SDS-PAGE of purified proteins expressed from stable transfected mink fetal cells.

<p>The proteins were purified by nickel affinity chromatography as described in Methods. Ten microliters of protein were loaded in the gel, separated by electrophoresis and stained with Coomassie blue.</p

Southern blot for detection of gene integration in stable transfected mink fetal cells.

<p>DNA was isolated from cell extracts and 10 µg of each DNA were run on agarose electrophoresis, transferred to Hybond N membrane and hybridized with an astrovirus specific probe. Transiently transfected and non-transfected cells were included as controls. The signals were revealed by radiography scanning in a phosphorimager. Hybridization signals were detected in extracts of cells stable transfected with constructs CPΔC of DK5790 and DK7627, CPΔN of DK5790 and DK7627 (left blot) and CP of DK5790 and DK7627 (right blot). The signals were stronger in stable than in transiently transfected cells, as exemplified with transient transfection (tt) of CPΔN of DK7627.</p

Primer pairs used for amplification of the full-length (CP), the N-terminus truncated (CPΔN) and the C-terminus truncated (CPΔC) fragments of ORF2 of mink astrovirus strains DK5790 and DK7627.

<p>F indicates the forward and R the reverse primer. The recognition site for <i>Eam</i>1104I is in italics. The AAG codon for continuing translation of the protein as fusion to the c-Myc tag is underlined (in SMC2R, SMC4R and SMC4RN). NA – not applicable.</p

Antibodies to the mink astrovirus capsid proteins determined by ELISA.

<p>Adult mink were injected with proteins CP (A), CPΔN (B) and CPΔC (C) combined with Freund’s adjuvant, with two-week interval. Control mink received PBS plus Freund’s adjuvant injection on each occasion. The results of an indirect ELISA to determine antibodies in sera of the mink are presented as mean OD values. Asterisk show statistically significant difference between the levels of antibody at two time points (p<0.05).</p

Virus shedding in fecal samples evaluated by real-time PCR.

<p>Samples collected from the litters at different days post-challenge with mink astrovirus were analyzed by real-time PCR and graded as high to moderate or low to negative for content of astrovirus as per copy number criteria described in Methods. The percentage of high/moderate virus shedders was higher in litters of non-immunized and CPΔC immunized mink.</p

In situ-PLA and IFA in stable transfected cells.

<p>(A) The full-length (CP), N-terminally truncated (CPΔN) and C-terminally truncated (CPΔC) ORF2 constructs were transfected into mink fetal (MF) cells, subjected to G418 selection and clonal cells were thereafter tested for expression of the corresponding proteins by in-situ PLA as described in Methods. Sera to homologous (left panel) and heterologous (right panel) astrovirus were used, and thereafter cells were stained in an in-situ PLA. The controls were CP transfected and mock-transfected cells incubated with pre-immune sera or homologous/heterologous serum, respectively and stained as mentioned above. Signals of protein expression were present in the cytoplasm demonstrating stable transfection and constitutive expression of the indicated forms of the capsid protein in MF cells. (B) Quantitative expression of all three constructs for homologous (upper panel), heterologous (middle panel) antibodies and difference between homo- and heterologus (lower panel) is shown, as determined using Duolink ImageTool. (C) BHK21 cells transfected as before with DK7627 constructs were incubated with homologous serum and stained for IFA. Signals of protein expression were present in the cytoplasm demonstrating stable transfection and constitutive expression of the indicated forms of the capsid protein in BHK21 cells.</p

(A) Strategy for amplification of the full-length and truncated ORF2 of mink astrovirus.

<p>The full-length (CP), and truncated CPΔN and CPΔC fragments are represented. (B) Amplicons of the full-length (CP) and N (CPΔN), and C- terminally truncated (CPΔC) ORF2 of mink astrovirus generated with primers as described in Methods.</p

Expression of forms of the capsid protein of astrovirus in transient and in stable transfected mink fetal (MF) cells.

<p>The primary antibody was polyclonal serum for genotype 1 astrovirus, i.e. homologous to strain DK5790. Western blotting was performed as described in Methods. A) Transient expression of the full-length CP of astroviruses DK5790 and DK7627. (B) Transient expression of CPΔN and CPΔC proteins of DK5790. (C) Transient expression of CPΔN and CPΔC proteins of DK7627. (D, F, H) Expression of protein from MF cells stable transfected with CP, CPΔN, and CPΔC, respectively, of astrovirus DK5790. (E, G, I) Expression of protein from BHK21 cells stable transfected with CP, CPΔN, and CPΔC, respectively, of astrovirus DK7627.</p

Groups of Danish brown hares individuals according to the presence of EBHSV and the variation in exon 2 DQA gene.

<p>Numbers (N) and percentages of Danish brown hares assessed for the four groups defined in this study: EBHSV negative (EBHSV⁻), EBHSV positive (EBHSV⁺), susceptible (EBHSV⁺ Dead), resistant (EBHSV⁺ not Dead).</p

Phylogenetic tree of the EBHSV isolates.

<p>Phylogenetic tree resulting from the Bayesian analysis, clustering the seven Danish isolates identified in this study combined with sequences available from the EMBL database. The topology of the clusters was similar for the NJ tree. Numbers on branches at the internodes of the clusters correspond to posterior probabilities from the Bayesian analysis. At the end of the branches the designation and origin of the EBHS viruses studied. Probabilities below 50% are omitted.</p