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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
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  • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
  • C12N2720/00011—Details
  • C12N2720/10011—Birnaviridae
  • C12N2720/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
  • C12N2720/00011—Details
  • C12N2720/10011—Birnaviridae
  • C12N2720/10051—Methods of production or purification of viral material
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  • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
  • C12N2720/00011—Details
  • C12N2720/10011—Birnaviridae
  • C12N2720/10061—Methods of inactivation or attenuation
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/30011—Nodaviridae
  • C12N2770/30034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • 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
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/30011—Nodaviridae
  • C12N2770/30051—Methods of production or purification of viral material
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  • C12N2796/00—Viruses not covered by groups C12N2710/00 - C12N2795/00

Definitions

• the present invention relates to methods for producing and propagating viruses, in particular fish viruses.
• the invention also relates to cell cultures for use in methods for producing and propagating viruses and to vaccines comprising viruses prepared by the methods thereof.
• IPNV infectious pancreatic necrosis virus
• PCNV pillar cell necrosis virus
• VNN viral nervous necrosis
• SPDV salmonid pancreatic disease virus
• HTNV infectious hematopoietic necrosis virus
• VHSV viral haemorrhagic septicaemia virus
• IHUNV infectious hypodermal and hematopoietic necrosis virus
• WSV white spot virus
• TSV Taura syndrome virus
• UDV infectious salmon anaemia virus
• ISAV infectious salmon anaemia virus
• Methods of vaccine manufacture known in the art include use of embryonic eggs (e.g. bird embryos), cell (tissue) cultures (TC), live animal inoculation and/or use of transgenic animals. Investigational methods using plant, insect cells or bacteria cultures are also known.
• EPC epithelioma papulosum cyprinid
• BF-2 bluegill fry caudal trunk
• GF-1 grouper Epinephelus coioides
• SSN-1 striped snakehead fry
• SAF-1 gilt headed seabream caudal fin
• RTG-2 rainbow trout gonad 2
• CHSE-214 Chinook salmon embryonic cells
• ANK Asian grouper strain K
• the first aspect of the present invention provides a method of propagating a virus comprising the steps:
• said cell culture is a Japanese eel cell culture.
• the cell culture may comprise a mixed cell line, i.e. a mixture of two or more cell lines, or a single cell line.
• the Japanese eel cell culture may comprise a single cell type or a mixture of two or more cell types.
• the Japanese eel cell culture may comprise an epithelial cell and/or a fibroblast cell.
• the cell culture comprises an epithelial cell and a fibroblast cell.
• the cell culture is the cell culture derived from Anguilla japonica designated Anguilla japonica-K deposited by the Applicant at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 16062701.
• the term "Anguilla japoniccT ⁇ A. japonica) or "Japanese eeF refers to a species of anguillid eel. This is commonly found in Japan, China, Taiwan and Vietnam and is a catadromous fish, meaning that it spawns in the sea but lives partly in freshwater.
• the term "Anguilla japonica-K (AJK), or alternatively "Anguilla genus K? is the name given to the novel cell culture according to the present invention.
• the infected cell culture may be incubated at a temperature of between 10 and 30°C, more preferably between 15 and 28°C. It will be appreciated that the optimum incubation temperature will depend on the type and strain of the virus that is to be propagated. For example, where the virus is IPNV, the culture temperature is preferably around 15°C. For example, where the virus is nodavirus, such as betanodavirus, the culture temperature is preferably around 28°C.
• the present inventors have found that tissues collected from Anguilla japonica can be cultivated in vitro and unexpectedly form a viable cell culture for the production and propagation of viruses at surprisingly high yields.
• the cell culture was able to produce and propagate these unexpectedly high viral yields at lower incubation temperatures than commonly observed for other cell lines or cell cultures used in the field.
• the cell culture has been called Anguilla japonica-K.
• the virus is a fish virus.
• a "fish virus” is any virus that is capable of causing symptoms or disease in fish.
• the virus may be a RNA virus or a DNA virus.
• the virus is a RNA virus.
