WO2005121325A1 - Virus (hsmi) d'inflammation musculaire squelettique et cardiaque - Google Patents

Virus (hsmi) d'inflammation musculaire squelettique et cardiaque Download PDF

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WO2005121325A1
WO2005121325A1 PCT/NO2005/000197 NO2005000197W WO2005121325A1 WO 2005121325 A1 WO2005121325 A1 WO 2005121325A1 NO 2005000197 W NO2005000197 W NO 2005000197W WO 2005121325 A1 WO2005121325 A1 WO 2005121325A1
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virus
fish
hsmi
heart
cells
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PCT/NO2005/000197
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Trygve Meum Eliassen
Inge Tom Solbakk
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Pharmaq As
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Priority to CA 2570050 priority Critical patent/CA2570050C/fr
Priority to GB0625374A priority patent/GB2429711B/en
Publication of WO2005121325A1 publication Critical patent/WO2005121325A1/fr
Priority to NO20065765A priority patent/NO343278B1/no

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • C12N7/08—Inactivation or attenuation by serial passage of virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12061—Methods of inactivation or attenuation
    • C12N2720/12064—Methods of inactivation or attenuation by serial passage

Definitions

  • HEART AND SKELETAL MUSCLE INFLAMMATION (HSMI ) VIRUS 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.
  • HSMIV Heart and Skeleton Muscle Inflammation Virus
  • the invention may be utilized to prevent and diagnose Heart and Skeleton Muscle Inflammation (HSMI) in fish.
  • HSMI Heart and Skeletal muscle inflammation
  • 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%.
  • IPNV Infectious Pancreatic Necrosis Virus
  • ISAV Infections Salmon Anaemia Virus
  • SPDV Salmon Pancreas Disease Virus
  • 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.
  • 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 na ⁇ ve 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 na ⁇ ve cohabitants.
  • 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.
  • HSMIV Heart and Skeleton Muscle Virus
  • 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.
  • 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 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 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.
  • HSMIV Heart and Skeleton Muscle Virus
  • 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.
  • 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.
  • the fish is Atlantic salmon (Salmo salar).
  • the virus is the strain deposited at ECACC under deposit number 04050401 or strains with related genotypic and/or phenotypic characteristics.
  • HSMIV 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.
  • 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.
  • 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.
  • 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.
  • the seed virus 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).
  • carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, and glucose
  • proteins such as albumin or casein
  • protein containing agents such as bovine serum or skimmed milk
  • buffers such as alkali metal phosphates
  • 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, ⁇ -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.
  • 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.
  • 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,
  • 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. ⁇ -glucan, oil adjuvants, liposomes, etc.
  • adjuvants like, but not limited to, e.g. ⁇ -glucan, oil adjuvants, liposomes, etc.
  • the vaccine herein described can be administrated by injection, dipping, bath or orally.
  • 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.
  • the invention comprises a virus as described above use as a therapeutic.
  • 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
