected and centrifuged twice at 4°C (10 min at 3200 × g and 5 min at 20800 × g in Eppendorf 5804R centrifuge). Pellet was resuspended in astrocyte growth medium and labelled with different concentrations of fluorescent lipophilic Vybrant® DiD labelling solution (DiD, Molecular Probes, Invitrogen) in µM: 20, 50, 100 and 200. Labelling was performed for 2 h at 37°C (Eppendorf Thermomixer Compact, 500 rpm). Afterwards, the unbound dye was removed by buffer exchange into Hepes 145 buffer (50 mM Hepes, 145 mM NaCl, pH 7.4; [33] (link) by using illustra NAP-5 columns with sephadex G-25 DNA grade (GE Healthcare)). Labelled virus (conc. 10¹⁰ copies per ml) was diluted in astrocyte growth medium, aliquoted and stored at −80°C. Astrocytes were infected with 10³–10⁷ TBEV.
Cytotoxicity of TBEV in astrocytes and Vero E6 cells was tested at various time intervals: 4 h, 18 h, 48 h, 3 days, 6 days, 10 days and 14 days p.i. with Countess™ Automated Cell Counter (Invitrogen) according to manufacturer’s instructions.