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DISEASES OF AQUATIC ORGANISMS

Vol. 47: 175–181, 2001 Published December 5
Dis Aquat Org

Detection of infectious salmon anaemia virus
(ISAV) by RT-PCR after cohabitant exposure in
Atlantic salmon Salmo salar
Aase B. Mikalsen1, Ann Teig1, Anne-Lill Helleman2, Siri Mjaaland1, Espen Rimstad1,*
1
Department of Pharmacology, Microbiology and Food Hygiene, The Norwegian School of Veterinary Science,
PO Box 8146, Dep., 0033 Oslo, Norway
2
GenoMar asa, Oslo Research Park, Gaustadalléen 21, 0349 Oslo, Norway

ABSTRACT: A reverse transcription polymerase chain reaction (RT-PCR) was used to study the early
phase of infectious salmon anaemia virus (ISAV) infection in Atlantic salmon Salmo salar L. The
detection threshold for the RT-PCR was estimated to be 0.01 to 0.1 TCID50. A protocol that closely
mimics the conditions in populations of farmed salmon was used. The major port of ISAV entry was
most likely the gills, but oral entry could not be excluded. The gills and heart were RT-PCR positive
5 d post infection and there was a rapid viraemic spread of the virus after entry. Ten or more days post
infection, most organs yielded RT-PCR positive samples. The viral load of the fish followed a 2-phase
curve with the first maximum at approximately 15 d and a minimum around 25 d. After 25 d, there
was a steady increase in viral load until all sampled organs eventually became positive. In an exper-
iment in which the transportation of material from field to diagnostic laboratory was simulated, the
transportation of whole fish was found to be optimal for the performance of RT-PCR.

KEY WORDS: Infectious salmon anaemia virus · ISAV · RT-PCR · Detection of ISAV
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INTRODUCTION prominent in some recent ISA outbreaks (Rodger &
Richards 1998). The differences in clinical appearance
Infectious salmon anaemia (ISA) is a multisystem and pathology are probably related to properties of
disease recognized in farmed Atlantic salmon Salmo the infectious agent, infectious salmon anaemia virus
salar L. in Norway, Canada and Scotland (Thorud & (ISAV), and/or of the host and/or environmental fac-
Djupvik 1988, Mullins et al. 1998, Rodger et al. 1998). tors. Confirmative detection methods such as RT-PCR
The disease may cause a rapid, dramatic increase in in addition to pathological examination are therefore
mortality in affected fish, but occasionally, the fish beneficial tools for the diagnosis of ISAV.
exhibit reduced weight gain, a dizzy appearance and a Currently there is no commercial vaccine or treat-
slow increase in mortality. The diagnosis of ISA has ment available for ISA. Therefore, control of the dis-
been based mainly on clinical observations and typical ease has been based on reducing exposure of farmed
changes in gross pathology, histopathology and haema- salmon to ISAV. The properties of ISAV that have been
tology, and by antigen detection tests on frozen tissue described so far have all been in concordance with
sections (Evensen et al. 1991, Falk & Dannevig 1995). properties of members of the Orthomyxoviridae (Falk
More recently, RT-PCR has been introduced (Rimstad et al. 1997, Mjaaland et al. 1997, Krossoy et al. 1999,
et al. 1999, Devold et al. 2000). Although the patho- Rimstad et al. 1999, 2001, Sandvik et al. 2000). How-
logical changes found in ISA-affected fish are typical ever, phylogenetically, ISAV is only distantly related to
in most cases, there are variations such as the pre- mammalian or avian orthomyxoviruses (Mjaaland et
dominant hepatic changes described in classical ISA al. 1997, Krossoy et al. 1999). The mammalian ortho-
(Evensen et al. 1991), while renal changes have been myxoviruses induce acute respiratory diseases. The
virus is present in the animal for a limited period of
*Corresponding author. E-mail: espen.rimstad@veths.no time, and there is no persistent carrier state of these

