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<strong>Spredning</strong> <strong>av</strong> <strong>ILA</strong> <strong>virus</strong><br />
Are Nylund<br />
Are Nylund, Institutt for Biologi<br />
Institutt for Biologi<br />
Universitetet i Bergen, 2008<br />
Universitetet i Bergen
Fordeling <strong>av</strong> genus Salmo
Biologien til Salmo spp.<br />
River<br />
Gyting<br />
Embryo<br />
Yngel<br />
Parr<br />
Ferskvanns patogener<br />
Gyrodactylus salaris<br />
Smolt<br />
Euryhaline patogener<br />
Ichthyobodo sp.<br />
Smolt<br />
Vekst, Atlantisk laks<br />
Sjø<br />
Marine patogener<br />
Lepeophtheirus salmonis<br />
Vekst, sjø-ørret<br />
Fjord
Nauplius 1<br />
0.5 mm<br />
Nauplius 2 - 0.6 mm
Sampling sites
ISA <strong>virus</strong> ?
Laks & Virus<br />
• Biologien til vert og parasitt/<strong>virus</strong> må være<br />
tilpasset.<br />
• Et arts/genus spesifikt <strong>virus</strong> vil alltid<br />
reflektere biologien til verten(e).<br />
• <strong>ILA</strong>V er et spesifikt <strong>virus</strong> for laksefisk.<br />
• Biologien til laksefisk vil derfor være<br />
<strong>av</strong>gjørende for spredning <strong>av</strong> disse to <strong>virus</strong>.
Stress
Virulens
Virulente<br />
og<br />
Avirulente<br />
(l<strong>av</strong>-virulente)
<strong>ILA</strong> <strong>virus</strong><br />
overflateproteiner<br />
Haemagglutinine - Esterase<br />
Fusjons protein
Evolusjonsmekanismer<br />
• Mutasjoner<br />
– Deletions, insertions, recombination,<br />
substitutions<br />
• Reassortments
Rekombinasjon<br />
RNA/DNA<br />
Seg 6
ISAV HE gene<br />
St<br />
HPR TMR Ct<br />
5`- - 3`<br />
1005 nt<br />
39 – 111 nt 69 nt 48 nt
HPR fra<br />
<strong>ILA</strong> <strong>virus</strong>
• Avirulent <strong>ILA</strong>V HPR0<br />
• HPR0<br />
• Canada<br />
• NIT DV KIRVDAI PPQL NQT FNTN QVEQ PS TSVL SIN FISM GV –NBr.<br />
• NIT DV KIRVDAI PPQL NQT FNTN QVEQ PA TSVL SIN FISM GV – NSc.<br />
• USA<br />
• NIT DV KIRVDAI PPQL NQT FNTN QVEQ PS TSVL SIN FISM GV - Maine<br />
• Scotland<br />
• NIT DV KIRVDAI PPQL NQT FNTN QVEQ PS NSVL SIN FISM GV – Scot.<br />
• Norway<br />
• NIT DV KIRVDAI PPQL NQT FNTN QVEQ PA TSVL SIN FISM GV – S&Fj
Avirulent <strong>ILA</strong>V<br />
Hvilken beydning?<br />
- <strong>ILA</strong> <strong>virus</strong> kan overleve i små populasjoner<br />
- <strong>Spredning</strong> via asymptomatiske bærere
Fusjons protein<br />
Segment 5<br />
Signal sequence<br />
Norway : MAFLTILVLF LLKEVLCEPC TCDNPTCLGL TIPRTGYVRS APGGVLLTET ITESPALAEW TTSRSRLEDS TWQGGEVKSG: 80<br />
Canada : .......... .F........ I.E......I ...QA.F... .......... ...R.Q.T.. ....PK..ET L.LD..T.N.: 80<br />
Norway : KVSQTLFEAI QGTQMENCAV KAVFDTSFVN LTRHDVVLGR VKVSPFGGEH DISKCGRKGL KVFICGGTTG YVTRGCPPEE: 160<br />
Canada : .......... .......... ...L..T... ..KQ.I...K I...E...DS .......... ........V. ..........: 160<br />
Norway : CRGRKGRMMS LEPTADCGVE KGFTTERIKT GKVDLDSCCT QHGCTKGIRV EVPSPVLVSA KCNEISFRVV PFHSVPDRLG: 240<br />
Canada : .K.K.....A ....T..... ..L..D.... .ML.IT.... .......... .........S ..Q.VT.... .......K..: 240<br />
Norway : FARTSSFTLR ANLANQHGWS KYSFNLRAFP GEEFIKCCGF TLGIGGAWFQ AYLNGEVQGD GAASAEDVKE KLNGIIDQIN: 320<br />
Canada : .........K ..FV.K.... ..N....G.. .......... ...V...... .....M.... .....D.... ..........: 320<br />
Norway : KVNLLLEGEI EAVRRIAYMN QASSLQNQVE IGLIGEYLNI SSWLETKTLT KTEEGLMKDG WCRSSNHCWC PPGTVGIPTI: 400<br />