• the virus is a double-stranded RNA virus.
• the virus is a single-stranded RNA virus, for example a positive-sense single-stranded RNA virus.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a genus selected from the group consisting of: Alphanodavirus; Betanodavirus; Aquabirnavirus; Avibirnavirus; Blosnavirus; Entomobirnavirus; Alphavirus; Rubivirus; Novirhabdovirus; Brevidensovirus; Whispovirus; Aparavirus; Brevidensovirus; and Isavirus.
• the virus may be any fish virus known to the skilled person, some of which include infectious pancreatic necrosis virus (IPNV), pillar cell necrosis virus (PCNV), nodavirus, salmonid pancreatic disease virus (SPDV), infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp white spot virus (WSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), and infectious salmon anaemia virus (ISAV).
• IPNV infectious pancreatic necrosis virus
• PCNV pillar cell necrosis virus
• SPDV salmonid pancreatic disease virus
• IHNV infectious hematopoietic necrosis virus
• VHSV viral haemorrhagic septicaemia virus
• IHHNV infectious hypodermal and hematopoietic necrosis virus
• WSV white spot virus
• the virus is one selected from the group: nodavirus; Infectious Pancreatic Necrosis virus (IPNV); and Salmon Pancreas Disease Virus (SPDV).
• the virus may be a recombinant virus.
• the fish virus is capable of being propagated in the cell culture according to the present invention.
• recombinant virus refers to a virus produced by recombining pieces of DNA e.g. using recombinant DNA. The recombination is effected by human intervention. The term may also refer to naturally occurring recombination between virus genomes in cell infected by more than one virus strain, e.g. by homologous crossing over of nucleic acid strands or by re- assortment of genomic segments.
• the cell culture is infected with viral stock at a multiplicity of infection (MOI) of between 0.1 and 10.
• MOI refers to the ratio of agents (e.g. phage or more generally virus, bacteria) to the number of infection targets (e.g. cells).
• agents e.g. phage or more generally virus, bacteria
• the multiplicity of infection or MOI is the ratio of the number of virus particles to the number of target cells present in a defined space.
• the agent is a fish virus and the target is an Anguilla japonica-K cell.
• the Anguilla japonica-K cells may be cultured at 30°C with L-15 medium (Invitrogen) supplemented with 10% L-glutamine and 1 ⁇ 7 ⁇ . of gentamicin, 7.5% foetal bovine serum (FBS). It will be appreciated that the culturing conditions e.g. media may vary depending on the virus that is to be propagated.
• L-15 medium Invitrogen
• FBS foetal bovine serum
• the Anguilla japonica-K cells may be cultured at 29°C with L-15 medium (Invitrogen) supplemented with 10% L-glutamine, 1 ⁇ / ⁇ of gentamicin and 7.5% foetal bovine serum (FBS). It will be appreciated that the culturing conditions e.g. media may vary depending on the virus that is to be propagated.
• L-15 medium Invitrogen
• FBS foetal bovine serum
• the Anguilla japonica-K cells may be cultured at 28°C with L-15 medium (Invitrogen) supplemented with 10% L-glutamine, 1 ⁇ / ⁇ of gentamicin and 7.5% foetal bovine serum (FBS).
• L-15 medium Invitrogen
• FBS foetal bovine serum
• the Anguilla japonica-K cells may be cultured at 27.5°C with L-15 medium (Invitrogen) supplemented with 10% L-glutamine, 1 ⁇ / ⁇ of gentamicin and 7.5%) foetal bovine serum (FBS).
• L-15 medium Invitrogen
• FBS foetal bovine serum
• the virus is harvested at a final titre between about 2 x 10 6 TCIDso/ml to about 2 x 10 11 TCID5o/ml.
• the final titre is at least about 2 x 10 6 TCIDso/ml.
• the final titre is at least about 2 x 10 7 TCIDso/ml, at least about 2 x 10 8 TCIDso/ml, at least about 2 x 10 9 TCIDso/ml or at least about 2 x 10 10 TCIDso/ml. It will be appreciated that the final titre that is harvested will depend on factors such as the virus type and strain.