  • 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.
  • GF-1 cell cultures are split to a cell density of approximately 2-5 x 10 4 cells/cm 2 , and inoculated with 5 ⁇ l 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 ⁇ m 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 ⁇ l HSMI supernatant per ml growth medium).
  • the development of the infection is normally delayed with 3-5 days compared with the 1 st passage.
  • 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.
  • 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.
  • Histological changes corresponding to HSMI were observed in heart tissue of all groups injected infected material.
  • 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.
  • 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.
  • 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.
  • lymphocytes 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).
  • sarcoplasm small vacuoles of the cytoplasm
  • the changes described in the skeletal muscle samples are typical of an inflammatory response to a muscle cell expressing a foreign antigen (non-self).
  • DNA vaccinated fish were viral proteins are expressed through transfection of muscle using plasmids encoding viral proteins (for example G-protein of VHS virus).
  • 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).
  • 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
  • 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.
  • IPNV Infectious Pancreatic Necrosis Virus
  • ISAV Infectious Salmon Anemia Virus
  • Stability at different pH's were determined by adding 170 ⁇ l HSMI virus solution to 830 ⁇ l 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 ⁇ l non-treated HSMI virus solution. The flasks were incubated for 3 weeks at 15 degree C. No control viruses were run in parallel.
  • control flask and the flask with HSMI virus solution exposed to pH 7.4 had the same development of infection.
  • 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.
  • 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.
  • 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 ⁇ l/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.
  • the serum was absorbed with a noninfected lysate of GF-1 cells. This was achieved the following way:
  • 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
  • FITC-labeled anti-rabbit serum 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 ⁇ l propidiumiodide (200 ⁇ /ml) per well with 50 ⁇ l buffer and dark incubated for 15 min. 8. The cells were washed in PBS and 50 ⁇ l PBS was added before microscopy.
  • DAKO FITC-labeled anti-rabbit serum
  • 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.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Cell Biology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention a pour objet un Virus isolé d'Inflammation Cardiaque et Squelettique (HSMIV - Heart and Skeleton Muscle Inflammation Virus) dérivé du poisson ; un HSMIV vivant, atténué ou inactivé, ou une partie de sa région fonctionnelle destinée à des vaccins, un antigène recombinant et un vaccin ; de même que l'ADN, les lignées cellulaires, les vaccins, les anticorps, les procédés et leurs utilisations. L’invention peut être utilisée pour empêcher et diagnostiquer l'Inflammation musculaire cardiaque et squelettique (HSMI - Heart and Skeleton Muscle Inflammation) chez le poisson.
PCT/NO2005/000197 2004-06-11 2005-06-10 Virus (hsmi) d'inflammation musculaire squelettique et cardiaque WO2005121325A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA 2570050 CA2570050C (fr) 2004-06-11 2005-06-10 Virus (hsmi) d'inflammation musculaire squelettique et cardiaque
GB0625374A GB2429711B (en) 2004-06-11 2005-06-10 Heart and skeletal muscle inflammation (HSMI) virus
NO20065765A NO343278B1 (no) 2004-06-11 2006-12-14 Isolert hjerte- og skjelettmuskelbetennelsesvirus (HSMIV), fremgangsmåte til fremstilling av HSMIV og medisinsk anvendelse av HSMIV.