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experiments in which fish have been injected intra. Groups A to C. 24. spleen. 1. i. while in Groups D to I organs were aseptically fresh water at 8 to 10°C in 150 l tanks with a water flow collected from each fish and placed in separate tubes. 1. tissue samples were col- lected from 2 cohabitant fish twice weekly and the tis- sues were tested for the presence of ISAV by RT-PCR. 20. (a) In Groups A to C.9% NaCl were ‘transported’. infection spreads in a host are not known. cedure for ISAV. gills. a total of 0.3 ml diluted cell culture highly sensitive detection method as it can be expected supernatant from ISAV-infected SHK-cells containing that the viral load in such fish would be low. DNase treatment prior to RT reaction. fish were labelled and injected with ISAV as in Trial I ural route of infection is not well described. used in the experimental infections. However these studies have been based on quently collected from 5 fish from each tank at Days 5. 1995. 1995). 1997). At Day 0. the infec. However. 5 fish from each Screenings and surveys of ISAV infection in salmon tank were labelled by fin cutting and given an intra- with a clinically healthy appearance would require a peritoneal injection with 0. However. 45. 30. and present diagnostic procedures may 72 h after addition of the virus. to the standard organs.5 × after the introduction of the virus to a farm. Heart. 49. mid-kidney ratory. On Ice 24 h Room temp 24 h Frozen at –20°C On Ice 24 h Room temp 24 h Frozen at –20°C ported’ Then –20°C Then –20°C On ice for 24 h. Tissue samples were collected from 5 fish mary target organs of ISAV and the rate with which the prior to adding the ISAV-injected fish and at 24. there are indications during sampling. Trial I at Day 0. RNA extraction methods. then –20°C Then –20°C Then –20°C On ice for 24 h. The Glesvaer strain of ISAV was that ISAV can cause long-term infection. 48 and ably be low. The initial phase of a primary Trial II: Thirty fish were kept in 1 tank and 5 of these ISAV infection in Atlantic salmon resulting from a nat. at Day 0. 2001 viruses in their hosts. an RT-PCR procedure was optimised Trial IV: Seventy-five fish were kept in 1 tank and 5 and used to monitor the early spread of ISAV within its of these fish were labelled and injected with ISAV as in host after cohabitant infection. and Standard RT-PCR. liver. samples were collected from the intestine in addition To address the problem of a sensitive diagnostic pro. 56 and 70. From Day 14. then –20°C modes of transporta- tion from the field to the diagnostic labo- b Organs. and also in Trial I: A total of 250 fish with 125 fish in each of 2 experimental settings (Nylund & Jakobsen 1995).176 Dis Aquat Org 47: 175–181. amounts of RNA added to Special care was taken to avoid cross-contamination the RT reaction. 40. Atlantic salmon presmolts and processed to simulate different modes of trans- with an average weight of approximately 40 g were portation from the field to the diagnostic laboratory. During the experiments. 10. The following different steps in tissue from the heart. Tissue samples were col- There have been some studies of the early patho. 48. 103 TCID50 of ISAV was added directly to the water at tious dose that the individual fish would be exposed to Day 0. Groups of 5 fish were sampled Group A Group B Group C and processed to On ice 24 h Room temp 24 h Frozen at –20°C simulate different Then –20°C Then –20°C On ice for 24 h. all tissues from the 2 sampled fish were RT-PCR positive. tissue not be sufficiently sensitive to detect these low levels. the fish were kept in bags. then –20°C . and. soon Trial III: Thirty fish were kept in 1 tank and 1. except for Trial IV. spleen and mid. In nated from a hatchery where ISA had never been diag. The pri. MATERIALS AND METHODS On Day 29. 15. 35. each tank prior to adding ISAV and at 2. 6. tanks were used in this trial. Groups of 5 fish were then collected Experimental infections. of 1 l min–1. Group D Group E Group F Group G Group H Group I organs were ‘trans. gills. whole fish were placed in separate nosed. observations in farms with infected fish. (b) in Groups D to I. Speilberg et adding the ISAV-injected fish and tissues were subse- al. lected from 5 fish from each tank at Day 0 prior to logical changes in ISA (Falk et al. Tissue samples were collected from 5 fish from and the viral load in the infected fish would presum.e. peritoneally with ISAV. the RT-PCR procedure were optimised before a stan- kidney from each fish was placed in separate tubes dard method was established: homogenisation of tissue. based on used in all experiments (Mjaaland et al. the initial phase of a natural ISAV infection. The fish origi. in 72 and 96 h after addition. immediately frozen at –70°C. The collection protocol is displayed in Fig. liver. 25. whole fish Sampling without additive Addition of 0. a Whole Fish Fig.3 × 103 TCID50. In this trial. All samples were aseptically removed.

