RT-qPCR of gills and gill swabs, and immunostaining on frozen gills from farm IV.

<p>RT-qPCR of gills and gill swabs, and immunostaining on frozen gills from farm IV.</p

Summary of RT-qPCR on gills.

<p>Summary of RT-qPCR on gills.</p

Immune marker RT-qPCR.

<p>Immune marker RT-qPCR.</p

Mx and γIP response to HPR0 infection at farm III.

<p>Across the three months, little variation in Mx expression (A) was seen. However, γIP was over 8-fold lower in the fish sampled in August compared to those sampled in September and October (B), suggesting the presence of a γIFN-induced response. In August 2009, all fish (n = 41) tested negative (neg.) for HPR0. In September 2009, 72% (58 out of 80) fish tested positive (pos.) (mean Ct 31.5, range 20.8–36.7). In October 2009, 5% (4 out of 79) fish tested positive (mean Ct 35.6, range 34.2–36.5). Ratios standardised relative to elongation factor 1α (ELF).</p

RT-qPCR on gills and fin swabs.

<p>RT-qPCR on gills and fin swabs.</p

Immunostaining of tissue sections.

<p>Immunostaining of formalin-fixed paraffin-embedded (A-C), and frozen (D-I) gill sections with massive and sparse infection ISAV HPR0 infection, demonstrating epithelial infection. Primary antibodies used were a rabbit antibody to ISAV NP (A-D, G), and a monoclonal antibody to ISAV HE (E-F, H-I). For detection of bound antibodies, alkaline phosphatase-conjugated antibody to rabbit Ig with Fast Red substrate (A-D; red colour), horseradish peroxidase-conjugated antibody to mouse Ig with DAB substrate (E-F; brown colour), and Alexa fluor 488-conjugated antibody to rabbit Ig (G) or mouse Ig (H-I), were used.</p

Western blot analysis of Nor98 isolates.

<p><b>A:</b> Representative western blots of PrP^res from Nor98 isolates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone.0066405.s004" target="_blank">Table S1</a>). Lanes 1–2, Norwegian sheep Nor98 (AFRQ/AFRQ); lanes 3–5, Italian goat Nor98 (ALRQ/ALHQ); lanes 6–7, Italian sheep Nor98 (AFRQ/AFRQ and ALRR/ALHQ, respectively); lane 8, Italian goat scrapie (ALRQ/ALRQ); lane 9, Italian sheep scrapie (ALRQ/AFRQ). All samples were digested at 200 µg/ml PK. Membranes were probed with P4 (top) and SAF84 (bottom). <b>B:</b> Effect of PK digestion on Nor98 PrP^Sc. WB analysis of healthy, Nor98 (Italian sheep, AFRQ/AFRQ) and scrapie (Italian sheep, ALRQ/ALRQ) sheep brain homogenate treated with 10, 50 and 200 µg/ml PK. Replica blots were probed with P4 (top) and SAF84 (bottom). MW marker was loaded as indicated (M). <b>C:</b> Epitope mapping of Nor98 (Norwegian sheep AFRQ/AFRQ) PrP^res produced after 200 µg/ml PK treatment. Membranes were probed with different mAbs, as indicated on the top of each blot. MW markers were loaded into the last lane of each blot. <b>A-C:</b> MW marker was loaded as indicated (M).</p

Epitope mapping analysis.

<p><b>A:</b> Representative blots of PrP^res from Nor98, GSS and VPSPr. Brain homogenates were treated with 100 µg/ml PK. Replica blots were probed with SAF32, 12B2, 9A2, 8G8, L42 mAbs. Case numbers are shown on the bottom of blots according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone-0066405-t001" target="_blank">Table 1</a>. <b>B:</b> Samples (ID #11, #1, #7, #13) were re-analysed at a lower PK concentration (50 µg/ml) to investigate partially cleaved epitopes. Replica blots were probed with 12B2, 9A2 and L42 mAbs. In these blots Nor98 is an internal control as it is recognised equally well by the 3 mAbs (see Fig. 4A). By comparison with Nor98, it is shown than i) in GSS A117V the L42 epitope is cleaved, the 12B2 is partially preserved and the 9A2 was fully preserved; ii) in VPSPr the 12B2 epitope is cleaved and the 9A2 partially preserved; iii) in GSS F198S the L42 epitope is partially preserved. <b>C:</b> Samples (ID #11, #1, #7, #13) were treated with 100 µg/ml PK and probed with 6D11, 8G8 and 12F10 mAbs. 6D11 and 8G8 bind to epitopes within the “core” of PrP^res fragments and reveal the actual quantity of PrP^res fragments in the samples analysed. By comparison, 12F10, which recognizes an epitope at the C-terminus of PrP^res fragments, only detected VPSPr, suggesting that its epitope is cleaved in all other samples.</p

Conformational stability of insoluble PrP^Sc.

<p><b>A:</b> Western blot analysis of VPSPr 129MV (#7) and 129VV (#3) and GSS P102L (#15 cerebellum) cases, showing separation of insoluble PrP^Sc by a solubility assay. Samples were centrifuged at 20000 g for 1 h in presence of 1% sarkosyl. Supernatant (S) and pellet (P) fractions were analysed with (+) or without (-) PK treatment (50 µg/ml), along with aliquots of samples before centrifugation (Tot). Note that PrP^res segregated into the insoluble fraction (compare lanes S+ and P+ in each blot). In each lane 0,7 mg TE were loaded. Membranes were probed with L42 mAb. <b>B:</b> Representative western blots of CSSA experiments in Nor98 (#1), VPSPr (#9), GSS F198S (#11), GSS A117V (#14) and GSS P102L (#16) cases. Lanes were loaded with insoluble PrP obtained as shown in Fig. 6A, with or without previous denaturation with increasing concentrations of GdnHCl, as shown on the top of each lane. Membranes were probed with L42. Molecular size markers are shown in kilodaltons on the right of each blot. <b>C:</b> Dose-response curves obtained by plotting the fraction of PrP^Sc remaining in the pellet as a function of GdnHCl concentration and best-fitted and using a four parameter logistic equation. Individual [GdnHCl]_1/2 values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066405#pone-0066405-t001" target="_blank">Table 1</a>.</p

Human and ovine cases used for comparative analyses.

a<p>Ovine polymorphism: amino acids at codon 136, 141, 154 and 171 of ovine PrP gene; human mutation at codon 102, 117 or 198 and polymorphism at codon 129 of human PrP gene.</p>b<p>Cer: cerebellum; Fr cx: frontal cortex.</p>c<p>Mean ± SEM.</p