Background
Results and discussion
Amplification and sequencing of cDNA of genomic segments of PRV from fish samples
PRV “isolate” | Fish species | Source |
---|---|---|
23 | Atlantic salmon | Farmed, Canada |
163 | Atlantic salmon | Farmed, Canada |
167 | Atlantic salmon | Farmed, Canada |
177 | Atlantic salmon | Farmed, Canada |
185 | Atlantic salmon | Farmed, Canada |
196 | Atlantic salmon | Farmed, Canada |
209 | Atlantic salmon | Farmed, Canada |
321 | Atlantic salmon | Farmed, Canada |
333 | Cutthroat trout | Wild, Canada |
340 | Cutthroat trout | Wild, Canada |
358 | Atlantic salmon | Farmed, Canada |
371 | Atlantic salmon | Farmed, Canada |
468 | Chum salmon | Wild, Canada |
480 | Steelhead trout | Farmed, Canada |
CGA337 | Atlantic salmon | Farmed, Chile |
CGA558 | Atlantic salmon | Farmed, Chile |
CGA8857 | Atlantic salmon | Farmed, Chile |
CGA280-5 | Atlantic salmon | Farmed, Chile |
The PRV conserved terminal nucleotide sequences
Reovirus genus | Reovirus species/strain | Conserved terminal nucleotide sequences | |
---|---|---|---|
5′ terminal nucleotides | 3′ terminal nucleotides | ||
PRV | Salmo/GP-2010/NOR [2]* | 5′-GAUAAA/U | UCAUC-3′ |
Orthoreovirus genus | Avian orthoreovirus-138 [1]* | 5′-GCUUUUU | UCAUC-3′ |
Nelson Bay orthoreovirus[1]* | 5′-GCUUUA | UCAUC-3′ | |
Mammalian orthoreovirus -1La [1]* | 5′-GCUA | UCAUC-3′ | |
Baboon orthoreovirus[1]* | 5′-GUAAAUUU | UCAUC-3′ | |
Reptilian orthoreovirus[1]* | 5′-GUUAUUUU | UCAUC-3′ | |
Aquareovirus genus | Aquareovirus A [1]* | 5′-GUUUUA | UCAUC-3′ |
Aquareovirus C [1]* | 5′-GUUAUU | UCAUC-3′ | |
Aquareovirus G [1]* | 5′-GUUUUA | UCAUC-3′ |
The PRV protein profile deduced from whole-genome sequence analysis
Genome segment* | Molecular size (bp)** | 5′UTR (bp)** | 3′UTR (bp)** | Protein name*** | Protein size (aa) & mass (kDa)1 | pI value | Predicted function |
---|---|---|---|---|---|---|---|
L1
| 3911 | 18 | 44 | Core shell (T = 1) [λ1] | 1282, 141.41 kDa | 5.47 | major inner capsid protein, Helicase, RNA triphosphatase |
L2
| 3935 | 18 | 44 | Core turret [λ2] | 1290, 143.75 kDa | 4.81 | core spike, guanylyl transferase |
L3
| 3916 | 7 | 48 | Core RdRp [λ3] | 1286, 144.24 kDa | 8.68 | minor inner capsid protein, RNA polymerase |
M1
| 2383 | 21 | 79 | Core NTPase [μ2] | 760, 86.09 kDa | 8.23 | minor inner capsid protein, nucleoside triphosphate phosphohydrolase |
M2
| 2179 | 26 | 89 | Outer shell (T = 13) [μ1] | 687, 74.26 kDa | 6.27 | outer capsid protein, membrane penetration, apoptosis |
M3
| 2403 | 83 | 61 | NS factory [μNS] | 752, 83.53 kDa | 5.00 | NS, genome packaging? |
S1
| 1081 | 28 | 60 | Outer clamp [σ3] | 330, 37.08 kDa | 7.43 | major outer capsid protein, dsRNA binding protein, translation control, modulation of cellular interferon, zinc-binding |
NS, p13 [σ1s] | 124, 12.99 kDa | 4.88 | NS, block cell-cycle progression, cytolytic in PRV | ||||
S2
| 1329 | 21 | 45 | Core clamp [σ2] | 420, 45.93 kDa | 9.02 | major inner capsid protein, morphogenesis? |
S3
| 1143 | 28 | 50 | NS RNA [σNS] | 354, 39.07 kDa | 7.76 | NS, genome packaging? |
S4