• Titre in the context of the present invention would be understood to mean the effective concentration of virus. Titre is commonly measured in TCIDso/ml (50% Tissue Culture Infective Dose) i.e. the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect (CPE) in 50% of inoculated tissue culture cells.
• TCIDso/ml 50% Tissue Culture Infective Dose
• CPE cytopathic effect
• final titre refers to the concentration of virus which has been harvested following full CPE by centrifugation of the suspension at for example 2500xg or more followed by recovery of the supernatant.
• the virus is harvested when a total cytopathic effect is observed in the cells.
• the virus is harvested from a supernatant and/or lysed cells.
• CPE cytopathic effect' or CPE in relation to a cell
• morphological changes are brought about as a consequence of viral replication which may be accompanied by accumulation of new viral particles in the cell or release of new viral particles in the supernatant.
• the second aspect of the present invention provides a cell culture suitable for the production of a virus, wherein the cell culture is a Japanese eel cell culture.
• the Japanese eel cell culture may comprise a single cell type or a mixture of two or more cell types.
• the Japanese eel cell culture may comprise an epithelial cell and/or a fibroblast cell.
• the cell culture comprises an epithelial cell and a fibroblast cell.
• the Japanese eel cell culture is the cell culture designated Anguilla japonica-K deposited with the European Collection of Authenticated Cell Cultures (ECACC) with accession number 16062701.
• ECACC European Collection of Authenticated Cell Cultures
• the virus is a fish virus.
• the virus may be a RNA virus or a DNA virus.
• the virus is a RNA virus.
• the virus is a double-stranded RNA virus.
• the virus is a single-stranded RNA virus, for example a positive-sense single-stranded RNA virus.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramyxovirus; Arterivi s; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a genus selected from the group consisting of: Alphanodavirus; Betanodavirus; Aquabirnavirus; Avibirnavirus; Blosnavirus; Entomobirnavirus; Alphavirus; Rubivirus; Novirhabdovims; Brevidensovirus; Whispovims; Aparavims; Brevidensovirus; and Isavirus.
• the virus may be any fish virus known to the skilled person, some of which include infectious pancreatic necrosis (TPNV), pillar cell necrosis virus (PCNV), nodavirus, salmonid pancreatic disease virus (SPDV), infectious hematopoietic necrosis virus (HTNV), viral haemorrhagic septicaemia virus (VHSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp white spot virus (WSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), infectious salmon anaemia virus (ISAV).
• TPNV pancreatic necrosis
• PCNV pillar cell necrosis virus
• SPDV salmonid pancreatic disease virus
• HTNV infectious hematopoietic necrosis virus
• VHSV viral haemorrhagic septicaemia virus
• IHHNV infectious hypodermal and hematopoietic necrosis virus
• WSV white spot virus
• TSV
• the virus may be one selected from the group: nodavirus; infectious pancreatic necrosis virus (TPNV); and salmon pancreas disease virus (SPDV).
• the virus may be a recombinant virus.
• the fish virus is capable of being propagated in the cell culture according to the present invention.
• the third aspect of the present invention provides use of a Japanese eel cell culture to multiply a viral stock.
• the term "viral stock” would be understood by the skilled person and generally refers to a solution comprising said virus.
• the Japanese eel cell culture may comprise a single cell type or a mixture of two or more cell types.
• the Japanese eel cell culture may comprise an epithelial cell and/or a fibroblast cell.
• the cell culture comprises an epithelial cell and a fibroblast cell.
• the Japanese eel cell culture is the cell culture designated Anguilla japonica-K deposited with the European Collection of Authenticated Cell Cultures (ECACC) with accession number 16062701.
• the virus is a fish virus.
• the virus may be a RNA virus or a DNA virus.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a genus selected from the group consisting of: Alphanodavirus; Betanodavirus; Aquabirnavirus; Avibirnavirus; Blosnavirus; Entomobirnavirus; Alphavirus; Rubivirus; Novirhabdovirus; Brevidensovirus; Whispovirus; Aparavirus; Brevidensovirus; and Isavirus.
