WO2005121325A1

WO2005121325A1 - Heart and skeletal muscle inflammation (hsmi) virus

Info

Publication number: WO2005121325A1
Application number: PCT/NO2005/000197
Authority: WO
Inventors: Trygve Meum Eliassen; Inge Tom Solbakk
Original assignee: Pharmaq As
Priority date: 2004-06-11
Filing date: 2005-06-10
Publication date: 2005-12-22
Also published as: GB0625374D0; NO343278B1; CA2570050C; GB2429711B; NO20065765L; CA2570050A1; GB2429711A

Abstract

The present invention relates to an isolated Heart and Skeleton Muscle Inflammation Virus (HSMIV) derived from fish; a live, attenuated or inactivated HSMIV, or parts of it functional for vaccine purposes, a recombinant antigen and vaccine; as well as DNA, cell lines, vaccines, antibodies, methods and uses thereof. The invention may be utilized to prevent and diagnose Heart and Skeleton Muscle Inflammation (HSMI) in fish.

Description

HEART AND SKELETAL MUSCLE INFLAMMATION (HSMI ) VIRUS .

The present invention relates to an isolated Heart and Skeleton Muscle Inflammation Virus (HSMIV) derived from fish; a live, attenuated or inactivated HSMIV, or parts of it functional for vaccine purposes; as well as DNA of the virus, cell lines, immunogenic or recombinant antigens, vaccines, antibodies, methods and uses thereof. The invention may be utilized to prevent and diagnose Heart and Skeleton Muscle Inflammation (HSMI) in fish.

BACKGROUND OF THE INVENTION

Heart and Skeletal muscle inflammation (HSMI) was first discovered in salmon farms in Norway in 1999. Since 1999 there have been an increasing number of outbreaks and the problem seems to be spreading along the Norwegian cost. Currently the disease is considered to have great economical impact for the Norwegian salmon farming industry. However, HSMI is expected to cause disease and mortality also in any area where salmonids are produced, like South America, North America, Europe and Asia, exemplified by Chile, USA, Canada, United Kingdom, Spain, France, Germany, Japan, and China.

Affected fish often show reduced appetite prior to abnormal swimming behaviour and in some cases, sudden death. Usually, no external lesions are recognized. At autopsy, the heart often appears pale and somewhat loose. In some cases the pericardial sac is filled with blood. Histological examinations indicate that most fish in affected net cages show severe lesions, although they seemingly appear to be clinically healthy. The most consistent findings are severe and extensive inflammation in heart and skeletal muscle. Outbreaks have been registered at all seasons, but with most outbreaks 5 to 9 months after seawater transfer, when the size of the fish is between 0,3-1,0 kg. So far the disease has only been diagnosed in Atlantic salmon (Salmo salar). The mortality has varied from slightly enhanced to 20%. The disease was first observed in 1999 (Kjaerstad 2002, Kongtorp 2004b), and was described as myocarditis and myositis in Atlantic salmon by T. Taksdal in 1999. Kjaerstad (2002) later described the disease as heart skeleton muscle inflammation, named after the histological symptoms that are somewhat similar to Pancreatic Disease (PD). The fish do, however, not have any pancreatic problems and infected fish were negative to PD using PCR (Kjaerstad 2002).

Watanabe et al. (2003) reported that diseased fish were also negative for Infectious Pancreatic Necrosis Virus (IPNV), Infections Salmon Anaemia Virus (ISAV), Salmon Pancreas Disease Virus (SPDV) by cell culture and PCR, and that infected fish were negative to bacterial diseases. Histological analysis showed vacuolization and degeneration of cells in most tissues. Although tissue changes in heart and muscle were significant, Watanabe's observations contrast the normal finding that only heart muscle and red skeleton muscle are affected. Transmission electron microscopy (TEM) identified four virus-like particles in fish from two sites. One virus, 80 nm, was localized in erythrocytes and similar to erythrocytic inclusion body syndrome (EIBS). The second and third were viruses earlier associated with hemorrhagic smolt syndrome (HSS) in Norwegian smolt farms. The fourth virus was a naked virus 80 nm, with an electron dense core, often associated with degenerated cells. Wantabe et. al. (2002), speculated that the fourth non-enveloped virus could be the causative agent for HSMI.

