Bivariate plot showing the relationship between the histoscore of the spongious part of the heart ventricle and the virus load (expressed as Cp values) in heart tissue for sample 1 (n = 40).

(a) Atrium at 8 weeks postchallenge. The foci show infiltration with lymphocytic cells (arrow) in proximity to a degenerate myofiber (asterisk) (H&E counterstain). (b) Ventricle at 8 weeks postchallenge. Shown is diffuse infiltration with inflammatory cells (lymphocytic cells and macrophages [arrow]) in and in proximity to a degenerate/necrotic myofiber (asterisk) (H&E counterstain).

(a) In situ hybridization showing the presence of the viral genome in degenerate cardiomyocytes (reddish coloration). Note that endothelial cells are negative (arrows) (counterstaining with hematoxylin). (b) In situ hybridization showing the granular staining pattern (arrows) of the sarcoplasm in an area with degenerate cardiomyocytes. Note the enlarged nucleus (counterstaining with hematoxylin).

Negative staining of electron micrographs of virus isolated from heart tissue homogenate as described in Materials and Methods. Shown are virus-like particles of approximately 50 nm with regular surface structures (a) and possibly with surface projections (b). Bars = 40 nm (a) and 50 nm (b).

Genome organization of PMCV. The genome includes three large ORFs. The predicted ORF1 and ORF2 resemble two overlapping open reading frames. ORF2 is translated as a fusion protein with ORF1 due to ribosomal −1 frameshifting or as an individual protein (start codon indicated). ORF3 is a nonoverlapping frame.

Histoscores for injection-challenged (Injected) and cohabitation-challenged (Cohabitants) salmon at different time postchallenge. No changes were observed by 4 weeks for the injected group, with gradual increases from 6 weeks up to 9 weeks. Fish challenged by cohabitation were collected at 6, 8, and 10 weeks postchallenge. Lower scores were found for this group, but distinct changes were present. Average values plus standard errors of the mean (SEM) are shown (n = 6 to 7 individuals per time point).

Sequential development of the viral load in heart specimens following experimental challenge of Atlantic salmon. The viral load of PMCV was measured by real-time PCR. Data were normalized against endogenous β-actin, and virus levels are given as relative copy numbers (the fish with the lowest viral load was set to 1). The results are presented as a box plot (whiskers and minimum to maximum). Fish were challenged by intramuscular injection (Injected) or infected by cohabitation (Cohabitants; stippled boxes).

(a to d) Phase-contrast micrographs of CPE in GF-1 cells at 0 (a), 6 (b), 14 (c), and 21 (d) days postinoculation with a heart homogenate sampled from Atlantic salmon clinically diagnosed with CMS. The diagnosis was confirmed by histopathology. (e) Virus replication expressed as Cp values at different times after infection of GF-1 cell cultures. There was moderate increase from day 7 to day 14 postinoculation and no further increase up to day 21. The experiment included three parallels at each time point (n = 3). P values are indicated (one-sided t test). d, day(s).

Sensitivities to nuclease digestion of three different RNA viruses were assessed by real-time PCR in order to determine the character of the PMCV genome. The following viruses were included: PMCV, IPNV (dsRNA), and ISAV (ssRNA). All treatments were performed in duplicate, and the results are given as Cp values, with ranges indicated by the error bars. Treatments were as follows: 1, untreated; 2, mung bean nuclease; 3, 2 μg RNase A, low-salt conditions; 4, 2 μg RNaseA, high-salt conditions. All treatments were done in duplicate. RNase A treatment at low salt concentration produced reductions of 99.96 and 99.83% for PMCV and IPNV, respectively, while both were resistant to mung bean nuclease. ISAV genome levels were reduced by 98.95% following mung bean treatment and >99.91% following RNase A treatment.

Virus replication expressed as Cp values at different times after infection of CHH-1 cell cultures in the cell-associated compartment (a) and in the supernatant (b). Cell cultures were infected with filtered, centrifuged homogenate from infected fish (heart) as described in Materials and Methods. The cells were washed after 4 h of incubation, and samples were collected sequentially, as indicated, postinfection. The 0-h time point is the first sampling after incubation and washing. There was a drop in cell-associated virus from 0 h to 1 day p.i. (a) and a similar increase in the supernatant (b). Similarly, there was an increase from 3 to 6 days p.i. for both cell-associated virus (a) and supernatant (b). The experiment included three parallels at each time point (n = 3). P values are indicated (two-sided t test).

Evolutionary relationships of members of the family Totiviridae. Phylogenetic analysis was based on a 480-aa sequence region encompassing the RdRp conserved motifs (right) of totiviruses, in line with the work published by Ghabrial and Nibert (13). Selected sequences were obtained from GenBank and aligned using the MUSCLE algorithm (version 3.8.31) with default settings (9). Neighbor-joining analysis was performed with 1,000 bootstrap replicates using Mega 4.0 software (36). Members of the family Totiviridae included in the tree are as follows: HvV190, Helminthosporium victoriae virus 190S (HVU41345); CeRV1, Chalara elegans RNA virus 1 (AY561500); CmRV, Coniothyrium minitans RNA virus (AF527633); EfV1, Epichloe festucae virus 1 (AM261427); GaRV-L1, Gremmeniella abietina RNA virus L1 (AF337175); HmTV1-17, Helicobasidium mompa totivirus 1 to 17 (AB085814); MoV1, Magnaporthe oryzae virus 1 (AB176964); SsRV1, Sphaeropsis sapinea RNA virus 1 (AF038665); SsRV2, Sphaeropsis sapinea RNA virus 2 (AF039080); BFTV1, Botryotinia fuckeliana totivirus 1 (NC_009224); MoV2, Magnaporthe oryzae virus 2 (AB300379); EbRV1, Eimeria brunetti RNA virus 1 (AF356189); LRV1-1, Leishmania RNA virus 1-1 (M92355); LRV2-1, Leishmania RNA virus 2-1 (U32108); LRV 1-4, Leishmania RNA virus 1-4 (U01899); TVV1, Trichomonas vaginalis virus 1 (U08999); TVV2, Trichomonas vaginalis virus 2 (AF127178); TVV3, Trichomonas vaginalis virus 3 (AF325840); ScV-L-A, Saccharomyces cerevisiae virus L-A (J04692); ScV-L-BC, Saccharomyces cerevisiae virus L-BC (U01060); UmV-H1, Ustilago maydis virus H1 (U01059); GLV, Giardia lamblia virus (L13218); IMNV, penaeid shrimp infectious myonecrosis virus (AY570982).