Canada : ..DT...... .......... .......... .......... ......T... ........N. ..Q.NT.... ..KPTIV...: 400<br />
Norway : GYVDNIKEVT GTSWWMVMIH YIIVGLIVVV LVVLGLKLWG CIRR: 444<br />
Canada : ....S..... .......... ........I. V..F...... .L..: 444<br />
Transmembrane region<br />
Fusion peptide<br />
Coiled-coil
Seg 3<br />
Seg 2 Seg 5<br />
5MR60/01 : ANLANQHGWSKYSFGHSVHKLSN--------------------QRA--------------------FPGEE<br />
CH01/08 : AGLANQHGWSKYNF----------------NKGKSANDIISDQRA--------------------FPGEE<br />
CH11/08 : AGLANQHGWSKYNF----------------N---------------------LRA--------------------FPGEE<br />
MR46/99 : ANLANQHGWSKYSF-----------------N--------------------QRAPGGVLLTETITFPGEE<br />
SF71/02 : ASLANQHGWSKYSF-----------------N---------------------HRAIPRTGYVRSA-FPGEE<br />
MR62/01 : ASLANQHGWSKYSF-----------------N---------------------QRAIPRTGYVRSA-FPGEE<br />
FM116/06 : ANLANQHGWSKYSF-----------------N---------------------LRA--------------------FPGEE<br />
Inserts in segment 5
Konklusjon<br />
• Evolusjon <strong>av</strong> <strong>ILA</strong> <strong>virus</strong><br />
kontrolleres <strong>av</strong> vel kjente<br />
mekanismer hvor rekombinasjon<br />
spiller en viktig rolle for evolusjon<br />
<strong>av</strong> overflateproteinene (HE og F<br />
proteinene).
Epizootiologi
Vertikal oveføring <strong>av</strong> <strong>ILA</strong> <strong>virus</strong><br />
• Tre separate forsøk
Forsøksoppsett<br />
• 8 egg-grupper representert ved 8 forskjellige<br />
hunner og 3 hanner<br />
• Hver egg-gruppe delt i 3<br />
– Ubehandlet<br />
– Behandlet (buffodin)<br />
– Behandlet 2 ganger (buffodin)<br />
• 24 grupper holdt frem til klekking<br />
(ca 520 døgn °C)<br />
• Uttak<br />
– Før befruktning<br />
– Øyerogn (319 og 433 døgn °C)<br />
– Yngel (509 og 538 døgn °C)
Prevalens <strong>av</strong> <strong>ILA</strong>V i øyerogn og yngel<br />
Gruppe 300-400<br />
døgn-ºC<br />
> 500<br />
døgn-ºC<br />
Lokalitet<br />
UD 20,7 % 11,5 % <strong>ILA</strong>B<br />
D 10,4 % 23,0 % Stamfiskanlegg<br />
DD 1,9 % 3,4 % Stamfiskanlegg<br />
Øyerogn<br />
Yngel
Vertikal overføring <strong>av</strong> <strong>ILA</strong>V<br />
vanlig produksjonssyklus
Resultater<br />
STAMFISK.<br />
295/352 stamfisk <strong>ILA</strong>V-positive (83.8%).<br />
ROGN.<br />
Finner <strong>virus</strong> i alle egg hos <strong>ILA</strong>V-positiv fisk.<br />
– Virus sitter hovedsakelig på utsiden.
Resultater<br />
YNGEL<br />
15/110 yngel <strong>ILA</strong>V-positive (13.6%).<br />
PAR<br />
2/120 Parr <strong>ILA</strong>V-positive (1.7%).<br />
Smolt<br />
23/150 smolt positive for <strong>ILA</strong>V (15,3 %)<br />
Postsmolt<br />
25/150 postsmolt positive for <strong>ILA</strong>V (16,7%)
Konklusjon<br />
• <strong>ILA</strong>V overføres vertikalt under en normal<br />
produksjonssyklus<br />
• Antall positive individer synes å øke med<br />
alder på fisken
ISAV i smolt i ferskvann<br />
2004 - 2005<br />
Smolt fra 30 anlegg undersøkt<br />
Smolt i 2 anlegg var negative for <strong>ILA</strong>V<br />
Konklusjon<br />
De fleste marine lakseanlegg har fisk som<br />
er positiv for <strong>ILA</strong>V
Fakta<br />
• <strong>ILA</strong> <strong>virus</strong> overføres vertikalt i<br />
produksjon <strong>av</strong> Norsk laks.<br />
• Er vertikal overføring viktig for<br />
spredning <strong>av</strong> <strong>ILA</strong>?