Kongtorp (2003 and 2004) showed that the disease could be transferred from diseased fish to healthy fish in cohabiting challenge study. Homogenated non-filtered material from diseased fish induced symptoms of disease, both in injected fish and naϊve fish. The diagnosis was verified by histological examination. However, Kongtorp was not able to propagate virus in cell cultures and was consequently barred from drawing any definite conclusions.

The literature describes the disease in infected fish, and confirms that homogenated infected material induces symptoms in fish, and that diseased fish spread the disease to naϊve cohabitants. However, identification of the causative agent and propagation of the virus in cell lines have so far not been reported. The current invention discloses a virus that is about 80 nm in diameter and propagates in the GF-1 cell line. The virus is named Heart and Skeleton Muscle Virus (HSMIV) as virus-containing material from the GF-1 cell line induces typical HSMI symptoms in fish, more particularly, Atlantic salmon. Moreover, the histological changes are identical to those observed in HSMI affected fish. The fact that the virus only propagates in vitro in the GF-1 cell line is surprising, considering that said cell line is derived from Grouper.

DESCRIPTION OF THE FIGURES

Fig 1 Tissue homogenate, injected i.p.; heart (left), muscle (right). Infiltration with mononuclear (lymphocytic) inflammatory cells is evident. Prominent interstitial infiltration in muscle tissue.

Fig 2 Tissue homogenate, injected i.p.; heart (left), muscle (right). Marked Infiltration with mononuclear (lymphocytic) inflammatory cells is evident heart. Interstitial infiltration in muscle tissue.

Fig 3 Cell homogenate, i.m. injection; heart (left) and muscle (right). Note area of inflammatory changes in both sections (arrows).

Fig 4 Cell homogenate, i.p.; heart (left), muscle (right). Mononuclear cell infiltrations, comparable to the other groups.

Fig 5 PBS injected group (cohabitants); heart (left), muscle (right). A few foci of inflammatory cells seen in the heart compact muscle layer (arrows). One single focus of inflammatory cells also in the muscle sampl (arrow).

Fig 6 Electron microscopy of cell homogenate, i.m. injection; heart.

Fig 7 Same specimen as in figure 6, but at higher magnification.

Fig 8 GF-1 negative control cells.

Fig 9 GF-1 cells inoculated with cell homogenate, 14 days post infection.

Fig 10 Overview (Fig 10a) and detail (Fig 10b) showing HSMIV infected GF-1 cells. Intracellular green fluorescence provides evidence for virus infection. These green signals are localized to small vacuole-like structures, also observed by light microscopy. The nucleus is stained with propidium iodide yielding red fluorescence. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated fish virus, herein named Heart and Skeleton Muscle Virus (HSMIV), which causes Heart and Skeleton Muscle Inflammation (HSMI) in fish. The virus has a diameter of about 80 nm, as judged by electron microscopy (Fig. 6 and 7).

The virus is expected to affect all members of the sub directory Teleostei. This is demonstrated by the its ability to cause disease in Atlantic salmon (Salmo salar) representing the taxa salmoniformes, family salmonidae, and its ability to propagate cells from Grouper (Epinephelus coioides) representing taxa Perciformes, family Serranidae. Hence, the virus according to the invention causes HSMI in fish of the directory Teleostei; preferably, Euteleostei; more preferably, Procanthopterygii or Acanthopterygii; even more preferably, Salmoniformes or Perciformes: even more preferably, Salmonidae or Serranidae; more preferably, Salmo or Epinephelus sp; more preferably, Salmo salar or Epinephelus coioides. In a most preferably embodiment, the fish is Atlantic salmon (Salmo salar).

In a preferred embodiment of the current invention, the virus is the strain deposited at ECACC under deposit number 04050401 or strains with related genotypic and/or phenotypic characteristics.

Another aspect of the present invention is an attenuated or inactivated HSMIV. More particularly, an attenuated or inactivated version of HSMIV as deposited at ECACC under deposit number 04050401 or strains with related genotypic and/or phenotypic characteristics. Methods for attenuating or inactivating a live virus are known to a person skilled in the art.

Yet another aspect of the current invention is a cell line comprising the virus described above. Several cell lines, like e.g. CHSE 214, BF-2, CHH-1, and TO have been evaluated for propagation of HSMIV, at temperatures from 15°C. The HSMIV did not propagate in any of the mentioned cell lines. Grouper Fin cell line (GF-1), from Epinephelus coioides having ATCC accession number No. PTA-859, was found to be capable of virus production. Given HSMIV ability to propagate in Grouper cells it is reasonable to assume that the virus may have the abilities to propagate in other cell lines from groupers and other species in the taxa Perciformes. Hence, the cell line is preferably derived from Perciformes, more preferably from Serrandiae, more preferably from Epinephelus sp, and most preferably from Epinephelus coioides.

