Avl buffer

Within the European Network for Diagnostics of Imported Viral Diseases (ENIVD) we gathered a wide range of emerging viruses, as inactivated culture supernatants or as purified viral DNA or RNA (Table 1). Viruses were inactivated by heat and/or gamma-irradiation, or by suspension in an RNA-extraction reagent (TRIzol, Life Technologies; TriFast, Peqlab; AVL buffer, Qiagen) [27] (link). The majority of viruses were grown in Vero E6 cell-cultures (kidney epithelial cell line derived from African green gonkey) (ATCC CRL-1586), but poliovirus (PV) was grown in L20B cells (a murine recombinant cell line) [28] (link). We also used six control samples from the QCMD EQA programme for 2010 and 2013 (WNV10-01, WNV10-07, WNV13-01, WNV13-10, WNV13-11 and DENV13-01). The WNV13-01 sample contained West Nile virus (WNV) at a concentration of 1.0×10⁷ copies/ml and the DENV13-01 sample contained Dengue virus (DENV) type 1 at a concentration of 1.0×10⁶ copies/ml. The WNV10-01 and WNV13-10 samples contained a mixture of flaviviruses (DENV type 1, 2 and 4, and Japanese encephalitis virus (JEV)). The WNV10-07 and WNV13-11 samples contained a mixture of DENV type 3, tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV), each at a concentration of 1.0×10⁶ copies/ml.

BMDCs were utilized for infection studies between days 6 and 10 of culture. Cultures from each bat were infected with EBOV-Makona at a MOI of 3 for 1 h, thereafter BMDCs were washed with fresh media and were seeded into 48-well plates at a concentration of 2.5 × 10⁵ cells/well. Mock uninfected control samples were also set up as a negative control. Cells were harvested at 1, 24, 48, and 72 h post-infection (hpi) and resuspended in RLT Buffer (Qiagen). Supernatants from cultures were also collected into AVL Buffer (Qiagen). RNA was extracted according to the manufacturer's instructions using RNeasy, for cell pellets, and Qiamp Kits, for cell supernatants (Qiagen). EBOV transcripts were quantified using a quantitative PCR (qPCR) assay targeting EBOV-VP30 with use of AgPath-ID One Step RT-PCR Kit (4387391, ThermoFisher). Reactions of 25 μl were formulated by addition of 3 μl of RNA sample to a master mix containing: 400 nm of forward and reverse primer, 200 nm of TaqMan Probe, 1 μl enhancer, 1X buffer, and 1X RT-PCR enzyme mix. The thermal profile included incubation at 15 min 45°C, 10 min 95°C, and 45 cycles of 15 s 95°C and 45 s 60°C. The sample CTs were compared to a standard curve, which was produced using EBOV in vitro transcripts of known concentrations that ranged from 10¹ to 10⁷ copies. This standard curve was used to quantify copy numbers within each sample.

Virus titer was measured by plaque assay on Vero E6 cells. Cells were plated in 6-well plates and grown to confluency. Virus was titrated from 10^-1 to 10^-6 in duplicate. Plaques were counted using neutral red stain; limit of detection was 25 PFU/ml.
RNA was isolated from WB using AVL Buffer and Viral RNA mini-kit (Qiagen, Valencia, CA). As previously described, primers/probe targeting the VP30 gene of ZEBOV-Makona were used for RT-qPCR^{39 (link)}. EBOV RNA was detected using the CFX96 detection system (BioRad Laboratories) using One-Step Probe qRT-PCR Kits (Qiagen) with the following cycle conditions: 50 °C for 10 minutes, 95 °C for 10 seconds, and 40 cycles of 95 °C for 10 seconds followed by 59 °C for 30 seconds. Threshold cycle values representing ZEBOV genome equivalents (GEq) were analyzed with CFX Manager Software, and data are shown as means ± SD of technical replicates. To create the GEq standard, RNA from ZEBOV stocks was extracted and the number of ZEBOV genomes calculated using Avogadro’s number and the molecular weight of the ZEBOV genome.

Patient samples were taken by clinical staff using appropriate personal protective equipment. Inactivation of samples occurred in their country of origin. Samples were then shipped to the Broad Institute or tested at the local center (Nigeria, Sierra Leone). Samples were inactivated in AVL buffer (Qiagen) or TRIzol (Life Technologies) following standard operating procedures. Samples were stored in liquid nitrogen or at −20 °C. RNA was isolated at the clinical site using the QIAamp Viral RNA Minikit (Qiagen) according to the manufacturer’s protocol. Poly(rA) and host rRNA were depleted using RNase H selective depletion, using 616 ng oligo (dT) (40 nt long) and/or 1000 ng DNA probes complementary to human rRNA. Samples then underwent RNase-free DNase using a kit (Qiagen) according to the manufacturer’s protocol. AMPure RNA clean beads (Beckman Coulter Genomics) were used to clean and concentrate samples. cDNA synthesis was performed using the Superscript III kit (Thermo Fischer) plus dNTPs, random primers, and SUPERASE-IN for first-strand synthesis. Then, the 10× second-strand buffer kit (New England Biolabs), plus Escherichia coli DNA ligase, E. coli DNA polymerase, Rnase H, and dNTPs were used for second-strand synthesis. Samples then underwent a final AMpure DNA beads clean-up^{30 (link)}.