| 1040 | 38 | 54 | Outer fiber [σ1] | 315, 34.60 kDa | 6.04 | outer capsid protein (virus attachment), cell tropism, pathways of viral spread in-vivo, virulence |
Whole-genome sequence comparison to other members of family Reoviridae
Phylogenetic analysis and sequence diversity of PRV genomic segment S1
PRV isolate | 163 | 167 | 196 | 209 | 358 | 371 | 5433* | 3817* | 1921* | 9326* | GP-2010 | 8286* | 7030* | 7243* | 280-5 | 8857 | 337 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
163 | - |
99.4
|
99.4
|
99.2
|
99.3
|
99.6
|
99.6
|
99.0
|
98.9
|
98.6
| 96.8 | 96.6 | 96.5 | 96.5 | 96.6 | 94.9 | 94.8 |
167 |
99.4
|
-
|
99.6
|
99.5
|
99.6
|
99.3
|
99.4
|
98.8
|
98.7
|
98.4
| 96.6 | 96.4 | 96.3 | 96.3 | 96.4 | 94.7 | 94.6 |
196 |
99.4
|
99.4
|
-
|
99.4
|
99.4
|
99.3
|
99.4
|
98.8
|
98.7
|
98.4
| 96.6 | 96.4 | 96.3 | 96.3 | 96.4 | 94.7 | 94.6 |
209 |
98.8
|
98.8
|
98.8
|
-
|
99.4
|
99.0
|
99.1
|
98.5
|
98.4
|
98.1
| 96.3 | 96.1 | 96.0 | 96.0 | 96.1 | 94.4 | 94.3 |
358 |
99.4
|
99.4
|
99.4
|
98.8
|
-
|
99.1
|
99.2
|
98.6
|
98.5
|
98.2
| 96.4 | 96.2 | 96.1 | 96.1 | 96.2 | 94.4 | 94.8 |
371 |
98.8
|
98.8
|
98.8
|
98.2
|
98.8
|
-
|
99.4
|
98.8
|
98.7
|
98.4
| 96.6 | 96.4 | 96.3 | 96.3 | 96.4 | 94.7 | 94.6 |
5433* |
99.7
|
99.7
|
99.7
|
99.1
|
99.7
|
99.1
|
-
|
99.2
|
99.1
|
98.8
| 96.9 | 96.9 | 96.8 | 96.8 | 96.7 | 95.2 | 95.1 |
3817* |
99.7
|
99.7
|
99.7
|
99.1
|
99.7
|
99.1
|
100.0
|
-
|
99.9
|
99.0
| 96.9 | 96.9 | 96.9 | 96.9 | 96.8 | 95.3 | 95.2 |
1921* |
99.4
|
99.4
|
99.4
|
98.8
|
99.4
|
98.8
|
99.7
|
99.7
|
-
|
98.9
| 96.9 | 96.9 | 96.8 | 96.8 | 96.7 | 95.2 | 95.1 |
9326* |
99.7
|
99.7
|
99.7
|
99.1
|
99.7
|
99.1
|
100.0
|
100.0
|
99.7
|
-
| 96.8 | 96.8 | 96.7 | 96.7 | 96.6 | 95.1 | 95.0 |
GP-2010 | 96.7 | 96.7 | 96.7 | 96.1 | 96.7 | 96.1 | 97.0 | 97.0 | 96.7 | 97.0 | - | 100.0 | 99.9 | 99.9 | 99.8 | 98.2 | 98.3 |
8286* | 96.7 | 96.7 | 96.7 | 96.1 | 96.7 | 96.1 | 97.0 | 97.0 | 96.7 | 97.0 | 100.0 | - | 99.9 | 99.9 | 99.8 | 98.2 | 98.3 |
7030* | 96.4 | 96.4 | 96.4 | 95.8 | 96.4 | 95.8 | 96.7 | 96.7 | 96.4 | 96.7 | 99.7 | 99.7 | - | 99.8 | 99.7 | 98.1 | 98.2 |
7243* | 96.7 | 96.7 | 96.7 | 96.1 | 96.7 | 96.1 | 97.0 | 97.0 | 96.7 | 97.0 | 100.0 | 100.0 | 99.7 | - | 99.9 | 98.3 | 98.4 |
280-5 | 96.4 | 96.4 | 96.4 | 95.8 | 96.4 | 95.8 | 96.7 | 96.7 | 96.4 | 96.7 | 99.7 | 99.7 | 99.4 | 99.7 | - | 98.2 | 98.3 |
8857 | 82.5 | 82.2 | 82.2 | 93.9 | 82.5 | 82.2 | 82.5 | 82.5 | 82.2 | 82.5 | 86.2 | 86.2 | 85.9 | 86.2 | 86.2 | - | 97.4 |
337 | 93.0 | 93.0 | 93.0 | 92.3 | 93.0 | 92.6 | 93.3 | 93.3 | 93.0 | 93.3 | 96.7 | 96.7 | 96.3 | 96.7 | 96.7 | 86.6 | - |
Divergence time estimation between Canadian and Chilean PRV and the Norwegian strains
Conclusions
Methods
Fish samples and processing
RT-PCR and nucleic acid sequencing
-
ASELF1a Forward – 5'- CGT GAC ATG AGG CAG ACA GT-3';
-
ASELF1a Reverse – 5'- CGG CCT TAA CAG CAG ACT TTG-3';
-
ASELF1a Probe – 5'-TGC TGT CGG TGT CAT CAA GGC T-3'; and
-
CSELF1α Forward – 5′- GGT CAC CAC CTA CAT CAA GAA GA-3′;
-
CSELF1α Reverse – 5′- CCA ACC AGA GAT GGG CAC AAA G-3′;
-
CSELF1α Probe – 5′-TGG CTA CAA CCC TGC CAC TGT C-3′.