Benzonase

All RNA extractions from fecal suspensions for AstV-screening by RT-PCR were done with the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) and RNA isolation from brain tissue was performed with TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturers’ protocols. To prepare samples for NGS, 500 μL of fecal suspensions were centrifuged at 16,000×g for 3 min. The supernatants were then centrifuged through Vivaclear MINI Clarifying filters with 0.8 μm PES (Sartorius, Göttingen, Germany) at 2,000×g for 5 min. Next, 280 μL of the filtrates were treated with 2 μL Benzonase (1U/μL) (Merck, Kenilworth, NJ, USA) for 2 h at 37 °C. The Benzonase was inactivated by adding ethylenediaminetetraacetic acid (EDTA) to a final concentration of 5 mM. Finally, RNA extraction was performed with the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) with the modification that the carrier RNA was omitted. RNA extracts were stored at –80 °C until further analysis.

As summarized in Figure 1, VMRP was treated prior extraction with benzonase (Novagen, Madison, WI, USA) (5 U/µL) and baseline-ZERO (EUROMEDEX, Souffelweyersheim, France (1 U/µL) nucleases for 2 h at 37 °C, in order to digest unprotected nucleic acids. Enzymes were inactivated with a final concentration of 3 mM EDTA and heating for 10 min at 65 °C. A plasma sample was spiked with VMRP diluted at a ratio 1:10, then centrifuged at low speed, filtrated through Spin-X^® centrifuge tube filter 0.45 µM (cellulose acetate membrane) (Costar, WA, USA) then treated by nucleases as described above.
Other plasma samples derived from the same pool and spiked with WRVS at a final concentration of 10⁴, 10³ or 10² viral gc/mLwere treated prior to extraction with benzonase (25 U/µL) for 2 h at 37 °C.
Total nucleic acids were extracted by the QIAamp^® Cador^® Pathogen kit (Qiagen, Courtaboeuf, France) with the substitution of carrier RNA by Linear Acrylamide (Ambion, Waltham, MA, USA) (10 µg per extraction). To get the RNA fraction, the extract was digested with TURBO DNase (Ambion) (10 U for 40 µL of nucleic acids) and purified with a Qiagen RNeasy Mini Kit.
All VMRP sample extracts were below the detection limit of the Qubit quantification system (LOQ = 200 pg for the dsDNA HS Assay Kit, Invitrogen, Waltham, MA, USA).

DNA was extracted from all samples using a QIAamp^® UCP Pathogen DNA Kit (Qiagen), following the manufacturer’s instructions. Human DNA was removed using Benzonase (QIAGEN, Hilden, Germany Sigma, St. Louis, Missouri) and Tween20 (Sigma) (Amar et al., 2021 (link)). Libraries were constructed using a Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, United States) (Miller et al., 2019 (link)). The library was quality-assessed by the Qubit dsDNA HS Assay kit, followed by High Sensitivity DNA kit (Agilent) on an Agilent 2100 Bioanalyzer. Library pools were then loaded onto an Illumina Nextseq CN500 sequencer for 75 cycles of single-end sequencing to generate approximately 20 million reads for each library. For negative controls, we also prepared Peripheral blood mononuclear cell (PBMC) samples with 105 cells/ml from healthy donors in parallel with each batch, using the same protocol, and sterile deionized water was extracted alongside the specimens to serve as non-template controls (NTCs) (Miller et al., 2019 (link)).

All samples were extracted with QIAamp^® UCP Pathogen DNA Kit (Qiagen). Tween 20 and Benzonase (Qiagen) are used to remove DNA from human samples according to the manufacturer’s instructions (Amar et al., 2021 (link)). A QIAamp^® Viral RNA Kit was used to extract RNA, and a Ribo-Zero rRNA Removal Kit (Illumina) was used to remove ribosomal RNA. cDNA was synthesized with reverse transcriptase and dNTP (Thermo Fisher). The construction of the selected DNA and cDNA library was done using Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) (Miller et al., 2019 (link)). Quality control for the library was performed using a Qubit dsDNA HS Assay Kit and a High Sensitivity DNA kit (Agilent) on an Agilent Bioanalyzer 2100. Then, the library pools were loaded onto an Illumina NextSeq CN500 sequencer for 75 cycles of single-end sequencing of 20 million reads for each library. Negative control (NC) is a peripheral blood mononuclear cell sample of 105 cells/mL prepared alongside each batch with the same protocol (Amar et al., 2021 (link)), and sterile deionized water was used as a nontemplate control (NTC) alongside the specimens (Li et al., 2018 (link)).