Another aspect of the invention is a vaccine comprising the virus of the invention or parts of the virus available for interaction with the immune system of the virus host. These particles, whole virus, or other parts or fragments thereof, can be used live, attenuated or inactivated to immunize fish against HSMI. Also the viral particles can be used for isolation of DNA and/or fragments thereof that can be used for DNA vaccines or recombinant vaccines to produce recombinant vaccines to immunize fish against HSMI.

Attenuation is established by serial passages of the virus in a culture of cells. For each step the viruses harvested from the previous culture step are inoculated to a medium containing a fresh culture. Culturing of the cells used is done according to methods known in the art.

For preparation of a live vaccine the seed virus, optionally attenuated as described above, can be grown on a cell culture, such as GF-1. The viruses thus grown may be harvested by collecting the tissue cell culture fluids and/or cells. The virus yield can be promoted by techniques that improve liberation of the infected particles from the grown substrate, e.g. sonication. The live vaccine may be prepared in the form of a suspension or may be lyophilized. In lyophilized HSMIV vaccine it is preferable to add one ore more stabilizers. Suitable stabilizers are for example carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, and glucose), proteins (such as albumin or casein) or degradation products thereof, protein containing agents (such as bovine serum or skimmed milk) and buffers (such as alkali metal phosphates). Optionally, one or more compounds having adjuvant activity may be added. Suitable adjuvants are for example aluminium hydroxide, phosphate or oxide, mineral oils, liposomes and saponines.

A vaccine according to the invention may alternatively comprise the HSMIV strain in inactivated form. Inactivated HSMIV are prepared from viruses which both replication and virulence have been abolished. In general this can be attained by chemical or by physical means. Chemical inactivation can be carried out by treatment of the viruses for example, but not limited to, with enzymes with formaldehyde, β-propiolactone, or ethyleneimine or a derivative thereof, with organic solvent (e.g. halogenated hydrocarbon) and/or detergent, e.g. Trition® or Tween®. Physiological inactivation can advantageously be carried out by subjecting the viruses to energy-rich radiation, such as UV light, gamma irradiation or X-rays. If necessary the inactivating agent can be neutralized; for example formaldehyde-inactivated preparations can be neutralized with thiosulphate. If required, the pH subsequently is returned to about pH 7. Generally, an adjuvant is also added to inactivated virus vaccines, and optionally one ore more emulsifiers, e.g. Tween® and Span®.

The vaccine according to the invention may be combined with other bacterial, viral and/or antigens to from multivalent vaccines. Antigenic material obtained from a bacterial source may be selected from, but is not limited to, live, attenuated or killed Aeromonas salmonicida, Vibrio salmonicida, V. anguillarum 01, V. anguillarum 02, V. ordalii, Moritella viscosa, Pisckirikettsia salmon is, Photohacterium damsela,

Flavobacterium psychrophilum, Flavobacterium columnaris and any combination thereof. Antigenic material obtained from a viral source includes, but is not limited to, glycoprotein (G) or nucleoprotein (N) of Viral Hemorrhagic Septicemia Virus (VHSV); G or N proteins of Infectious Hematopoietic Necrosis Virus (IHNV); VP1, VP2, VP3 or N structural proteins of Infectious Pancreatic Necrosis Virus (IPNV); G protein of Spring Viremia of carp (SVC); a membrane-associated protein, tegumin or capsid protein or glycoprotein of channel catfish virus (CCV). Other viruses include fish pancreatic disease virus (FPDV), Iridoviruses, Infectious Salmon Anaemia virus (ISAV).

Antigenic material obtained from a parasitic source includes, but is not limited to, one or more of the surface antigens of lchthyophthirius or any other parasitic organism known to infect or infest fin-fish.

Antigenic material obtained from a fungal source includes, but is not limited to, fungal pathogens known to infect or infest fin-fish, for example, Saprolegnia, Branchiomyces sanguinis, Branchiomyces demigrans and lcthyophonus hoferi.

The vaccine may also comprise adjuvants like, but not limited to, e.g. β-glucan, oil adjuvants, liposomes, etc. The vaccine herein described can be administrated by injection, dipping, bath or orally.