To verify results. The other 1-tube method had an end-point the ISAV genomic segment encoding hemagglutinin dilution at 5 × 10– 5 (2a). introduced in the RT-step. To avoid non-specific re- actions in the cDNA synthesis. containing cell culture fluid was then diluted 10-fold and Gibco). length of giving consistent positive results at 1 × 10– 6 dilution product: 155 bp). Lane 2: 5 × 10– 4. Lane 7: 1 × 10– 6. 1997. 2. Random hexamers (pd [N]6 Amersham Pharmacia) (3) When the different RT-PCR methods using the were used as primers for the cDNA synthesis and ISAV. Quantification of the virus was performed by seeding SHK-1 cultures with 10- fold dilutions of the cell culture fluid. optimisation of the method. Uppsala. Cloning and sequencing were performed to confirm the ISAV specificity of the PCR product generated using primers binding to the hemagglutinin encoding segment primers. mide. Ready-To-Go® specific primer pair-targeting genomic segment 8 was used RT-PCR beads were found to be the most sensitive.: Detection of ISAV 177 MgCl2 concentration. Sensitivity of RT-PCR. PCR products were resolved by electrophoresis on a 3% NuSieve 3:1 agarose (FMC Bioproducts. From this suspension. 2 µg RNA was added and incubated at 42°C for a M 1 2 3 4 5 6 7 30 min for the cDNA synthesis. Optimisation of RT-PCR detection of ISAV infection nized in DEPC-treated phosphate buffered saline (PBS) (10 × volume of tissue sample weight) in sterile The standard RT-PCR procedure was a result of an plastic bags. respectively . length of product: 194 bp) (Rimstad et al. cycling conditions of time and RESULTS temperature. was used as positive control. 2-tube RT-PCRs versus 1-tube RT-PCRs. less sensitive and showed variable end-point dilutions. From each sample. a tissue sample from an ISAV negative fish that went through all steps in the procedure was used as a negative control while RNA from ISAV- infected SHK-1 cell culture. The virus. (a) Titan™ One Tube RT-PCR System. RT-PCR products were elec- 3H6F8 and secondary anti-mouse antibodies for im. The following comments 400 µl were mixed with 800 µl TRIzol® and RNA can be made: extracted according to the manufacturer’s instructions. (b) Ready-To-Go® negative control fish. incubating at 15°C Fig. 260 nm (OD260) and the OD260 /OD280 ratio. Lane M: molecular size marker (123 basepair ladder. ME) and visualized under UV light after ethidium bromide stain- ing. Lane 4: 5 × 10– 5. Lane 6: 5 × 10– 6. Lane 3: 1 × 10– 4. using a roller. Cycling conditions were 94°C for 5 min followed by 35 cycles of 94°C for 30 s. 2 µg RNA RT-PCR Beads. 2001). Lane 1: 1 × 10– 3. respectively. in Lanes 1 to 7 (a). (2) Nonspecific DNA fragments were observed with One-step RT-PCR was carried out using Ready-To-Go® concentrations other than 1. For each run. trophoresed in agarose gel and stained with ethidium bro- munofluorescent staining (Falk et al. 1997). with the tubes kept on ice. After RNA extraction. and thus eventual contamination with genomic were estimated by measuring the optical density at DNA was not considered to cause problems. Lane 5: each dilution was mixed with a tissue suspension from a 1 × 10– 5. while the 2-tube method was was used (forward primer: ACTTGGGAACCAATGAC.5 mM MgCl2. Sweden). To measure the sensitivity of the RT-PCR. TGC. the ISAV specific primers were added after the cDNA synthesis had terminated. 1998). reverse primer: CTTCACCGAAAAACCGGTAA. Rockland. Lanes 8 to 14: same dilutions of samples as from each tissue suspension was tested by RT-PCR. optimised conditions were compared. (1) Treatment of the extracted total RNA with RNase- The RNA was dissolved in 20 to 200 µl DEPC-treated free DNase did not increase the sensitivity of the RT- water and the concentration and purity of the RNA PCR. a primer pair targeting (Fig. Comparison of 2 different 1-tube RT-PCR procedures for 1 wk and then subsequently staining with the Mab after end-point dilution of target. 55°C for 15 s and 72°C for 30 s and a final extension at 72°C for 7 min. which subse- RT-PCR Beads (Amersham Pharmacia. 2b). as has been done previously for the segment b M 8 9 10 11 12 13 14 8 product (Mjaaland et al. Tissue samples were thawed on ice and homoge. for the PCR amplification (Mjaaland et al. a cell culture harvest containing 4 × 105 TCID50 ml–1 ISAV was used. quently was chosen as the standard concentration. Mikalsen et al.

