Atlantic salmon & viral research

Ann-Inger Sommer

ABSTRACT: The main topics of the research on viral diseases in Atlantic salmon (Salmo salar) at Fiskeriforskning today are: Studies of viral characteristics associated with virulence, antiviral immune defence in salmon and interactions between virus and host.

Infectious salmon anaemia virus (ISAV)

We have participated in the ISA research since it was recognised as a problem in Norway, but finished our last ISAV project activity 4 - 5 years ago. It took several years of research before the virus was isolated, but ISA was studied in A. salmon naturally infected or challenged by injection and cohabitation. Cells permissive for ISAV infection and production were strongly needed, and cultures of immune cells (macrophages) and several cell lines were investigated. Transmission electron microscopy studies of ISAV infected cells were performed and diagnostic methods like indirect fluorescent antibody technique (IFAT) on tissue imprint, haemadsorption centre assay (HACA), and RT-PCR have been established. (Sommer & Mennen, 1996, 1997)

Infectious pancreatic necrosis virus (IPNV)

There are several good reasons why IPNV is central in our viral research today: The economical loss due to IPN is large in the salmon farming industry, and outbreaks may occur both in A. salmon juveniles in fresh-water and in post-smolts after transferred to sea-water. An IPNV infection may persist without any signs of disease, but this carrier situation represents a risk of reactivation with new outbreaks in post-smolts after transfer to seawater. Vaccines are available that show high efficacy after experimental IPNV challenge. Still IPN is a serious disease problem in A. salmon and the protective effect in the field is uncertain.

Some of these issues have been studied in a recently finished project funded by Norwegian Research Council (project 141887/120):

“Virulence studies of Norwegian field isolates of IPNV”

K. Julin, S. Mennen & A-I. Sommer have been participating in this project at Fiskeriforskning, in co-operation with C. Koren, Fiskehelse Nord and K.E. Christie, Intervet Norbio AS.

Virulence and genomic variations of IPNV isolates involved in 19 IPN outbreaks of different mortality in the Northern region of Norway have been studied. The reported mortality varied from 1 – 45 %, although all post-smolts were vaccinated against IPN. Several factors may vary out in the field, which make the mortality rates alone unsuitable to predict the virulence of strains involved.

Specific amino acid (aa) ”patterns” associated with high, low or moderate virulence, located mainly in the variable region of viral protein VP2, was first identified in 3 reference strains A, B and N1. They were compared to each other after 2 and 10 passages in a CHSE-214 cell line, and to the mortality registered after challenge. The putative aa motifs were used for grouping of 17 different field isolates of IPNV according to virulence.

The IPNV field isolates were collected during 2001 from IPN vaccinated post-smolt in verified IPN outbreaks starting about 3 – 9 weeks after seawater transfer. Although all sampled fish came from different smolt producers, fish farms or locations, the variation in the genotype of IPNV strains involved in mortality was low. About 75 % of the isolates associated with IPN mortality shared the putative aa motif indicating high virulence, while none showed the low virulence motif (unpublished results).

In vivo bath-challenge experiments were performed on non-vaccinated smolt under equal conditions, with selected field isolates that have identical VP2 aa motif indicating high virulence, but different mortality in the field. In addition some other strains with motifs indicating moderate or low virulence were included. Correlation between accumulated mortality and aa motifs indicating different virulence were studied: Field strains of similar genotype associated with IPN outbreaks of varying mortality in vaccinated post-smolt, showed no significant differences in mortality after bath challenge of non-vaccinated post-smolt under equal conditions. This means that other factors than virulence significantly affects the outcome of an IPNV infection. No strains with low virulence aa motif were detected, so the described characteristics of the genome associated with virulence seem to be required for an outbreak (unpublished results).

These well characterised IPNV strains of different virulence are now valuable tools for studies of host – virus interactions (carriers, reactivation and antiviral defence mechanisms).

Persistent IPNV infection and antiviral immune defence

Several projects at FF have been dealing with the important IPNV carrier situation: Previously we have shown that IPNV infect head kidney macrophages from A. salmon and are carried in these cells in vivo (Johansen & Sommer 1995 a). Effects an IPNV carrier condition may have on health, physiology and immunological responses of the fish, and on other diseases have also been studied (Eggset et al 1997, Damsgård et al 1998, Johansen & Sommer 1995 b, 2001). There is a balance between the antiviral mechanisms of the leucocytes and the carrier virus that might be affected by stress. Effects of different types of stress on development and reactivation of IPN, especially environmental factors due to intensive production regimes, have been studied. This topic was presented in another lecture at the workshop (by Johansen & Toften).

Antiviral immune defence are studied in a strategic research program (143286/140): “Virological investigations on emerging disease conditions in domestic animals and fish” funded by the Norwegian Research Council, 2001-2005. It consists of 5 different projects co-ordinated by the Veterinary Institute, Oslo. The project at Fiskeriforskning is:

“Cellular defence mechanisms against viral infections in Atlantic salmon”

L-H. Johansen, A. Johansen, K. Julin, S. Mennen, E. Sandaker, K. Steiro & A-I. Sommer have been/are participating in this project at Fiskeriforskning, in co-operation with J.B. Jørgensen at Norwegian College of Fisheries. Different interactions between virus and leucocytes, where IPNV is serving as a suitable test organism, are studied. For instance are antiviral defence mechanisms and virulence investigated, to see whether IPNV of high or low virulence differ in ability to establish an infection, persist and be reactivated. Virus infection and production in leucocytes/macrophages are also studied in vitro, to reveal some of the viral strategies to omit antiviral defence and possible mechanisms behind this.