Another aspect of the current invention is an antibody selectively binding the HSMIV as described above. The antibody may also comprise a marker, e.g. a radiolabel, fluorescent tag, or an enzyme. Furthermore, the antibody may be polyclonal or monoclonal, or any fragment thereof.

A further aspect of the invention is a diagnostic kit comprising the disclosed antibody or the virus, whole or parts, described above.

Another aspect of the invention is a method of isolating HSMIV comprising: (a) identifying a fish suffering from HSMI, (b) producing a homogenate comprising the HSMI virus, (c) inoculating a suitable cell strain, (d) isolation of virus particles. The homogenate is preferably filtered.

Yet another method of the current invention is a method of producing HSMIV comprising the steps of culturing the cell line as described above and isolating the virus particles.

The described vaccine can be used to prevent HSMI, or related diseases, in fish. Hence, the invention comprises a virus as described above use as a therapeutic. Furthermore, the current invention also relates to a method of preventing HSMI comprising administering the vaccine described above to a fish in need thereof. Another aspect of the invention is use of the virus as herein described for the manufacture of a medicament for preventive treatment of Heart and Skeleton Muscle Inflammation Virus in a fish in need thereof. The particular type fish may be as described above.

The virus of the current invention is isolated from Norwegian fish specimens. EXAMPLES

By way of example the following experiments demonstrate, inter alia, that homogenate from diseased fish has the ability to propagate GF-1 cell line, and that the homogenate from the infected GF-1 cell line has the ability to infect Atlantic salmon with HSMI. The following examples are meant to illustrate how to make and use the invention. They are not intended to limit the scope of the invention in any manner or to any degree.

Example 1

Isolation and propagation of HSMI virus

Isolation

A total of 10 hearts isolated from Atlantic salmon diagnosed for HSMI was homogenised using a porcelain mortar and quarts sand. The homogenate was diluted 1:3 in EMEM with gentamicin. The homogenate was centrifuged with 2000 x g for 10 minutes at 4 degrees C and 0.45 urn filtrated prior to inoculation of cells. The remaining homogenate was dispatched in cryo vials and frozen at -80 degrees C.

Virus propagation

Inoculation of GF-1 cell cultures (1^st passage HSMI virus)

GF-1 cell cultures are split to a cell density of approximately 2-5 x 10⁴ cells/cm², and inoculated with 5 μl HSMI homogenate per ml growth medium (1:200 dilution) within two hours after splitting. The infected GF-1 cell cultures are incubated 12-18 days at 15 degrees C. The first signs of infection appear normally within a week as big vacuoles in the cytoplasm of the cells (Figure 8 & 9). The number of infected cells, and the size and number of vacuoles increase until the end of the incubation period when almost 100% of the cells are affected.

The infected GF-1 cell cultures are harvested by freeze-thawing three times before the supernatant is clarified by filtration with 0.45 μm filter and dispatched in cryo vials and stored at -80 degree C. 2^nd passage HSMI virus

The infection is performed like the 1^st passage, but the inoculation volume is five fold the volume in the 1^st passage (25 μl HSMI supernatant per ml growth medium). The development of the infection is normally delayed with 3-5 days compared with the 1^st passage.

Example 2

Challenge of Atlantic salmon with HSMI In order to examine if the cultivated viral agent could be introduce the symptoms and disease of HSMI Atlantic salmon, Atlantic salmon (50g) were injected with infected GF- 1 cell homogenate prepared from 1 passage of the viral agent, prepared as described in Example 1. The cell homogenate was injected intrapertoneally (ip) or intramuscularly (im) in doses of 0.1 ml/fish.

Additionally heart tissue homogenate collected from fish diagnosed with HSMI, was injected intrapertoneally (ip) in doses of 0.2 ml per fish, as positive control, and one group was injected phosphate buffered saline (PBS) in doses of 0.1 ml per fish. All groups were cohabiting in the same tank with seawater. The temperature during the study was 12°C. 8 weeks post vaccination, heart, kidney, liver, spleen and muscle were sampled and fixed in buffered formaline.and glutaraldehyde.

Histopathology (see Example 3) was used for confirming the presence of the disease.

Results

No abnormal behavior and mortality were observed during the trial.

Histological changes corresponding to HSMI were observed in heart tissue of all groups injected infected material.

