Percent mink positive for viral DNA (PCR) and AMDV antibodies (ELISA) in three different age groups; juvenile (24 of 56 mink and 14 of 55 mink, respectively); one year old (28 of 47 mink and 26 of 45 mink, respectively) and mink two years or older (15 of 18 mink and 16 of 18 mink, respectively).

<p>Percent mink positive for viral DNA (PCR) and AMDV antibodies (ELISA) in three different age groups; juvenile (24 of 56 mink and 14 of 55 mink, respectively); one year old (28 of 47 mink and 26 of 45 mink, respectively) and mink two years or older (15 of 18 mink and 16 of 18 mink, respectively).</p

Free-ranging mink sampled for AMD antibody and AMDV virus detection (small circles) and virus sequence analysis (crosses).

<p>1a) Percentage positive mink (by PCR)/total mink within each area (areas within ellipses are calculated together). 1b) Crosses are labelled with UNC number and phylogenetic group.</p

Percentage of positive and negative mink for detection of AMDV antibodies (ELISA) or AMDV DNA (PCR).

<p>Percentage of positive and negative mink for detection of AMDV antibodies (ELISA) or AMDV DNA (PCR).</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

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

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

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

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