however. Hearts and gills kidney and intestine in a second fish and in the heart of were the only RT-PCR positive organs at the first sam- pling. Subsequently.) and same organs were tested as in trial I. The number in 10 each organ column represents how many of the 10 samples 0 were RT-PCR positive. The fish that were not collected of 1 fish. The co. (b) samples from the heart . 2001 Sensitivity of RT-PCR The number of organs that tested ISAV positive by RT-PCR peaked at Day 15 when 94% of the samples The standard RT-PCR had an estimated detection were positive. having a samples increased until all samples were positive at limit between 1 and 10 TCID50. positive. only 5 fish were sampled various times during the experiment: (a) samples from all organs. This estimate was organ that produced negative samples (3/10 liver sam- based on end-point dilution trials of cell culture fluid ples were negative) (Fig. 3b shows results for the heart). test whether ISAV could be detected in the fish before In infection Trial I. for tissue sampling were all dead by Day 87. No significant In Trial III.1 TCID50. 3a). At this sampling. At 24 h. The ‘Day’ column represents the days elapsed after ISAV-injected fish were added to the tank. from some or all fish. 60 50 40 30 Table 1.i. the percentage of positive The 2-tube RT-PCR system was less sensitive. positive RT-PCR was found.178 Dis Aquat Org 47: 175–181.01 and 0. RT-PCR results from organs of cohabitating fish in 20 Trial I. habitating fish started to die at Day 33. All tissue samples collected from fish prior to the In Trial II. the fish that were injected with Day 5. 3. For difference in the time distribution of RT-PCR results or the tissue samples collected 2 h after addition of ISAV. 3 and 4 to addition of ISAV to the tanks were negative in RT-PCR. and mid-kidney samples from one fish. The ISAV started to die at Day 24 post-injection (p. 2. mid- ing fish in Trial I are shown in Table 1. The viral load then de- containing ISAV in which each dilution was mixed creased until Day 25 when 30% of all samples were with a tissue suspension from a negative control fish. RT-PCR positive results were obtained in the gill. which was the first sampling day in Trial I. heart PCR results for the different organs from the cohabitat. Day 56 and the level remained at 100% until the end of the experiment. in 100 Percent positive by RT-PCR 90 a all of the samplings. while no samples were positive at 6 h. clinical appearance or mortality of ISA was observed RT-PCR positive results were found in the gill sample between the 2 tanks. ISAV was added directly to the water. 80 and mid-kidney samples were RT-PCR negative in all 70 of the 10 fish sampled on Days 5. the liver was the only limit between 0. The RT. This 2-phase shape of the curve was also typical when RT-PCR results for each organ were Experimental infections displayed separately (Fig. Percentage of organs with positive RT-PCR results at *At Day 70. The ‘Individuals’ column displays the 0 5 10 15 20 25 30 35 40 45 49 56 70 total number of fish that produced positive RT-PCR results 100 Percent positive by RT-PCR 90 b Day Heart Gill Liver Spleen Kidney Individuals 80 5 3 5 0 0 0 5 70 10 5 0 4 5 100 100 60 15 100 100 7 100 100 100 50 20 4 0 100 100 0 100 25 5 8 0 2 0 9 40 30 2 100 2 2 2 100 30 35 4 2 7 9 3 9 20 40 7 5 6 6 7 8 45 9 5 6 6 100 100 10 49 9 7 8 9 9 9 0 56 100 100 100 9 100 100 0 5 10 15 20 25 30 35 40 45 49 56 70 70* 5 5 5 5 5 5 Days Sum 730 670 650 730 660 10500 Fig. The heart was also the only organ that yielded positive RT-PCR. 20 and 25. The liver samples were RT-PCR negative in all of the 10 fish sampled on Days 5 and 25. in heart. at trial Day 5. no all 10 fish died before cohabitating fish died. fish were collected at Days 1.