Activation of innate antiviral defence and prophylactic treatment

Different types of immune stimulants are used to study mechanisms involved in preventing viral infections both in vivo and in vitro. Antiviral effects of Poly I:C and CpGs have been studied (Nygaard et al. 2000, Jørgensen et al. 2001, Jensen et al. 2002, Jørgensen et al. 2003, Johansen et al. 2004. The CpG results were presented in another lecture at the workshop (by Jørgensen).

In a present experimental trial with IPNV carriers we are investigating if it is possible to inhibit reactivation, or even ”cure” a persistent infection, by immune stimulation. A real time RT-PCR that can detect the usually very low amount of IPNV found in a carrier situation is now established.

Methods to measure effects on antiviral mechanisms.

There is a need for methods to measure effects of different prophylactic treatment, physiological or environmental influences, farming conditions etc. For several years the challenge model of IPN developed at Fiskeriforskning has been a valuable tool for in vivo testing of prophylactic treatments. It has proven very useful for several research projects and for the vaccine industry.

We have previously established an anti-viral assay (AVA), which is a standard cell protection assay measuring the inhibition of IPNV-induced cytolysis by interferon-like cytokine (ILC) activity (Jørgensen et al 2001). More sensitive is the Mx 1 promoter reporter gene assay for quantification of A. salmon type-1 IFN. CHSE-214 cells are transient transfected with a rainbow trout Mx 1 promoter linked to a luciferase reporter (Johansen et al. 2004). Stable CHSE cell transfectants, with Mx 1 promoter and zeosin-resistance plasmids, have also been established and have been successfully passed through 50 cell splits. These cells have recently been tested and compared to the transient transfected cells and will be taken into use now, as well.

Publications from the presented projects

Damsgård, B., Mortensen, A. and Sommer, A.-I. (1998) Effects of infectious pancreatic necrosis virus (IPNV) on appetite and growth in Atlantic salmon, Salmo salar L. Aquaculture 163, 185-193.

Eggset, G., Mortensen, A., Johansen, L.-H. and Sommer, A.-I. (1997) Susceptibility to furunculosis, cold water vibriosis, and infectious pancreatic necrosis (IPN) in post-smolt Atlantic salmon (Salmo salar L.) as a function of smolt status by seawater transfer. Aquaculture 158, 179-191.

Jensen, I., Albuquerque, A., Sommer, A-I. & Robertsen, B. (2002) Effect of poly I.C on the expression of Mx proteins and resistance against infection by infectious salmon anaemia virus in Atlantic salmon. Fish & Shellfish Immunology 13, 311-326

Johansen, A., Collet, B., Sandaker, E. & Jørgensen, J.B. (2004) Quantification of Atlantic salmon type-1 interferon using an Mx1 promoter reporter gene assay. Fish & Shellfish Immunology 16, 2, 173-184

Johansen, L.-H. and Sommer, A.-I. (1995 a) Multiplication of infectious pancreatic necrosis virus (IPNV) in head kidney and blood leucocytes isolated from Atlantic salmon, Salmo salar L. Journal of Fish Diseases, 18, 147-156

Johansen, L.-H. and Sommer A.-I. (1995 b) In vitro studies of infectious pancreatic necrosis virus infections in leucocytes isolated from Atlantic salmon (Salmo salar L.) Aquaculture, 132, 91-95.

Johansen, L-H. & Sommer, A-I. (2001) Infectious pancreatic necrosis virus infection in Atlantic salmon Salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or Vibrio salmonicida. Diseases of Aquatic Organisms 47 (2), 109-117.

Jørgensen, J. B., Johansen, A., Stenersen, B. & Sommer, A.-I. (2001) CpG- oligodeoxynucleotides and plasmid DNA stimulate Atlantic salmon (Salmo salarL.) leucocytes to produce supernatants with antiviral activity. Developmental and Comparative Immunology 25, 313-321.

Jørgensen, J. B., Johansen L.-H., Steiro K & Johansen A. (2003). CpG DNA induces protective antiviral immune responses in Atlantic salmon (Salmo salar L.).J ournal of Virology 77, 21, 11471-11479

Nygaard, R., Husgárd, S., Sommer, A.-I., Leong, J. C. & Robertsen, B. (2000) Induction of Mx protein by interferon and double-stranded RNA in salmonid cells. Fish & Shellfish Immunology 10, 435-450.

Sommer, A.-I. & Mennen, S. (1996) Propagation of infectious salmon anaemia virus in Atlantic salmon (Salmo salar L.) head kidney macrophages. Journal of Fish Diseases, 19, 179-183.

Sommer, A.-I. & Mennen, S. (1997) Multiplication and haemadsorbing activity of infectious salmon anaemia virus in the established Atlantic salmon cell line. Journal of General Virology, 78, 1891-1895