Anti-HEV antibody titres in positive canine serum samples.

<p>Positive canine serum samples were prepared in dilutions of 1:100, 1:200, 1:400, 1:800; 1:1600 and 1:3200 and used in an ELISA assay. The corrected OD450 was obtained by subtracting the background signal from the VLP coated well OD450 value. The positive threshold was determined by calculating the mean OD450 of buffer coated wells with the highest serum dilution, plus 3 standard deviations.</p

Viruses detected in dogs with and without gastroenteritis.

<p>qPCR was performed on nucleic acid extracted from stools of 228 dogs. Of these dogs, 47 were admitted to veterinary clinics with primary gastroenteritis, 64 dogs were admitted to veterinary clinics with other clinical disease, and 117 dogs were healthy animals. Primer probes specific for canine norovirus (CNV), canine parvovirus (CPV) and canine enteric coronavirus (CECoV) were used to assay for the enteric viruses.</p

Evaluation of cross-reactivity between antibodies against human and canine noroviruses, and between different CNV strains.

<p>Canine serum was pre-incubated with serial diutions of either pooled human norovirus VLPs from genogroups I and II (GI/GII) or pooled CNV VLPs. The ability to detect pooled CNV was analysed using ELISA (A). Canine serum was then pre-incubated with serial dilutions of each of the CNV strains VLPs separately. ELISAs were again used to analyse the ability to detect CNV strain 170 (B) and strain HK (C). No C33 seropositive sample of adequate titre was available for the blocking assay.</p

Western blot analysis of serum sample reactivity with HEV, vesivirus 2117 and human norovirus G1.1 VLPs.

<p>Three types of VLP were separated by SDS-PAGE. One gel was stained with Coomassie Blue to identify VLP protein at the expected molecular weight. Additional gels were used for western blotting with canine serum samples positive by ELISA for HEV (samples A and B). A pig serum sample (kind gift from S. Emerson) and a human serum sample known to be positive for anti-HEV antibody were used as a positive control for the HEV VLPs. Canine sample C, previously confirmed positive for anti-vesivirus antibody by ELISA, was used as a positive control for the vesivirus VLPs. Canine sample D was used as a negative control for all VLPs.</p

SDS-PAGE analysis of purified calicivirus VLPs.

<p>VLPs from three CNV strains and an unrelated calicivirus, vesivirus 2117, were analysed by SDS-PAGE. The molecular weights of VP1 of CNV isolates C33 and 170 are larger than that of the third isolate HK. This is attributed to the length of C33 and 170 VP1 sequences being 52 and 50 amino acids respectively longer than HK VP1.</p

Serological titres to CNV.

<p>Ten dogs seropositive to CNV from both the 1999–2001 and the 2012–2013 cohorts were randomly selected to determine anti-CNV antibody titre. Corrected OD450 values are plotted, calculated by subtracting the OD450 value of buffer-only coated wells. The positive threshold was calculated from the mean plus three times the standard deviation of the OD450 reading of buffer-only.</p

Relationship of CNV antibody status to age.

<p>Age was known for 93 dogs in the 2012–2013 cohort. (A) Box plot of age distribution of dogs relative to seroconversion to CNV. The box represents the interquartile range, with the band inside the box representing the median age. The whiskers are the minimum and maximum of all data. (B) Histogram representing the percentage of dogs that have seroconverted to CNV in each age group. Numbers inside bars indicate the quantity of samples associated with each data point.</p

Seropositive samples by ELISA to three different strains of CNV.

<p>Canine serum samples that were seropositive to pooled CNV VLPs were screened against individual CNV VLP strains; 170, C33 and HK. (A) Venn diagrams represent seroprevalence of each CNV strain in the 1999–2001 and the 2012–2013 cohorts. The number of dogs seropositive to one strain alone or combinations of strains are represented as percentages. (B) The relative OD450 value to each CNV strains of each dog are represented by heatmaps. Each column represents a single dog. Positive threshold value was established from the mean OD450 of coating buffer alone plus three standard deviations. Relative increase in OD450 values above the positive threshold were calculated to enable fair comparison between experiments. A relative increase of <1 indicates a seronegative sample, represented by a white box. The degree of relative increase for samples is represented by increasing darkness of the corresponding box.</p

Overview of CNV strains used to design primer-probes and produce virus-like particles (VLP).

<p>Six CNV sequences were used to design the primer-probe set used for qPCR detection. The strain GVI.1/HKU_Ca035F/2007/HKG had not been reported at time of design. Three of the most diverse CNV sequences were selected for VLP production.</p

Western blotting of purified VLPs using seropositive canine serum.

<p>Five different VLPs were separated by SDS-PAGE (2 µg each). Gel A was stained with Coomassie blue to identify purified VLP protein at the expected molecular weight. Gel B was used for western blotting with a canine serum sample which was seropositive to CNV strain 170 and HK by ELISA.</p