The histological and the electronmicroscopic examinations confirmed that the virus produced in GF-1 cell line, was HSMIV. Example 3 Histology

Laboratory examination Four different groups of fish were injected with 3 different preparations of infective material and 1 group with PBS (see Table below for more details). The infected material was either tissue homogenate from a diseased fish (heart) that was injected intraperitoneally (i.p.). A cell homogenate originating from a GF-1 cell culture infected with organ material from a diseased fish was split in two and injected either i.p. or intramuscularly (i.m.). All fish were kept in the same tank, also the PBS group. The PBS group would in principle serve as an indication of whether the virus could be transferred horizontally in water.

Organ samples from heart, skeletal muscle (red muscle), spleen, liver, kidney and pyloric caeca with pancreas were collected at 8 weeks post infection and were received submerged in 10% phosphate-buffered formalin. The samples were subsequently trimmed and processed according to standard procedures for paraffin embedment. Sections were laid on standard slides for routine staining with haematoxylin and eosin.

At submission, information was provided as to the origin of the different samples. It was indicated that the different groups were treated differently but no details were revealed. The assessment was thus carried out without knowledge of the type of treatment/ challenge of the different groups.

The histological changes were observed in a Zeiss Axioplan equipped with a digital camera for recording of images. The assessment of histological changes was carried out according to standard procedures and the changes observed will be presented in a descriptive manner.

Results

The findings are summarised in Table 1 below.

The most prominent changes were observed in heart and red skeletal muscle (along the side line of the fish). The changes are described in more detail below, but were characterised by an infiltration of mononuclear inflammatory cells in the stratum compactum of the heart and the red skeletal muscle. The inflammatory foci were made up as a mixture of lymphocytes and lymphocyte-like cells and macrophages. The changes were focal and multifocal in distribution in the heart and to some extent also in the red muscle specimens. The inflammatory changes in heart and skeletal muscle were frequently found in conjunction with degenerated and necrotic muscle cells. There were no obvious pathological changes in any of the other organs.

Table 1

Skeletal Pyloric Inocolum Marking Heart Liver Spleen Kidney muscle caeca

Tissue Vkj+øh ++ ++ homogenate i.p.

Cell homogenate Vkj+FF +++ +++ i.m.

Cell homogenate Vkj+FF ++ ++ i.m.

Cell homogenate Vkj+Hkj ++ i.p.

Cell homogenate Vkj+Hkj ++ ++ i.p.

PBS Vkj +

PBS Vkj

Detailed description of histomorphological findings

There was no distinct difference between the group given tissue homogenate i.p. and the two groups injected with cell homogenate either i.p. or i.m., as can be seen from Table 1. In one of the cell homogenate groups, there was a tendency of one of the groups having more pronounced inflammatory changes, both in the heart and the muscle tissue.

The most prominent finding in the submitted samples was an infiltration of inflammatory cells in stratum compactum of the heart (Figs 1-3). This was observed in all groups injected with infective material, although with some variation between groups. Frequently, the changes in the compact layer of the heart were accompanied by an intense infiltration of inflammatory cells in the epicard (Fig 3). In association with the inflammatory changes, there was also a prominent vacuolation of cells in the compact layer of the myocardium (Fig 2).

There is also indication of degenerative changes in the myocardial cells in some areas, for the main part associated with areas of heavy infiltration of inflammatory cells (Figs 3, 4).

In the PBS group, the histomorphological changes were more moderate in distribution and the number of inflammatory cells in the majority of the sections examined (Fig. 5). This was seen as a reduction of the area involved in inflammatory changes in the compact layer and the number of inflammatory cells was less than for the other groups (Fig 5). The infiltration was as small foci.

In one of the fish from the group given cell homogenate i.p., no changes were found in the skeletal muscle sample.

Discussion and Conclusion

At higher magnification it is well revealed that the cells making up the inflammatory changes are dominated by mononuclear cells, mainly lymphocytes and lymphocyte-like cells (see figures below). The histomorphological changes seen in the parenchymal cells of the heart in Figure 2, opens up for the possibility of other cells than the myocytes being involved in the changes observed. Relatively large cells with a rounded nucleus show small vacuoles of the cytoplasm (sarcoplasm). There are no striations found in these cells by light microscopy, but this is not excluding these cells as belonging or originating from the same cells as the myocytes.