Genotyping<br />
Molekylær epizootiology
ISA <strong>virus</strong> in the North Atlantic<br />
Canada 1997<br />
(Chile)<br />
USA 2002<br />
100<br />
Nordland 1999<br />
+ Chile<br />
Sør Trøndelag 1996<br />
Skottland 1998<br />
98<br />
100<br />
Skottland 1998<br />
Nova Scotia 2003<br />
99<br />
99<br />
Maine, USA 2004<br />
Færøyene 2002<br />
85<br />
0.1<br />
Færøyene 2002
Mønster ved<br />
horisontal<br />
<strong>Spredning</strong><br />
Lokale<br />
reservoar
Mønster ved<br />
vertikal<br />
spredning<br />
Stamfisk<br />
reservoar
NB:<br />
NBC<br />
Alle disse<br />
anleggene har<br />
mottatt smolt<br />
basert på egg fra<br />
stamfiskanlegg<br />
NBC
Hardangerfjord<br />
ISA<br />
02.02.04<br />
?<br />
ISA<br />
16.07.04
6FM86/04<br />
6H88/04<br />
6H96/04<br />
6T79/03<br />
6R111/05<br />
6N89/04<br />
6H99/04<br />
6H36/98<br />
6SF41/98<br />
6N5/89<br />
6H45/99<br />
6H40/98<br />
6MR46/99<br />
6SF18/96<br />
6MR60/01<br />
6H51/00<br />
6H56/00<br />
6H101/04<br />
6H17/96<br />
AF388582<br />
ISA276859<br />
AF395337<br />
6NT84/04<br />
6ST82/04<br />
6ST25/97<br />
6T90/04<br />
6ST28/97<br />
6ST27/97<br />
6N9/93<br />
AF388581<br />
6ST110/05<br />
6Scot43/98<br />
6N29/97<br />
6N32/98<br />
6H97/04<br />
6SF83/04<br />
6SF47/99<br />
6T33/98<br />
6T22/96<br />
6SF48/99<br />
6FM100/04<br />
6SF109/05<br />
6MR104/05<br />
6H1/87<br />
6H8/93<br />
6H7/92<br />
6FM106/05<br />
6FM98/04<br />
6FM108/05<br />
6FM107/05<br />
6T73/02<br />
6T74/03<br />
6N75/03<br />
6T91/04<br />
6T10/93<br />
6H2/89<br />
6T85A/04<br />
6T37/98<br />
6SF14/95<br />
6NT38/98<br />
6H6/91<br />
6F72b/02<br />
6F72d/02<br />
6F72e/02<br />
6F72/02<br />
6FM114/05<br />
T041005<br />
6SF94/04<br />
6NS2003<br />
AF294881<br />
usa-tot<br />
6MR61/01<br />
6SF71/02<br />
6H112A/04<br />
6SF70/02<br />
6SF63/01<br />
6SF57/00<br />
6SF54/00<br />
6MR62/01<br />
6ST26/97<br />
6ST44/99<br />
6ST30/97<br />
6H93/04<br />
6MR52/00<br />
6N49/99<br />
6ST21/96<br />
6NT78/03<br />
6ST77/03<br />
6NT92B/04<br />
6NT87/04<br />
6NT81/03<br />
6MR103/05<br />
6MR105/05<br />
6MR102/05<br />
6NT95/04<br />
AY059402<br />
6Can31/97<br />
AF404342<br />
H101/04<br />
H93/04<br />
Forskjeller<br />
26 nucleotider<br />
13 amino syrer
<strong>ILA</strong> <strong>virus</strong> i Chile
66<br />
81<br />
93<br />
99<br />
100<br />
NT141/08<br />
T124/07<br />
77 T151/08<br />
T148/08<br />
T129/07<br />
NBC ST77/03<br />
MR103/05<br />
ST26/97<br />
ST30/97<br />
N49/99<br />
ST21/96<br />
65<br />
ST44/99<br />
H93/04<br />
CH09/08<br />
94 AM941715 CH18/08, CH19/08, CH20/08, CH21/08, CH22/08<br />
87 98CH01/08, CH11/08, CH12/08, CH15/08, CH1608<br />
CH03/08, CH04/08<br />
CH05/08<br />
80 CH08/08<br />
86 CH06/08<br />
NBC<br />
78<br />
T131/07<br />
ST25/97<br />
ST28/97<br />
ST27/97<br />
ST110/05<br />
NBC<br />
57<br />
SF83/04<br />
N127/07<br />
N29/97<br />
N9/93<br />
90<br />
86 NBC<br />
R111/05<br />
MR118/06<br />
H101/04<br />
66<br />
H51/00<br />
66<br />
H56/00<br />
74<br />
H17/96<br />
MR46/99<br />
Scotland<br />