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
NO20042454A NO20042454D0 (no) 2004-06-11 2004-06-11 Novel fish virus
NO20042454 2004-06-11
US57882104P 2004-06-14 2004-06-14
US60/578,821 2004-06-14

Publications (1)

Publication Number Publication Date
WO2005121325A1 true WO2005121325A1 (fr) 2005-12-22

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Family Applications (1)

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PCT/NO2005/000197 WO2005121325A1 (fr) 2004-06-11 2005-06-10 Virus (hsmi) d'inflammation musculaire squelettique et cardiaque

Country Status (4)

Country Link
CA (1) CA2570050C (fr)
GB (1) GB2429711B (fr)
NO (1) NO343278B1 (fr)
WO (1) WO2005121325A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2452363A (en) * 2007-06-28 2009-03-04 Pharmaq As Fish virus which causes Cardiomyopathy Syndrome
WO2009108067A2 (fr) * 2008-02-27 2009-09-03 Thia Medica As Prévention et traitement de l’inflammation hsmi
WO2012066481A1 (fr) * 2010-11-15 2012-05-24 Pharmaq As Nouvel agent étiologique
JP2013506672A (ja) * 2009-10-02 2013-02-28 トラスティーズ・オブ・コロンビア・ユニバーシティ・イン・ザ・シティ・オブ・ニューヨーク 魚類レオウイルス免疫原性組成物
US8562802B1 (en) 2006-02-13 2013-10-22 Life Technologies Corporation Transilluminator base and scanner for imaging fluorescent gels, charging devices and portable electrophoresis systems
WO2015028565A1 (fr) * 2013-08-30 2015-03-05 Veterinærinstituttet Procédé de propagation d'un virus
WO2021122507A1 (fr) 2019-12-16 2021-06-24 Intervet International B.V. Vaccin inactivé à base d'orthoréovirus pisciaire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KONGTORP R T ET AL: "Heart and skeletal muscle inflammation in Atlantic salmon, Salmo salar L.: a new infectious disease", JOURNAL OF FISH DISEASES, vol. 27, no. 6, 27 May 2004 (2004-05-27), pages 351 - 358, XP008053704, ISSN: 0140-7775 *
KONGTORP R T ET AL: "Pathology of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar", DISEASES OF AQUATIC ORGANISMS, vol. 59, no. 3, 11 June 2004 (2004-06-11), pages 217 - 224, XP008053700, ISSN: 0177-5103 *
TRYGVE MEUM ELIASSEN, INGE TOM SOLBAKK, OYSTEIN EVENSEN , KJERSTI GRAVNINGEN: "Isolation of Heart and Skeletal Muscle Inflammation Virus (HSMIV) from Salmon", 2004, XP002348508, Retrieved from the Internet <URL:http://www.pharmaq.no/Posters/HSMIV_poster.pdf> [retrieved on 20051010] *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8562802B1 (en) 2006-02-13 2013-10-22 Life Technologies Corporation Transilluminator base and scanner for imaging fluorescent gels, charging devices and portable electrophoresis systems
GB2452363B (en) * 2007-06-28 2012-08-08 Pharmaq As Novel fish pathogen
GB2452363A (en) * 2007-06-28 2009-03-04 Pharmaq As Fish virus which causes Cardiomyopathy Syndrome
NO346433B1 (no) * 2007-06-28 2022-08-08 Pharmaq As Nytt fiskepatogen
WO2009108067A2 (fr) * 2008-02-27 2009-09-03 Thia Medica As Prévention et traitement de l’inflammation hsmi
WO2009108067A3 (fr) * 2008-02-27 2009-11-12 Thia Medica As Prévention et traitement de l’inflammation hsmi
EP2482824A4 (fr) * 2009-10-02 2013-06-19 Univ Columbia Compositions immunogènes à base de réovirus pisciaire
EP2482825A4 (fr) * 2009-10-02 2013-06-26 Univ Columbia Compositions diagnostiques à base de réovirus pisciaire
JP2013506672A (ja) * 2009-10-02 2013-02-28 トラスティーズ・オブ・コロンビア・ユニバーシティ・イン・ザ・シティ・オブ・ニューヨーク 魚類レオウイルス免疫原性組成物
US9366667B2 (en) 2009-10-02 2016-06-14 The Trustees Of Columbia University In The City Of New York Piscine reovirus diagnostic compositions
US9395356B2 (en) 2009-10-02 2016-07-19 The National Veterinary Institute Piscine reovirus immunogenic compositions
DK179132B1 (en) * 2009-10-02 2017-11-27 Univ Columbia Piscine reovirus immunogenic compositions
EP2640832B1 (fr) 2010-11-15 2017-12-20 Pharmaq AS Nouvel agent étiologique
WO2012066481A1 (fr) * 2010-11-15 2012-05-24 Pharmaq As Nouvel agent étiologique
WO2015028565A1 (fr) * 2013-08-30 2015-03-05 Veterinærinstituttet Procédé de propagation d'un virus
CN105683367A (zh) * 2013-08-30 2016-06-15 坊玛药物公司 用于增殖病毒的方法
WO2021122507A1 (fr) 2019-12-16 2021-06-24 Intervet International B.V. Vaccin inactivé à base d'orthoréovirus pisciaire

Also Published As

Publication number Publication date
CA2570050A1 (fr) 2005-12-22
GB0625374D0 (en) 2007-01-31
NO343278B1 (no) 2019-01-14
GB2429711B (en) 2008-05-21
GB2429711A (en) 2007-03-07
NO20065765L (no) 2007-01-11
CA2570050C (fr) 2013-03-05

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