The changes described in the skeletal muscle samples are typical of an inflammatory response to a muscle cell expressing a foreign antigen (non-self). The same type of reaction is seen in DNA vaccinated fish were viral proteins are expressed through transfection of muscle using plasmids encoding viral proteins (for example G-protein of VHS virus). The lesser degree of infiltration of inflammatory cells seen in the PBS group is most likely caused by the fact that these fish were infected (through the water) at a later stage compared to the fish injected with infective material. It is reasonable to assume that fish that become infected after injection will start shedding the virus to the water after some time (weeks). The characteristic changes but of less intensity seen at 8 weeks in the PBS group, is a strong indication of this being a contagious disease. Also the fact that the changes can be reproduced not only with material from infected fish (tissue homogenate) but also from cell culture homogenate is in strong favour of this being caused by a viral infection.

The changes seen are typical for what has been described for HMSB in Atlantic salmon in Norway. These changes are possibly caused by a pathogen of viral origin, the proposed conclusion being based on the distribution of changes, the involvement of the epicardium, the complete lack of suppuration in the areas involved and the changes observed in the striated skeletal muscle. The mononuclear and lymphocyte-dominated picture is also in favour of these changes being caused by a virus. There is indication of the myocytes (of the heart and skeletal muscle) being involved, but it cannot be ruled out that other cell populations are also targeted.

In summary, the observed changes in the myocard and the skeletal muscle are typical of HSMB.

Example 4 Electron microscopy

Laboratory examination

Organ samples from heart were sectioned in thin slides and fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). They were further postfixed with 2% osmium tetroxide and 1.5% potassium ferrocyanide, followed by staining with 1.5% uranylacete. Upon dehydration through increasing alcohol concentrations the specimens were embedded in Epon plastic resin. Ultrathin sections of heart from fish injected i.m. with cell homogenate and PBS (Vkj-FF and Vkj, respectively) (heart were cut and poststained with 0.2% lead citrate. Sections were examined in a Philips CM 100 electron microscope at 80 kV. Results

Electron micrographs from (Vkj-FF, Table 1) revealed large amounts of viral like particles of approximately 80 nm. No viral like particles were revealed in Vkj (table 1 ). (Data not shown).

The results shows that heart samples from fish injected intra muscular with cell homogenate contains large amount of viral like particles, while no viral like particles could be found in the PBS group. This corresponds well with the above histomorphological findings.

Example 5

Characterisation of HSMI virus

Chloroform sensitivity

Sensitivity to chloroform was determined by adding of chloroform to the virus solution to a final concentration of 10% (v/v). The mixture was shaken for 35 minutes at 20 degree C and then centrifuged at 2000xg for 10 minutes to remove the chloroform. Residual infections virus was detected by inoculation of a GF-1 cell culture. A control consisted of the virus solution only. Infectious Pancreatic Necrosis Virus (IPNV) and Infectious Salmon Anemia Virus (ISAV) were also included as negative (non-sensitive) and positive (sensitive) virus controls, respectively.

Results

The infectivity of the HSMI virus and ISAV was lost following exposure to chloroform, indicating the presence of an envelope containing essential lipids. In contrast, IPNV infectivity was not affected by the same treatment.

Table 1

Virus Chloroform test Control Treated

HSMI + virus

IPN virus + +

ISA virus + Stability at pH 3.0, 5.4, 7.4, 9.0 and 10.9

Stability at different pH's were determined by adding 170μl HSMI virus solution to 830μl L-15 adjusted to the respective pH's. The mixtures were incubated for 4 hours at 4 degree C, and checked for residual infections virus by inoculation of GF-1 cell culture flasks. Two control flasks were included; one was inoculated with 1.0 ml of L-15 with pH 3.0, another with 170 μl non-treated HSMI virus solution. The flasks were incubated for 3 weeks at 15 degree C. No control viruses were run in parallel.

Results

The infectivity of the HSMI virus solution was lost when it was exposed to pH 3.0.

During three weeks incubation no vacuoles were observed. No negative effect on the

GF-1 cell growth was observed in the flask inoculated with L-15 with pH 3.0.

Exposure to pH 5.4 reduced the infectivity of the HSMI virus solution and it was detected as a delay in the appearance of the first cells with vacuoles (8 days after inoculation).

The control flask and the flask with HSMI virus solution exposed to pH 7.4 (pure L-15) had the same development of infection. In both flasks the first cells with vacuoles appeared at 6 days pi, and at 14 days pi 100% of the cells were infected with numerous big vacuoles.