75 H2/89<br />
H6/91<br />
SF14/95<br />
FM114/05<br />
NT115/05<br />
SF94/04<br />
61<br />
97<br />
F72/02<br />
F72b/02<br />
H112A/04<br />
SF70/02<br />
70<br />
SF63/01<br />
SF57/00<br />
SF54/00<br />
MR61/01<br />
T121/07<br />
92 T126/07<br />
97<br />
FM98/04<br />
T91/04<br />
96<br />
T73/02<br />
T10/93 100 T74/03<br />
NT134/08<br />
T130/07<br />
0.01<br />
Segment 6
0.01<br />
CH01/07<br />
CH16/98<br />
EU130923<br />
CH11/08<br />
CH07/08<br />
ST25/97<br />
ST28/97<br />
ST27/97<br />
NBC<br />
SF83/04<br />
90<br />
T73/02<br />
T74/03<br />
94<br />
T10/93<br />
83<br />
T122/07<br />
T129/07<br />
73<br />
N127/07<br />
68<br />
98<br />
Faeroe Island<br />
Nova Scotia, EF217314<br />
FM107/05<br />
Faeroe Island<br />
76<br />
98<br />
94<br />
MR61/01<br />
SF71/02<br />
100<br />
MR62/01<br />
SF57/00<br />
98<br />
FM116/06<br />
94<br />
H51/00<br />
H02/89<br />
99<br />
H06/91<br />
98<br />
90<br />
80<br />
H45/99<br />
SF41/98<br />
95<br />
H36/98<br />
H40/98<br />
85<br />
MR46/99<br />
67<br />
N75/03<br />
T126/07<br />
98<br />
92<br />
FM86/04<br />
H56/00<br />
75<br />
MR60/01<br />
Scotland 1998<br />
84<br />
H93/04<br />
MR52/00<br />
98<br />
94<br />
ST26/97<br />
ST21/96<br />
Chile<br />
Segment 5
HPR<br />
CH01/08 : NITDVK--------------------------------------------TSVLSNIFISMGVAG<br />
CH02/08 : NITDVK--------------------------------------------TSVLSNIFISMGVAG<br />
CH03/08 : NITDVK--------------------------------------------TSVSSNIFISMGVAG<br />
CH04/08 : NITDVK--------------------------------------------TSVSSNIFISMGVAG<br />
CH05/08 : NITDVKIRVDAIPPQLNQTF--------------------------------------GVAG<br />
CH06/08 : NITDVKIRVDAIPPQLNQTF--------------------------------------GVAG<br />
CH08/08 : NITDVKIRVDAIPPQLNQTF--------------------------------------GVAG<br />
CH09/08 : NITDLK-----------------------TFNTNQVEQPATSVLSNIFISMGVAG<br />
CH10/08 : NITDLK-----------------------TFNTNQVEQPATSVLSNIFISMGVAG<br />
CH11/08 : NITDLK-----------------------TFNTNQVEQPATSVLSNIFISMGVAG<br />
CH12/08 : NITDVK---------------------------------------------TSVLSNIFISMGVAG<br />
CH15/08 : NITDVK---------------------------------------------TSVLSNIFISMGVAG<br />
CH16/08 : NITDVK---------------------------------------------TSVLSNIFISMGVAG<br />
CH17/08 : NITDVK---------------------------------------------TSVLSNIFISMGVAG<br />
CH18/08 : NITDVK---------------------------------------------TSVLSNIFISMGVAG<br />
CH19/08 : NITDVK---------------------------------------------TSVLSNIFISMGVAG<br />
CH20/08 : NITDVK---------------------------------------------TSVLSNIFISMGVAG<br />
CH21/08 : NITDVK---------------------------------------------TSVLSNIFISMGVAG<br />
CH22/08 : NITDVK---------------------------------------------TSVLSNIFISMGVAG<br />
HPR0 : NITDVKIRVDAIPPQLNQTFNTNQVEQPATSVLSNIFISMGVAG
Konklusjon<br />
<strong>ILA</strong> <strong>virus</strong> fra Chile er lik <strong>ILA</strong> <strong>virus</strong> fra<br />
kontemporære utbrudd i Norge.<br />
Dette kan kun forklares med spredning via<br />
befruktede egg (vertikal overføring).