of transportation of samples from the fish farm to the Trial IV was conducted to simulate the transport of diagnostic laboratory. alter- tivity would be even better when using RT-PCR in field natively the use of RNase inhibiting transport media cases for which the ISAV strain was not cell culture could be used. and perhaps the relative sensi. the cumulative number of positives. cent antibody test was not possible as no estimated The present RT-PCR procedure was found to be detection limits for that test. In our study. only the heart sample of 1 fish was Trial IV was performed to simulate different modes positive and at 72 h no samples were positive. was chosen because a highly sensi. but the sensitivity of the method would have studied. and performing single previously (Mjaaland et al. Devold et al. standard RT-PCR was estimated to performance of the RT-PCR. 1998). the situation was rather different as extraction. The main use of these RT. and the risk of contamination was kept at a low tion. from each gill and intestine was found to be positive. At 48 h. Both gills and/or oral entry are possible. however. In Trial I. 1997. No organ was thus ISAV and the sensitivity of the test has not been crucial found to be better suited than others as a target for RT- (Rimstad et al. in which 0. After decreased accordingly.1 TCID50. It was thus tive RT-PCR. one RT-PCR positive gill detects ISAV RNA. and at 24 h one sample the infectivity of the virus. graded during regular transportation of whole fish. and is thus not recom- be 0. However. was in the range 57 to 63% of total breaks in which the samples tested have a high load of samples tested for each organ. A comparison of the sensitivity of the fluores. In the present procedure a relatively short stage of infection when most other organs yielded amplicon. If the size of the fish prohibits the transport of than virus isolation. observed when 0. B. Several RT-PCR level by minimising the manual handling by running procedures for detection of ISAV have been described RT and PCR in the same tube. samples should probably be treated like Group E. The optimisation of the proce. and from Groups B and E positive possible to compare the effect of different handling RT-PCRs were achieved for most tissue samples. This indicates that the ISAV transport medium for organ samples. the samples in Groups D and F were kept on ice DISCUSSION for 24 h during the process. any RT-PCR only addition of ISAV to the water. For procedures on the RT-PCR performance prior to RNA Groups D and F. the potential toxicity of these adapted. This finding demonstrated the importance of test- dure did affect the sensitivity as illustrated by the ing several organs for the presence of ISAV in regular 100-fold difference in the detection limit between the RT-PCR diagnostic procedures. All monitored prior to sample collection to ensure the fish from Groups A. 1999). but gave mostly positive RT- PCR. This difference indicates that the combination of dissection of samples and stor- Both the study of the pathogenesis of ISAV infection age at 0 to 4°C for 24 h probably activated proteolytic and the detection of ISAV infection in salmon require activity and RNA degradation.01 to 0. 2000. For the process of whole fish did not strongly influence the Groups G to I. Mikalsen et al. when used for ISAV infec. The results showed that a normal transport only a few positive RT-PCRs were observed. are available (Falk et al. This procedure had a negative effect on the for the optimised. . However. In these groups some samples also pro. the RT-PCR RNA in tissue is rather stable and not severely de- results in most samples were seen as smearing in addi. tive method was desirable. negative results.9% NaCl was added as result of the RT-PCR. and not nested PCR. C and E yielded at least 1 posi. samples were negative. the detection limit medium. presence of ISAV at the time of sampling. In tion to the band representing the ISAV specific Groups D and F. robust. A similar effect was robust methods and thus benefit from the use of a sen. Griffiths & Melville 2000). PCR. a laboratory-adapted whole fish to the diagnostic laboratory. However. A procedure producing a The standard RT-PCR procedure enabled us to con- longer amplicon spanning most of a genomic segment duct experiments in which the route of entry of ISAV could have been more informative as to the viability of and the initial spread of ISAV in the fish could be the virus. and does not give information on sample was found after 2 h. most sam- duced DNA fragments with low density and some ples were RT-PCR negative. for all PCRs has been in the confirmation of disease out. solutions may be problematic in the transport process. the heart standard. indicating the methods in which these reactions were run in separate importance of testing this organ especially in the early tubes. optimised RT-PCR in which the RT and PCR was the only organ that gave positive RT-PCR results were run in the same tube and the detection limit of from one or more fish in all samplings. In the present study. to the simulated transportation. 155 bp. organs tested. in which the organs were also dissected prior to the simulated transportation.: Detection of ISAV 179 a third fish. The ISAV infection status was samples from the field to the diagnostic laboratory.9% NaCl was added as transport sitive RT-PCR. Compared with Group E. for which organs were dissected prior sequence. then tissue virus strain was used. implying a higher sensitivity mended.