• the virus may be any fish virus known to the skilled person, some of which include infectious pancreatic necrosis (TPNV), pillar cell necrosis virus (PCNV), Nodavirus, salmonid pancreatic disease virus (SPDV), infectious hematopoietic necrosis virus (JJTNV), viral haemorrhagic septicaemia virus (VHSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp white spot virus (WSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), infectious salmon anemia virus (ISAV).
• TPNV pancreatic necrosis
• PCNV pillar cell necrosis virus
• SPDV salmonid pancreatic disease virus
• JJTNV infectious hematopoietic necrosis virus
• VHSV viral haemorrhagic septicaemia virus
• IHHNV infectious hypodermal and hematopoietic necrosis virus
• WSV shrimp white spot virus
• the virus may be one selected from the group: nodavirus; Infectious Pancreatic Necrosis Virus (IPNV); and Salmon Pancreas Disease Virus (SPDV).
• IPNV Infectious Pancreatic Necrosis Virus
• SPDV Salmon Pancreas Disease Virus
• the virus may be a recombinant virus.
• the fish virus is capable of being propagated in the cell culture according to the present invention.
• the fourth aspect of the present invention provides an isolated Japanese eel cell infected with a virus.
• the Japanese eel cell may be an epithelial cell or a fibroblast cell.
• the Japanese eel cell is from the cell culture designated Anguilla japonica-K deposited with the European Collection of Authenticated Cell Cultures (ECACC) with accession number 16062701.
• the virus is a fish virus.
• the virus may be a RNA virus or a DNA virus.
• the virus is a RNA virus.
• the virus is a double-stranded RNA virus.
• the virus is a single-stranded RNA virus, for example a positive-sense single-stranded RNA virus.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a genus selected from the group consisting of: Alphanodavirus; Betanodavirus; Aquabirnavirus; Avibirnavirus; Blosnavirus; Entomobirnavirus; Alphavirus; Rubivims; Novirhabdovims; Brevidensovirus; Whispovims; Aparavims; Brevidensovirus; and Isavirus.
• the virus may be any fish virus known to the skilled person, some of which include infectious pancreatic necrosis virus (IPNV), pillar cell necrosis virus (PCNV), nodavirus, salmonid pancreatic disease virus (SPDV), infectious hematopoietic necrosis virus (HfNV), viral haemorrhagic septicaemia virus (VHSV), infectious hypodermal and hematopoietic necrosis virus (HfHNV), shrimp white spot virus (WSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), and infectious salmon anaemia virus (ISAV).
• IPNV infectious pancreatic necrosis virus
• PCNV pillar cell necrosis virus
• SPDV salmonid pancreatic disease virus
• HfNV infectious hematopoietic necrosis virus
• VHSV viral haemorrhagic septicaemia virus
• HfHNV infectious hypodermal and hematopoietic necrosis virus
• WSV shrimp
• the virus is one selected from the group: nodavirus; Infectious Pancreatic Necrosis virus (IPNV); and Salmon Pancreas Disease Virus (SPDV).
• the virus may be a recombinant virus.
• the fish virus is capable of being propagated in the cell culture according to the present invention.
• the fifth aspect of the present invention provides a vaccine comprising a virus produced by a method according to the invention, for use in the prophylaxis or treatment of disease in a fish.
• the virus is in an inactivated form.
• the virus is present at a titre greater than 1 x 10 6 TCIDso/ml, greater than 1 x 10 7 TCID5o/ml, greater than 1 x 10 8 TCIDso/ml, greater than 1 x 10 9 TCIDso/ml, or greater than 1 x 10 10 TCIDso/ml.
• Cell -based vaccines have a number of advantages over egg-based vaccines which include faster production times, easy isolation of the virus and avoidance of egg-based allergy reactions.
• cell lines and cultures can be grown in synthetic media which minimises the risks associated with animal serum.
• disadvantages of cell-based methods include increased cost and reduced viral yields.
