Paxgene blood tubes

Blood was received either in EDTA or PAX gene blood tubes (Qiagen). Samples in EDTA tubes were processed within 2 h of being drawn from the patient. Blood in PAX gene tubes was maintained at room temperature and processed within 10 days of being taken. The blood was centrifuged (VWR, Radnor, PA, USA) at 1600 g for 10 min at 4 °C, after which the plasma was decanted into DNA LoBind tubes (Eppendorf, Stevenage, UK). The tubes were then spun again (Thermo Fisher) at 1880 g for 10 min at 4 °C to remove remaining blood cells.

Bull Terriers with their characteristic egg-shaped head were founded as a dog breed in the 1850s in the United Kingdom. Originally, there were no size standards in this breed and smaller dogs were bred as a variety of the regular Bull Terrier. Eventually, two sub-populations formed and the Miniature Bull Terrier with a maximum height of 35.5 cm was recognized as an independent breed in 1991 by the American Kennel Club (AKC) and in 2011 by the European Fédération Cynologique Internationale (FCI). Therefore, Bull Terriers and Miniature Bull Terriers share a common ancestral gene pool, but represent independent closed populations today.
This study included samples from 385 Miniature Bull Terriers (43 cases / 200 controls / 38 unknown phenotype / 104 excluded). The study also included 75 Bull Terriers (74 unknown phenotype / 1 excluded), 28 American Staffordshire Terriers (19 cases / 9 controls), 90 French Bulldogs (3 with impaired laryngeal function / 8 controls / 79 unknown phenotype), 12 Pugs (1 with impaired laryngeal function / 2 controls / 9 unknown phenotype), 10 Staffordshire Bull Terriers (4 cases / 6 controls) and >1000 dogs from many different breeds, which were assumed to be free of early-onset LP (Table 1, S1 Table, S5 Table, S6 Table). EDTA blood samples were taken for DNA isolation. PAXgene blood tubes (Qiagen) were used to collect blood samples for RNA isolation.

Total RNA from liver biopsy samples was isolated using the RNeasy Mini kit (Qiagen, Redwood City, CA) by following the manufacture’s protocol. Total RNA from whole blood collected in PAXgene blood tubes (Qiagen) was isolated using the PAXgene Blood miRNA kit (Qiagen) with on-column DNase I digestion using RNase-free DNase according to the manufacturer’s instructions. RNA concentrations were measured using a Nano Drop 8000 spectrophotometer (Thermo Scientific, Waltham, MA). Liver and blood derived mRNA samples were then reverse transcribed using random primers and the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Changes in the transcript level of various PRRs, adaptor molecules, transcription factors, and effector molecules and cytokines (Supplementary Table S1) in the liver and blood during the course of acute WHV infection were determined using real-time PCR and woodchuck-specific primers and probes (Supplementary Table S2), as described previously (10 (link)). A fold-change of ≥2.1 from the pre-inoculation baseline was considered a positive result for increased molecule expression. Expression results for the above molecules in woodchuck liver are provided in Supplementary Tables S3 and S4.

Whole blood was collected into PAXgene blood tubes (Qiagen, Redwood City, CA) and stored at -80°C until use. Total RNA was isolated with on-column DNase I digestion using the PAXgene Blood miRNA kit (Qiagen). Total RNA was further isolated from liver tissue using the RNeasy Mini kit (Qiagen) with on-column DNase I digestion using RNase-free DNase (Qiagen). Following reverse transcription of mRNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) using oligo(dT), complementary (c) DNA samples were amplified on a 7500 Real Time PCR System instrument (Applied Biosystems) using TaqMan or SYBER Green Gene Expression Master Mix (Applied Biosystems). Target genes investigated (S1 Table) included markers for: (i) type I IFNs and ISGs; (ii) NK-cells; (iii) APCs; (iv) Th-cells; (v) CTLs; and (vi) T_regs. The woodchuck-specific primers and probes are presented in S2 Table. 18S rRNA expression was used to normalize target gene expression. Transcript levels of target genes were calculated as a fold-change relative to the pre-inoculation (baseline) level in blood and liver at week -2 (or at week 0 prior to inoculation in blood of woodchuck F7394 and woodchucks M7392 and M7249 for selected genes) using the formula 2^-ΔΔCt. A fold-change of ≥2.1 from this baseline was considered a positive result for the presence of increased target gene expression.