TECPR2 domain structure and p.R1337W mutation associated with NAD in Spanish water dogs.

<p>(<b>A</b>) TECPR2 possesses three N-terminal WD (tryptophan-aspartic-acid dipeptide) repeats (red), a polylysine tract (green), and six C-terminal tectonin beta-propeller repeat (TECPR) domains (blue). (<b>B</b>) The arginine at position 1337 that is substituted by a tryptophan residue (red) is located in the sixth TECPR domain. Note that the mutation affects a conserved amino acid residue in all known TECPR2 orthologs. Highly conserved residues are marked in green.</p

Association of the <i>TECPR2</i> missense mutation with the NAD phenotype in Spanish water dogs.

<p>Association of the <i>TECPR2</i> missense mutation with the NAD phenotype in Spanish water dogs.</p

Histology of NAD in Spanish water dogs.

<p>(<b>A</b>, <b>B</b>) Histology of the brain stem (cuneate nucleus) stained with hematoxylin and eosin revealed numerous large granular axonal swellings (spheroids; arrow). Note the hypereosinophilic central target-like core structure of distinct spheroids (<b>A</b>). Single neurons displayed an accumulation of a finely or coarse granular, intensely eosinophilic material associated with the soma (arrow) displacing the Nissl substance. A high proportion of neurons adjacent to affected areas displayed a normal morphology with equally distributed Nissl substance (arrowhead, <b>B</b>).</p

Pedigree of the collected Spanish water dogs with NAD.

<p>Note the inbreeding loops and the likely common ancestors appearing 8 to 9 generations ago. Only for the numbered animals DNA was available. Affected animals are shown with black symbols; genotyped carriers of the causative mutation are indicated with half-filled symbols; females are shown as circles and males as squares.</p

Distribution of cervical and thoracic spinal cord spheroids in Spanish water dogs with NAD compared to mostly affected areas in human hereditary spastic paraparesis (HSP).

<p>In NAD affected Spanish water dogs (left), spheroids and neuronal loss were restricted to the sensory, ascending pathways localized to the grey matter of the spinal cord dorsal horn and single large spheroids were detected within the cuneate and gracile fasciculus. This might explain the clinical signs as gait disturbances, proprioceptive deficits, decreased spinal reflexes and urinary incontinence. In other human forms of NAD, HSP and <i>Pla2g6</i> knock-out mice (right), spinal cord spheroid formation is accentuated in the sensory pathways including the gracile fasciculus as well as the corticospinal tracts. Furthermore, also the descending motor pathways including the ventral horns as well as the descending pyramidal tracts are affected. Note that spinal cord histology of TECPR2 associated HSP in humans is unknown. Ascending, sensory pathways (red; transmission of sensory signals from the periphery (red arrow) via dorsal horn (DH) towards the brain): Dorsal funiculus composing of gracile fasciculus (GF) and cuneate fasciculus (CF); Spinocerebellar tracts with: dorsal spinocerebellar tract (DST) and ventral spinocerebellar tract (VST); Descending, motor pathways (blue; signal transmission via the ventral horn (VH) neurons towards the muscles; blue arrow): Pyramidal tracts with lateral corticospinal tract (LCT) and ventral corticospinal tract (VCT).</p

Reverse genetics protocols for the rescue of SBV.

<p>A. Schematic representation of the antigenome plasmids used to rescue SBV. B and C. Schematic representation of the strategies used for the rescue of SBV in BSR-T7/5 and 293T cells as described in the text. The two methods are very similar with the exception that BSR-T7/5 stably express the T7 RNA polymerase and therefore these cells are transfected only with the SBV antigenome plasmids. On the other hand 293T cells are transfected with the antigenome plasmids and an expression plasmid for the T7 RNA polymerase.</p

Virulence of SBV mutants in suckling NIH-Swiss mice.

<p>A. 3 and 7-day old mice were inoculated intracerebrally with either 100 or 400 PFU with the indicated viruses or cell culture media as a control. Survival plots show that SBVΔNSs possesses an attenuated phenotype while SBVp32 is more virulent than sSBV. B. Immunohistochemistry of brain sections derived from NIH-Swiss mice inoculated with sSBV or SBVp32 and killed at various time points post-infection as indicated in the figure. Immunohistochemistry was performed using an SBV N antiserum as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003133#s4" target="_blank">Materials and Methods</a>. At the early time points SBV antigens are detected only in sections derived from SBVp32 infected mice (Bar = 500 µm).</p

<i>In vitro</i> phenotypic characterization of SBV and sSBV.

<p>A. Comparison of plaques produced by wild type SBV and sSBV rescued using BSR-T7/5 and 293T cells. B. Growth kinetics of SBV and sSBV. CPT-Tert and BFAE cells were infected at a MOI of 0.05 for 90 min. Supernatants were collected at the indicated times post-infection and virus titer was measured using standard plaque assays in CPT-Tert cells. C. The presence of SBV in CPT-Tert and BFAE cells infected with wild type and rescued virus was confirmed by western blotting using antibodies against the SBV N (nucleocapsid) protein.</p

SBVSΔNSs induces the production of IFN.

<p>A. IFN protection assay. 2fGTH cells were infected with the indicated viruses and supernatants were collected 24 h post infection. Supernatants were UV treated to remove infectious virus and fed to CPT-Tert cells after serial dilution. CPT-Tert cells were later infected with EMCV and the presence of CPE monitored and compared to cells supplemented with known amounts of universal IFN. B. The induction of IFN-β mRNA was investigated by RT-PCR from RNA extracted from 2fGTH cells infected with virus as indicated or transfected with Poly I∶C as a positive control. To control for the presence of residual genomic DNA all the samples were amplified after reverse transcription performed without reverse transcriptase (top panel, indicated as+/−RT). The quality of the extracted RNA was verified by the amplification of the 45S ribosomal RNA (middle panel). The presence of virus was confirmed by the amplification of part of the SBV S segment (bottom panel). C. IFN protection assay performed in primary ovine trophoblast cell (oTr-1) and primary ovine endothelial cells as described in A. D. Survival plots of 7 day old IFNAR^(−/−) mice inoculated intracerebrally with sSBV, SBVSΔNSs or cell culture media as a control. Data indicate that SBVΔNSs is as virulent as sSBV in these mice that lack an intact IFN system.</p

Sequences of 3′ and 5′ UTRs of SBV obtained by RACE PCR.

<p>A schematic representation of the genomes of the 3 viral segments is shown. Sequences on the top of each segment (sSBV) indicate the sequence of the UTRs of the plasmids used for reverse genetics. The middle sequences indicate the UTRs sequences inferred by RACE PCR (RACE1, RACE 2 etc). The bottom sequences indicate SBV UTRs reported in GenBank (HE649914, HE649913 and HE649912). Positions highlighted in red correspond to nucleotides inferred for the construction of sSBV sequences based on the AKAV sequence. Scores represent positions in the genome segments for which there was no sequence available in the submitted GenBank sequences. Note that 54 nucleotides previously reported in the 5′ UTR of the M segment that appeared to be a sequence artifact (i.e. not part of the SBV genome) were not, as expected, detected by RACE and are not shown in the figure. Numbers shown in this figure take into account the corrected 3′ and 5′ UTRs.</p