• the cell culture of the present invention provides improvements to existing cell-based methods for producing fish vaccines.
• the sixth aspect of the present invention provides a kit for propagation of a virus, the kit comprising a Japanese eel cell culture and instructions for use.
• the Japanese eel cell culture may comprise a single cell type or a mixture of two or more cell types.
• the Japanese eel cell culture may comprise an epithelial cell and/or a fibroblast cell.
• the cell culture comprises an epithelial cell and a fibroblast cell.
• the cell culture is the cell culture designated Anguilla japonica-K deposited with the European Collection of Authenticated Cell Cultures (ECACC) with accession number 16062701.
• the virus is a fish virus.
• the virus may be a RNA virus or a DNA virus.
• the virus is a RNA virus.
• the virus is a double-stranded RNA virus.
• the virus is a single-stranded RNA virus, for example a positive-sense single-stranded RNA virus.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a genus selected from the group consisting of: Alphanodavirus; Betanodavirus; Aquabirnavirus; Avibirnavirus; Blosnavirus; Entomobirnavirus; Alphavirus; Rubivirus; Novirhabdovirus; Brevidensovirus; Whispovirus; Aparavirus; Brevidensovirus; and Isavirus.
• the virus may be any fish virus known to the skilled person, some of which include infectious pancreatic necrosis virus ( PNV), pillar cell necrosis virus (PCNV), nodavirus, salmonid pancreatic disease virus (SPDV), infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp white spot virus (WSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), and infectious salmon anaemia virus (ISAV).
• PNV pancreatic necrosis virus
• PCNV pillar cell necrosis virus
• SPDV salmonid pancreatic disease virus
• IHNV infectious hematopoietic necrosis virus
• VHSV viral haemorrhagic septicaemia virus
• IHHNV infectious hypodermal and hematopoietic necrosis virus
• WSV white spot virus
• the virus is one selected from the group: nodavirus; Infectious Pancreatic Necrosis virus (IPNV); and Salmon Pancreas Disease Virus (SPDV).
• the virus may be a recombinant virus.
• the fish virus is capable of being propagated in the cell culture according to the present invention.
• the seventh aspect of the present invention provides a method of producing a virus comprising the steps:
• the cell culture is a Japanese eel cell culture.
• the Japanese eel cell culture may comprise a single cell type or a mixture of two or more cell types.
• the Japanese eel cell culture may comprise an epithelial cell and/or a fibroblast cell.
• the cell culture comprises an epithelial cell and a fibroblast cell.
• the Japanese eel cell culture is the cell culture designated Anguilla japonica-K deposited with the European Collection of Authenticated Cell Cultures (ECACC) with accession number 16062701.
• the virus is a fish virus.
• the virus is a RNA virus.
• the virus is a double-stranded RNA virus.
• the virus is a single-stranded RNA virus, for example a positive-sense single-stranded RNA virus.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a genus selected from the group consisting of: Alphanodavirus; Betanodavirus; Aquabirnavirus; Avibirnavirus; Blosnavirus; Entomobirnavirus; Alphavirus; Rubivirus; Novirhabdovirus; Brevidensovirus; Whispovirus; Aparavirus; Brevidensovirus; and Isavirus.
• the virus may be any fish virus known to the skilled person, some of which include infectious pancreatic necrosis virus (IPNV), pillar cell necrosis virus (PCNV), nodavirus, salmonid pancreatic disease virus (SPDV), infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp white spot virus (WSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), and infectious salmon anaemia virus (ISAV).
• IPNV infectious pancreatic necrosis virus
• PCNV pillar cell necrosis virus
• SPDV salmonid pancreatic disease virus
• IHNV infectious hematopoietic necrosis virus
• VHSV viral haemorrhagic septicaemia virus
• IHHNV infectious hypodermal and hematopoietic necrosis virus
• WSV white spot virus
• the virus is one selected from the group: nodavirus; Infectious Pancreatic Necrosis virus (IPNV); and Salmon Pancreas Disease Virus (SPDV).
• the virus may be a recombinant virus.