For the flasks inoculated with HSMI virus solution exposed to pH 9.0 and 10.9 the first cells with vacuoles appeared the day after inoculation and within 10 days 100% of the cells were affected with numerous big vacuoles. However, the cell growth in the flask inoculated with pH 10.9 exposed HSMI virus seemed to be negatively affected by the high pH.

Temperature stability

Aliquots of HSMI virus solution were heated for 30 minutes at 25, 37, 45, 60, 70 and 80 degree C and then cooled immediately by immersion in iced water. 170 μl of each aliquot were checked for residual infections virus by inoculation of GF-1 cell culture flasks. A control flask was inoculated with 170 μl of the same virus stock that had been stored cool. The flasks were incubated for 3 weeks at 15 degree C. No control viruses were run in parallel. Results

The HSMI virus was stabile for temperatures up to 60 degree C. No difference was observed between GF-1 flasks inoculated with HSMI virus solution that had been incubated at temperatures up to 60 degree C and the control flask. In all these flasks the first cells with vacuoles appeared at 6 days pi, and at 14 days pi 100% of the cells were infected with numerous big vacuoles. No cells developed vacuoles in the flasks inoculated with the virus solution heated to 70 and 80 degree C.

Example 6

HSMI virus vaccine

The vaccine efficacy is documented by injecting the inactivated, attenuated, or recombinant vaccine into salmon. One group is receives sterile saline as control. Six weeks post injection the fish is challenged to HSMI by bath, injection or by cohabitation. The efficacy is evaluated based on different mortality and pathological features between the vaccinated and the control group. The vaccinated fish will perform significantly better than the control group.

Example 7

HSMIV is immunogenic in rabbits

Immunoserum from rabbit was produced by injection of an oil-emulsion vaccine comprising a formalin-inactivated virus supernatant from GF-1 cells. The cells were inoculated as described above and the virus supernatant was harvested 2 weeks after infection after 2 repetitive freeze-thaw cycles. After a 24 hour inactivation at room temperature the viruses were emulsified in an oil emulsion (water-in-oil) and injected subcutaneously into the rabbit. The rabbit was subjected to 3 booster-immunisations at 14 days intervals. Blood samples were collected before each immunisation. Serum was aspirated from the sample tubes after centrifugation (3000 rpm, 15 min) and stored at minus 25° Celsius. Serum harvested 2 weeks after the last immunization was employed in the following studies.

Detection of HSMI virus in cell culture employing immunoenzymatic or immunofluorescence methods was executed the following way: Cells were seeded in 24-well or 96-well cell cultivation plates and infected according to standard protocols known to persons skilled in the art. Viruses were added 2 hr after cell seed. A first passage of virus was employed at a concentration of 5 μl/ml. Cells were incubated at 15° Celsius and fixed after 14 +/- 2 days when cell changes characterized as CPE in the form of vacuoles were observed.

To avoid cross-reactions with cellular components commonly contained in antigen preparations, the serum was absorbed with a noninfected lysate of GF-1 cells. This was achieved the following way:

1. Cells from two 25 cm² flasks were harvested by adding 2 ml PBS to each of the flasks and scraping out the cells. 2. 2 μl rabbit-anti-HSMIV per ml of uninfected GF-1 cell suspension were added. 3. The suspension was incubated o/n at 4°. 4. The suspension was centrifuged and the cell fraction was discarded, while the supernatant was kept for further studies.

Detection of HSMIV in cell cultures Cell fixation 1. The cell medium was aspirated and the cells washed twice in PBS. 2. 4% paraformaldehyde in PBS was added (30-50 μl/well using 96-well plates) and incubated at r/t for 30 min. 3. The paraformaldehyde solution was removed and the cells washed twice in PBS.

