Characterization of a novel calicivirus causing systemic infection in atlantic salmon (Salmo salar L.): proposal for a new genus of caliciviridae.

The Caliciviridae is a family of viruses infecting humans, a wide range of animals, birds and marine fish and mammals, resulting in a wide spectrum of diseases. We describe the identification and genetic characterization of a novel calicivirus replicating in Atlantic salmon. The virus has a high prevalence in farmed salmon and is found in fish suffering from several diseases and conditions and also in presumable healthy fish. A challenge and vaccination trial shows that the calicivirus replicates in Atlantic salmon and establishes a systemic infection, which can be reduced by vaccination with formalin-inactivated virus preparation. The virus, named Atlantic salmon calicivirus (ASCV), is found in two genetically distinct variants, a cell culture isolated and a variant sequenced directly from field material. The genomes are 7,4 kb and contain two open reading frames where typical conserved amino acid motifs and domains predict a gene order reminiscent of calicivirus genomes. Phylogenetic analysis performed on extracted capsid amino acid sequences segregated the two ASCV variants in a unique cluster sharing root with the branch of noroviruses infecting humans and the unassigned Tulane virus and St-Valérien like viruses, infecting rhesus monkey and pig, respectively, with relatively large distance to the marine calicivirus subgroup of vesiviruses. Based on the analyses presented, the ASCV is predicted to represent a new genus of Caliciviridae for which we propose the name Salovirus

Sequential development of ASCV viral load in kidney specimens following experimental challenge of Atlantic salmon.

<p>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.</p

Primers.

<p>Primers used for real-time PCR against cell cultured isolate and field strains of ASCV and primers used in virus genome cloning and sequencing.</p><p>*Nucleotides matching cell cultured isolate only are underlined.</p><p>Primers.</p

Evolutionary relationships of ASCV with members of the family Caliciviridae.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107132#s2" target="_blank">Materials and Methods</a>. 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.</p

Virus replication expressed as Cp values at different times after infection of GF-1 cell cultures.

<p>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).</p

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).

<p>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).</p

Negative staining of electron micrographs of ASCV isolated from cell culture.

<p>Shown are virus-like particles of approximately 42 nm with indications of protrusions and depressions on the viral surface. Bar  = 100 nm.</p

ASCV screening of field samples, sample set B.

<p>RNA from heart (HE), head kidney (HK) tissue and pylorus caeca (PC) tissue of Atlantic salmon with experienced symptoms of malabsorption and floating faeces in the fish pens. Real-time PCR Cp-values using ASCV field isolate specific primers in parallel with primers specific for PRV and PMCV, the causative agents for HSMI and CMS, respectively. >35 denotes samples with uncertain CP-values above 35, but with melting point temperature indicating a specific product. – denotes a negative sample.</p><p>*- Cp-values of parallel samples. Nd – not done due to availability of sample.</p><p>ASCV screening of field samples, sample set B.</p

Prevalence of detection of ASCV cell culture isolate in challenge and vaccine trial.

<p>RNA from various organ tissue of naïve and vaccinated Atlantic salmon challenged with ASCV cell culture isolate tested for presence of the virus using real-time PCR. The number of fish with detectable ASCV in each organ of the number tested is given. wpc – weeks post challenge, Nd – not done, Na – no available sample.</p><p>Prevalence of detection of ASCV cell culture isolate in challenge and vaccine trial.</p

Genome organization of ASCV.

<p>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.</p