• the fish virus is capable of being propagated in the cell culture according to the present invention.
• the cell culture is transduced with viral stock at a Multiplicity of Infection (MOI) of between 0.05 and 10, between 0.1 and 10 or between 0.1 and 1. It will be appreciated that the MOI will depend on factors such as the virus type and strain.
• MOI Multiplicity of Infection
• the virus is recovered at a final titre of the virus is between about 2 x 10 6 TCIDso/ml and about 2 x 10 11 TCIDso/ml. In some embodiments, the final titre is at least about 2 x 10 6 TCID5o/ml. In other embodiments the final titre is at least about 2 x 10 7 TCIDso/ml, at least about 2 x 10 8 TCIDso/ml, at least about 2 x 10 9 TCID 50 /ml or at least about 2 x 10 10 TCIDso/ml. It will be appreciated that the final titre that is harvested will depend on factors such as the virus type and strain. Preferably, the virus is harvested when a total cytopathic effect (CPE) is observed in the cells. Preferably, the virus is harvested from a supernatant.
• CPE total cytopathic effect
• Anguilla japonica-K cell culture of the present invention was able to produce higher viral yields that other known cell lines or cultures, such as SSN cells or CCH-1 cells.
• SSN cells or CCH-1 cells For example, for SPDV, a titre of 2xl0 7 TCIDso/ml was produced by CCH-1 cells, whereas a titre of more than two orders of magnitude was produced by cells of the invention (see Example 4).
• Anguilla japonica-K cell culture of the present invention also grows more rapidly when compared to salmonoid cell cultures.
• the eighth aspect of the present invention provides a method of identifying the presence of a virus in a sample, the method comprising:
• sample refers to for example bacterial, viral cells, fish cell, tissue or bodily fluid which can be obtained from any environmental source such as an animal medium that may or may not have been in contact with an animal or an artificial source such as tissue culture material.
• animal is a fish.
• the Japanese eel cell culture may comprise a single cell type or a mixture of two or more cell types.
• the Japanese eel cell culture may comprise an epithelial cell and/or a fibroblast cell.
• the cell culture comprises an epithelial cell and a fibroblast cell.
• the cell culture is the cell culture designated Anguilla japonica-K deposited with the European Collection of Authenticated Cell Cultures (ECACC) with accession number 16062701.
• ECACC European Collection of Authenticated Cell Cultures
• the method of identifying the presence of a virus in a sample may be a method of diagnosing the presence of a virus the sample. It would be appreciated that where the sample comprises a virus, the method would further comprise the steps of infecting the Japanese eel cell culture and growing the virus in the Japanese eel cell culture.
• step of incubating may be optional, depending on the virus, e.g. whether the virus is lytic or lysogenic.
• the virus is a fish virus.
• the virus may be a RNA virus or a DNA virus.
• the virus is a RNA virus.
• the virus is a double-stranded RNA virus.
• the virus is a single-stranded RNA virus, for example a positive-sense single-stranded RNA virus.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramyxovirus; Arterivi s; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramyxovirus; Arterivi s; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a genus selected from the group consisting of: Alphanodavirus; Betanodavirus; Aquabirnavirus; Avibirnavirus; Blosnavirus; Entomobirnavirus; Alphavirus; Rubivirus; Novirhabdovims; Brevidensovirus; Whispovims; Aparavims; Brevidensovirus; and Isavirus.
• the virus may be any fish virus known to the skilled person, some of which include infectious pancreatic necrosis virus (TPNV), pillar cell necrosis virus (PCNV), nodavirus, salmonid pancreatic disease virus (SPDV), infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp white spot virus (WSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), and infectious salmon anaemia virus (ISAV).
• TPNV pancreatic necrosis virus
• PCNV pillar cell necrosis virus
• SPDV salmonid pancreatic disease virus
• IHNV infectious hematopoietic necrosis virus
• VHSV viral haemorrhagic septicaemia virus
• IHHNV infectious hypodermal and hematopoietic necrosis virus
• WSV white spot virus
• the virus is one selected from the group: nodavirus; Infectious Pancreatic Necrosis virus (TPNV); and Salmon Pancreas Disease Virus (SPDV).