Permeabilization of cells

Permeabilization solution: Freshly made 0.1 % triton X-100 in 0.1 % trisodiumcitrate- dihydrate. 1. Permeabilization solution added and incubated 10 min. 2. The cells were washed twice in PBS

Immunostaining 1. Unspecific binding sites were blocked with 5 % powdered milk in Tris-buffer (TBST) for 20 min (50 μl per well in 96-well plates). 2. Gently washed in TBST. 3. The primary antibody rabbit-anti HSMIV diluted in 2.5 % powdered milk in TBST was added and the cells were incubated o/n at 4° C. Because of the absorption (as described above) the serum had an initial dilution of 1 :500. The serum was further diluted to 1:1000, 1:1500, 1:2000, and 1:2500. 4. The cells were washed 3x10 min. in TBST. 5. FITC-labeled anti-rabbit serum (DAKO) diluted 1 :40 in 2.5 % powdered milk/TBST was added and dark incubated at r/t for 30 min. 6. The cells were washed twice in TBST. 7. The cells were contrast stained by adding 2.5 μl propidiumiodide (200 μ/ml) per well with 50 μl buffer and dark incubated for 15 min. 8. The cells were washed in PBS and 50 μl PBS was added before microscopy.

Result:

Incubation with absorbed serum showed a positive reaction for HSMIV infected cells at all dilutions of the serum. On the other hand, uninfected cells gave no signal. See figures 10a and 10b.

This study demonstrated that HSMI virus cultured in GF-1 cells produces an immunogenic response in HSMIV infected rabbits. The rabbit serum comprises antibodies specifically reacting with virus components produced during replication in cell culture. This support the commercial use of the current invention as a vaccine.

REFERENCES

1. Kjaerstad A. (2002). Hjerte og skjellett muskel betennelse - en alvorlig sykdom? Fiskehelse (4) 19-20.

2. Kongtorp R.T., Kjaerstad A., Guttvik A., Skjelstad H., Bruheim T., Taksdal T. and Falk K. (2003). Heart and skeletal muscle inflammation in Atlantic salmon: A "new" infectious diseae? Diseases of Fish and Shellfish, Proceedings 11^th European Association of Fish Pathologists, Malta September 2003, p- P-56. 3. Kongtorp R.T., Kjaerstad A., Taksdal T., Guttvik A. and Falk K. (2004) Hjerte- og skjellettmuskelbetennelse (HSMB) - en "ny" sykdom hos atlantisk laks?

4. Kongtorp R.T. (2004b) Hjerte- og skjellettmuskelbetennelse (HSMB). In. Hjerterapporten. 2004. Rapport om hjertelidelser hos laks of regnbueørret p. 58. [Online] Oslo < http://www.vetinst.no/Arkiv/Pdf-filer/Hierterapporten FISK 20Q4.pdf> [assessed April 30 2004]

5. Taksdal T. (1999) Diagnoser fra vetennaerinstituttet. Norsk Veterinaertidskrift 111 (10) 646 and 647.

6. Watanabe K., Devoid M., Myhr E., Lyngøy A., Isdal E., Fridell F. and Nylund A. (2003) Hjerte- og skjellettmuskelbetennelse (HSMB): Ny virussykdom hos laks? Fiskehelse (2) 23-30.

Claims

WHAT IS CLAIMED IS:

1. An isolated Heart and Skeleton Muscle Inflammation Virus (HSMIV) that causes Heart and Skeleton Muscle Inflammation (HSMI) in fish.

2. The virus of claim 1 , wherein the virus has a diameter of about 80 nm.

3. The virus of claim 1 , wherein said virus is the strain deposited at ECACC under Deposit No. 04050401 or strains with related genotypic and/or phenotypic characteristics.

4. The virus of claim 1 , wherein said virus is the strain deposited at ECACC under Deposit No. 04050401.

5. The virus of claim 1, wherein said virus is attenuated or inactivated.

6. A cell line comprising the virus of claim 1 to 5.

7. The cell line of claim 6, wherein the cell line is derived from Epinephelus coioides.

8. The cell line of claim 6, wherein said cell line is GF-1 (ATCC PTA-859).

9. A vaccine comprising the virus of anyone of claims 1 to 5, or parts thereof.

10. The vaccine of claim 9 further comprising a suitable pharmaceutical carrier.

11. An isolated antibody that selectively binds a virus as claimed in anyone of claims 1 to 5 or to a component thereof.

12. The antibody of claim 11 comprising a marker.

13. A diagnostic kit comprising the antibody of claim 11.

14. A method of producing HSMIV comprising a. identifying a fish suffering from HSMI b. producing a homogenate comprising the HSMI virus c. inoculating a suitable cell strain d. Isolation of virus particles.

15. A virus according to claims 1-5 for medical use.

16. Use of the virus of claim 1 or parts thereof for the manufacture of a medicament for preventive treatment of Heart and Skeleton Muscle Inflammation Virus in a fish in need thereof.