Hva med<br />
<strong>ILA</strong><br />
i Troms ?
Toppsundet 2007
97<br />
FM107/05<br />
FM106/05<br />
FM98/04<br />
Nord-Norge 2007<br />
FM108/05<br />
Nordland 2003, HPR 4a<br />
94<br />
92<br />
Troms III, 2007, HPR 31<br />
Troms III, 2007, HPR 31<br />
Toppsundet 2007<br />
98<br />
87<br />
T74/03<br />
T73/02<br />
T91/04<br />
Gullesfjord 1993<br />
Stamfisk 2005<br />
Utgruppe<br />
Stamfisk 2005<br />
0.01
Nord-Norge 2007<br />
100<br />
Stamfiskanlegg 2004, <strong>av</strong>irulent<br />
Sogn og fjordane 2004, <strong>av</strong>irulent<br />
100<br />
100<br />
Nordland 1997<br />
Nordland 1998<br />
Nordland IV, 2007<br />
Nordland IV, 2007<br />
Hordaland 1992<br />
Hordaland 1987<br />
Utgruppe<br />
0.001
Toppsundet 2007<br />
Salaks 2007
95<br />
ST1997, Egg: ukjent<br />
ST1997, Egg: A<br />
ST1999, Egg: A<br />
Nord-Norge 2007<br />
97<br />
85<br />
MR2000, Egg: A<br />
H2004, Egg: A + B<br />
N1999, Egg: A + D<br />
ST1996, Egg: ukjent<br />
NT2003, Egg: A +B<br />
96<br />
NT2004, Egg: A + B<br />
ST2003, Egg: ukjent<br />
88<br />
NT2004, Egg: ukjent<br />
86<br />
85<br />
NT2003, Egg: A +B<br />
Troms I, 06.06-2007<br />
Troms I, 26.06-2007<br />
Troms II, 28.06-2007<br />
Stamfisk A, 2005<br />
Stamfisk A, 2005<br />
To anlegg<br />
Salaks<br />
Stamfisk A<br />
NT2004, Egg: A + B<br />
T10/93<br />
T73/02<br />
Utgruppe<br />
0.01
Lønskog-07<br />
NBC-C<br />
99<br />
ST28/97 ST27/97, Sifjord-07<br />
SF83/04<br />
Kværnbukta-07, Sifjord-07<br />
98<br />
NBC ST25/97<br />
MR118/06<br />
83<br />
96 Chile-08<br />
R111/05<br />
H99/04<br />
NBC<br />
T79/03 FM86/04<br />
96<br />
97<br />
80<br />
T73/02<br />
97<br />
T10/93<br />
99<br />
T74/03<br />
98<br />
Småvik -07<br />
100<br />
81<br />
83<br />
FM108/05<br />
Toppsundet-07<br />
Salangslia-07<br />
99<br />
100<br />
Digerneset-08<br />
Toppsundet-07<br />
Storvika-07, Skardbergvika-08, Kråkerø-08<br />
Storevika-08, Lekangsund-08, Ystevika-08<br />
NBC<br />
Kråkholmen-08<br />
Kjeiprød-08<br />
Salangslia-07<br />
ST21/96<br />
Ytre Strætet-08<br />
ST44/99<br />
0.01
Nt/åå Ytre s Kjeiper T149 T145 T144 T140 T130 T129 T124 BFC-X<br />
T122/07 2/1 2/1 1/0 1/0 1/0 1/0 9/3 1/1 1/0 2/0<br />
Ytre Str 2/2 1/1 1/1 1/1 1/1 9/4 2/2 1/1 2/1<br />
Kjeiperø 1/1 1/1 1/1 1/1 9/4 2/2 1/1 2/1<br />
T149/08 0/0 0/0 0/0 8/3 1/1 0/0 1/0<br />
T145/08 0/0 0/0 8/3 1/1 0/0 1/0<br />
T144/08 0/0 8/3 1/1 0/0 1/0<br />
T140/08 8/3 1/1 0/0 1/0<br />
T130/08 9/4 8/3 9/3<br />
T129/07 1/1 2/1<br />
T124/07 1/0<br />
BFC-X
<strong>ILA</strong> <strong>virus</strong> knopper <strong>av</strong><br />
fra endotelceller i<br />
blodkarene
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