Novel Picornavirus in Lambs with Severe Encephalomyelitis

Using metagenomic analysis, we identified a novel picornavirus in young preweaned lambs with neurologic signs associated with severe nonsuppurative encephalitis and sensory ganglionitis in 2016 and 2017 in the United Kingdom. In situ hybridization demonstrated intralesional neuronotropism of this virus, which was also detected in archived samples of similarly affected lambs (1998–2014).

instructions, and afterward purified them 2 times with 1.2x volume Agencourt Ampure XP Beads. After performing quality control on a High Sensitivity-Chip (Agilent Technologies, https://www.agilent.com), we quantified all libraries using KAPA Library Quantification Kit (Roche, https://www.kapabiosystems.com/) and sequenced them on an Ion Torrent platform (Ion Torrent PGM or Ion S5 XL, Thermo Fisher Scientific).

Data Analysis
We classified the resulting raw reads using the metagenomic software pipeline RIEMS.
We extracted reads belonging to the family Picornaviridae and assembled them de novo using the 454 Newbler software v2.6 and v3.0 (Roche). Thereafter, we mapped the whole dataset using Newbler v3.0 against the assembled full genome sequence for validation of the sequence, with a mean sequencing depth of 45 and 20 for the two OvPV genome sequences generated in the native spinal cord material. We assembled OvPV genomes of archived samples in an iterative mapping and assembly approach, with a mean sequencing depth of 340 to several thousand. We used EMBOSS Needle Pairwise Alignment v6.3.1 (https://www.ebi.ac.uk) to determine pairwise full genome sequence identities, as well as pairwise amino acid sequence identities for P1, P2, P3, and 3D pol . For phylogenetic analysis of full genomes, we aligned sequences using MAFFT v7.308 (https://mafft.cbrc.jp), followed by phylogenetic analysis with IQ-TREE v1.6.5 including search for the best-fit model with ModelFinder and 100,000 ultrafast bootstraps for statistical support. We constructed the phylogenetic tree using representative complete coding sequences of each species belonging to Sapeloviruses and Enteroviruses, based on the ICTV Master Species List 2016 v1.3 or, if not yet classified, the National Center for Biotechnology Information reference sequences. For phylogenetic analysis of peptide sequences, we aligned sequences using ClustalW (https://www.genome.jp/tools-bin/clustalw) with the BLOSUM scoring matrix, again followed by phylogenetic analysis with IQ-TREE v1.6.5 including search for the best-fit model with ModelFinder and 100,000 ultrafast bootstraps for statistical support.

Real-Time Reverse Transcription-PCR (RT-qPCR)
The designed RT-qPCR amplifies a 110-bp-long target in the 5-UTR region. The sequences of primers and probes are as follows: OvPV-215-F (5- OvPV-249-FAM (5-FAM-TAGACTGATCCCTGCGCTGGCTCAC-BHQ1-3). We performed the RT-qPCR using the QuantiTect Probe RT-PCR Kit (QIAGEN) in a 12.5 L scale with 5 M primers, 1.25 M probe, and 2.5 L sample. The thermal profile was as suggested by the manufacturer, with 50°C for 30 min, followed by 95°C for 15 min, and 45 cycles of 94°C for 15 s and 60°C for 60 s. We applied a primer-probe-system detecting -actin as an internal control (2). In case of the strongly degraded RNA extracted from archived FFPE samples, we performed an additional PCR (semi-nested) amplifying an 80 bp product. For the semi-nested PCR, we used the OvPV forward primer in combination with OvPV-319-R 5-TAGATTCAGTGCACGAAGCC-3.

Viral Culture
We obtained the following cultured cells from the Collection of Cell Lines in Veterinary inoculation, we homogenized sample material (brain and spinal cord) in 1 mL of minimum essential medium (MEM) using the TissueLyser (QIAGEN) at 3000 Hz for 1-2 min. After centrifuging them for 5 min at 10,000 rpm, we inoculated different volumes (100 L, 50 L, and 10 L) of the supernatant onto cells in a 24-well plate format. We incubated the cells at 37°C in a 2.5% CO2 atmosphere and checked for cytopathic effects once a day. After 3-6 days, depending on the viability of the cells, we performed a freeze-thaw-cycle and transferred 100 L of the culture lysate to a new passage of cells. In total, we conducted 3 blind passages and tested for viral replication using the established RT-qPCR.

In Situ Hybridization (ISH)
We performed colorimetric ISH manually on 5 m sections of FFPE tissue on Superfrost Plus slides (Fisher Scientific, https://www.fishersci.com) using the RNAscope 2.5 Red assay kit (Cat #322360, Advanced Cell Diagnostics, Inc., https://acdbio.com). We designed V-Picornavirus-O1, ACD Cat #555011, as 35ZZ paired probe sets targeting region 967-3210 of the viral genome (INSDC accession no. LR216011). We pretreated each 5 m section of FFPE tissue with heat and protease before probe hybridization for 2 h at 40°C. Negative controls used for validation of signal included an unrelated (GC-content matched) probe and an uninfected animal. We performed negative controls on serial sections. We counterstained slides with hematoxylin and mounted them with EcoMount (Biocare Medical, https://biocare.net). We digitized slides using an Olympus VS120 scanner (https://www.olympus-lifescience.com) and a 40× objective with brightfield illumination.