ments. that fish during the first days after infection is very low. Aspehaug V. The rapid spread and replication are made Devold M. the infectious dose to which the fish were response. combined with the observed 100% mortality ples were found.) show histo- of the viral load. viral load shortly after infection as observed in Trial III. positive RT-PCR results were obtained in heart tion for the fish population in the tanks. limited number of tissues tested at each sampling. it has been shown that was not monitored. both between the organs. and remained at 100% RT-PCR posi. Dannevig BH (1995) Spleen tive samples after Day 56. indicating that the virus load of the of fish not collected during the experiment. Mx proteins with exposed in the tanks may have been very high and selective antiviral activity. The steady increase in 48 and 72 h. in our experiments the fish did not clear the virus. which is possibly PCR results before Day 5 after cohabitation with ISAV. The gills 49. RT-PCR for diagnosis of infectious salmon anaemia virus endothelial cells (Hovland et al. Dis Aquat Org 40:9–18 Evensen O. there was a marked increase in the total 118549/121 from the Norwegian Research Council and from number of RT-PCR positive organs. The ISAV some fish occurred at Day 25 of the experiment or is a negative-stranded RNA virus. Rimstad et al. which was probably due to the these infection routes were possible. the buildup of a high infectious dose in the tank tively. Thorud KE. 40 and the heart was the only other positive organ. Olsen YA (1991) A morphological mum viral load of 30% RT-PCR positive samples was study of the gross and light microscopic lesions of infec- found at Day 25. Atlantic salmon. a consequence of the host’s immune response. However. the titre of ISAV in the tank water However. Krossoy B. infection. thus the cause of the observed lack of protection. indicating that the fish had initially cleared ISAV. Acknowledgements. and that maximum expression in liver is seen that the gills are the major route of entry of ISAV in 2 d post stimulation (Robertsen et al. This phase of early replication is followed by a exposure to ISAV. This drop was possibly due to both tious anaemia in Atlantic salmon (Salmo salar). when a et al. viral load. Res Vet innate and. The early peak indicates that after the initial drop in viral load in a pri- mary infection the virus spreads and replicates rapidly LITERATURE CITED in the fish.e. Trial III unless the amount of ISAV shed from infected and a second increase in viral replication followed. temporary decrease in the viral load. while In the tissue samples collected on Days 25. in Atlantic salmon. However. peaked at Day 15 with 94% positive. spe. this speculation suggests that the innate im- mune response affetcs ISAV replication in situ. RT-PCR. Sci 51:215–222 cific immune responses inhibiting the replication of the Falk K. Again. i. Dannevig BH (1995) Demonstration of infectious virus. After entry. but the nature of the before. The stranded RNA viruses. 1997). A decrease in (ISAV) in carrier sea trout Salmo trutta after experimental the viral load followed the peak at Day 15 and a mini. How- injected fish could appear to contradict the results of ever. but with some variations replicating virus or the passive transport of virus. however. In mammals and birds. At Day 5 in Trial I. final level of 100% positive samples occurred. which may be low virus shedding is consistent with the decrease in attributed to reinfection from cohabitant fish. 1 (heart) and no other positive organ sam. In the samplings at ably the cause of reinfection. the viral load displays an The fish in Trials I and II were infected by cohabita. individual fish were RT-PCR negative for all organs are therefore important as the initial route of entry for sampled. 1979). Nylund A (2000) Use of possible by the large presence of target cells. our findings support the suggestion for Mx. The technical assistance of Lise Berven is greatly appreciated. In the experi- and mid-kidney samples of 3/5 and 2/5 fish. 35. especially against negative. 5/10 gill samples were RT-PCR positive. producing a 2-phase curve and kidney of Atlantic salmon (Salmo salar L. respec. salmon anaemia (ISA) viral antigens in cell cultures and tissue sections. rather low. 1999). The virus load increased again after the mini. After Day 5. This may be caused by the innate immune However. The continued to the terminal stage of ISA. as has been indicated in earlier findings (Totland the infection. This 2-phase curve was also observed chemical changes early in the course of experimentally . are important in the restriction initial protective response that cleared the infection in of initial viral replication (Haller et al. Vet Res 26:499–504 mum at Day 25. the lack of positive RT. Falk K. 1996. when the viral load in the tank most likely was response restricting the ISAV replication is unknown. 2001 Since the RT-PCR cannot distinguish between an active for the individual organs. Press CM. Landsverk T. 1994). double-stranded RNA induces transcription of mRNA In conclusion. This result indicates that viraemic spread of the due to shedding of virus from infected fish was prob- virus occurs rapidly after infection. the increase GenoMar asa. indicated fish had decreased below the detection level of the that a protective immune response was not induced. initial decline before rapid replication and dispersion tion and thus it is not possible to pinpoint the time of occurs. The results from later samplings. considering the time after exposure. indicate Twenty-four hours after the addition of virus to the that reinfection was an important cause of ISAV infec- water. This study was supported by grant no.180 Dis Aquat Org 47: 175–181.

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