A novel cause of chronic viral meningoencephalitis: Cache Valley virus

Wilson, Michael R.; Suan, Dan; Duggins, Andrew; Schubert, Ryan D.; Khan, Lillian M.; Sample, Hannah A.; Zorn, Kelsey C.; Rodrigues Hoffman, Aline; Blick, Anna; Shingde, Meena; DeRisi, Joseph L.

A 250 ll sample of CSF and a <50mg piece of surplus brain biopsy tissue were collected in RNase/DNase-free Lobind tubes (Sigma-Aldrich, St Louis, MO) and snap frozen in liquid nitrogen. The samples were submitted for unbiased mNGS under a research protocol (#13-12236) for the identification of potential pathogens approved by the institutional review board of the University of California, San Francisco (UCSF). A common issue in metagenomic sequencing is that essentially all reagents used during the library preparation process contain some extraneous nucleic acid, including material commonly present in laboratory reagents. 9,10 The process of acquiring a human sample can also introduce unwanted nucleic acid from skin or environmental contamination. To control for irrelevant sequences, an unrelated, uninfected human CSF sample ("control") was prepared in parallel and sequenced on the same run in addition to a water control. Samples were processed for FIGURE 1: Neuroimaging from acute presentation. (A) Coronal T2-weighted/fluid-attenuated inversion recovery brain magnetic resonance imaging (MRI) demonstrating T2 hyperintensity in the right hippocampus and amygdala (arrow). (B) Axial diffusionweighted imaging brain MRI demonstrating restricted diffusion in the right hippocampus and amygdala (arrow). (C, D) Sagittal (C) and coronal (D) T1-weighted postcontrast brain MRI demonstrating contrast enhancement 2 weeks after the acute presentation (arrows). mNGS analysis as previously described. 11 complementary DNA (cDNA). The cDNA was converted to Illumina (San Diego, CA) libraries using the NEBNext Ultra II DNA library preparation kit (E7645) according to the manufacturer's recommendation and amplified with 11 PCR cycles. The libraries were subjected to an additional step to selectively deplete library amplicons whose sequences corresponded to the human mitochondrial genome. This was accomplished via Depletion of Abundant Sequences by Hybridization (DASH), a novel molecular technique using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated nuclease Cas9 in vitro. 11, 13 The pooled library was size-selected using Ampure beads, and concentration was determined using a Kapa Universal quantitative PCR kit (Kapa Biosystems, Woburn, MA). Samples were sequenced on an Illumina HiSeq 4000 instrument using 140/140 base pair paired-end sequencing.