Table 1.
Primers.
Figure 1.
Phase-contrast micrographs of CPE in ASCV infected GF-1 cells at 21 days post inoculation (A), parallel cell culture inoculated with 2,5x volume (B) and non-infected control cells (C).
Figure 2.
Virus replication expressed as Cp values at different times after infection of GF-1 cell cultures.
A) Cell-associated compartment. No detectable virus genome was found at 0 dpi. Samplings 0–14 dpi: Significant changes between all samplings separated one week or more in time (P<0,001–0,05), 14 dpi: significant change to 24 dpi (P<0,05). B) Supernatant. Samplings 0–7 dpi: Significant changes compared to all samplings in the study (P<0,001–0,01), 14 dpi: significant change to 21 (P<0,01) and 24 dpi (p<0,05) (One way ANOVA). The cells were washed after 4 h of incubation with virus, and samples were collected sequentially, as indicated, post infection. The 0 h time point is the first sampling after incubation and washing. The experiment included three parallels at each time point (n = 3).
Figure 3.
A) The genome includes two ORFs. The predicted ORF1 spans almost the complete genome (numbers define nucleotide of start/end of ORF's and genome). A predicted small ORF2 is included in the 3′ end of the genome. B) Translated ORF1 amino acid sequence (numbers define amino acids of start/end of ORF1) includes several motifs that are conserved in calicivirus (positions indicated by arrows) and conserved protein domains (grey shading). The two ORFs and conserved amino acid motifs and domains confirm that the ORFs and predicted encoded non-structural proteins and capsid of ASCV are organized in an equal order as for the other caliciviruses.
Figure 4.
Evolutionary relationships of ASCV with members of the family Caliciviridae.
Phylogenetic analysis was based on sequence regions encompassing the capsid or putative capsid of representatives of the five known genera of caliciviruses, suggested new genera and unassigned published viruses. Selected sequences were obtained from GenBank and present study and Neighbor-joining analysis was performed with 1000 bootstrap replicates, as described in Materials and Methods. Members of the family Caliciviridae included in the tree are as follows: Vesicular exanthema of swine virus (VESV) serotype A48 (NP786889); Steller sea lion vesivirus V810 (ABP88255); VESV-like calicivirus Pan-1 (AAC61759); San Miguel sea lion (SMSV) serotype 1 (AAG13639); Walrus calicivirus (NP786921); Feline calicivirus (FCV) Urbana (NP78331); Feline calicivirus (FCV) F9 (from AAA79327); Canine calicivirus (NP786912); European brown hare syndrome virus (EBHSV) GD (from CAA93445); Rabbit hemorrhagic disease virus (RHDV) FRG (NP740333); Newbury agent 1 (from YP529550); Calicivirus strain NB (AAT35531); Bat sapovirus (Bat-SaV) TLC58-HK (from AFJ39355); Porcine enteric sapovirus (PEC) Cowden (from NP051035); Sapovirus (SaV) Mc10 (YP052971); Sapovirus (SaV) Manchester (CAA60262); Calicivirus chicken Bavaria04V0021 (from ADN88287); Snow Mountain virus (AAN08112); Lordsdale virus (CAA60255); Southampton virus (AAA92984); Tulane virus (ACB38132); St Valerién calicivirus AB90 (from ACQ44559). Only bootstrap values of 60% and above have been displayed in the output.
Table 2.
Pairwise comparison of capsid amino acid sequences.
Figure 5.
Negative staining of electron micrographs of ASCV isolated from cell culture.
Shown are virus-like particles of approximately 42 nm with indications of protrusions and depressions on the viral surface. Bar = 100 nm.
Figure 6.
Sequential development of ASCV viral load in kidney specimens following experimental challenge of Atlantic salmon.
The viral load of ASCV was measured by real-time PCR. Results are presented as a box plot (whiskers and minimum to maximum). Fish were challenged by intramuscular injection as non-vaccinated (blank boxes, representing 9–10 individuals per sampling) or vaccinated (patterned boxes, representing four individuals each, *three samples were negative and not included). Significant differences are indicated by brackets.
Table 3.
Prevalence of detection of ASCV cell culture isolate in challenge and vaccine trial.
Figure 7.
Histoscores in heart at 12, 14 and 16 weeks following experimental challenge of Atlantic salmon.
Different organ compartments (atrium (A), ventricle (V) and epicard (E)) have been grouped according to score intensity at the different time points and are separated in score groups as indicated. 0) No pathological changes observed. 1) Mild pathological changes; limited (countable) mononuclear inflammatory cells infiltrating the epicardium. Multifocal to diffuse cell infiltration, can involve parts of/entire epicardium. 2) Moderate pathological changes; high number (uncountable) of inflammatory cells in the epicardium, extending into the compact layer of the heart. In compact layer, multifocal or diffuse changes typically oriented along small blood vessels. A few focal changes in the spongious layer. 3) Severe pathological changes; intense infiltration of inflammatory cells in the epicardium and compact layer, typically with a diffuse distribution pattern, and involving the spongious layer in a multifocal pattern. Degeneration/necrosis of muscle fibers. Inflammatory changes comparable to ventricle can be seen in atrium.
Table 4.
ASCV screening of field samples, sample set A.
Table 5.
ASCV screening of field samples, sample set B.