• the virus may be a recombinant virus.
• the fish virus is capable of being propagated in the cell culture according to the present invention.
• the identifying step comprises one selected from the group comprising, for example: polymerase chain reaction (PCR); reverse transcription polymerase chain reaction (RT-PCR), Western Blot analysis, Northern Blot analysis, Southern Blot analysis, enzyme- linked immunosorbent assay (ELISA); electrophoretic mobility shift assay (EMSA), and/or FACS analysis.
• PCR polymerase chain reaction
• RT-PCR reverse transcription polymerase chain reaction
• Western Blot analysis Northern Blot analysis
• Southern Blot analysis Southern Blot analysis
• enzyme- linked immunosorbent assay ELISA
• ESA electrophoretic mobility shift assay
• FACS analysis electrophoretic mobility shift assay
• the tenth aspect of the present invention provides a method of producing an immunogenic composition comprising the steps: providing the isolated cell culture of the present invention;
• the virus is a fish virus.
• the virus may be a RNA virus or a DNA virus.
• the virus is a RNA virus.
• the virus is a double-stranded RNA virus.
• the virus is a single-stranded RNA virus, for example a positive-sense single-stranded RNA virus.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a family selected from the group consisting of: Nodaviridae; Birnaviridae; Togaviridae; Rhabdoviridae; Iridoviridae; Reoviridae; Ortomixovirus; Paramixovirus; Arterivims; Picornavirus; Nimaviridae; Dicistroviridae; Parvoviridae; and Orthomyxoviridae.
• the virus may be a virus of a genus selected from the group consisting of: Alphanodavirus; Betanodavirus; Aquabirnavirus; Avibirnavirus; Blosnavirus; Entomobirnavirus; Alphavirus; Rubivirus; Novirhabdovirus; Brevidensovirus; Whispovirus; Aparavirus; Brevidensovirus; and Isavirus.
• the virus may be any fish virus known to the skilled person, some of which include infectious pancreatic necrosis virus ( PNV), pillar cell necrosis virus (PCNV), nodavirus, salmonid pancreatic disease virus (SPDV), infectious hematopoietic necrosis virus (HTNV), viral haemorrhagic septicaemia virus (VHSV), infectious hypodermal and hematopoietic necrosis virus (HTHNV), shrimp white spot virus (WSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), and infectious salmon anaemia virus (ISAV).
• PNV pancreatic necrosis virus
• PCNV pillar cell necrosis virus
• SPDV salmonid pancreatic disease virus
• HTNV infectious hematopoietic necrosis virus
• VHSV viral haemorrhagic septicaemia virus
• HTHNV infectious hypodermal and hematopoietic necrosis virus
• WSV shrimp white spot virus
• the vims is one selected from the group: nodavirus; Infectious Pancreatic Necrosis virus (IPNV); and Salmon Pancreas Disease Virus (SPDV).
• the virus may be a recombinant virus.
• the fish virus is capable of being propagated in the cell culture according to the present invention.
• Figure 1 shows the results of size separation by agarose gel electrophoresis of the products of PCR analysis of the Anguilla japonica-K cell culture deposited under ECACC accession number 16062701;
• Figure 2 is a graph showing the growth curve for Infectious Pancreatic Necrosis Virus isolate IPN NVI 015 (GenBank accession numbers AY379740 and AY379741) in Anguilla japonica-K cell culture (ECACC accession number 16062701) in terms of the virus titre at 5, 6, 7 and 8 days post-harvest, infected at an MOI of 1;
• Figure 3 is a graph showing the growth curve for Salmon Pancreas Disease Virus (SPDV) in Anguilla japonica-K cell culture (ECACC accession number 16062701) in terms of the virus titre at 5, 6, 7, 12 and 14 days post-harvest, infected at an MOI of 0.5; and
• SPDV Salmon Pancreas Disease Virus
• Figure 4 is a graph showing the growth curve for Nodavirus in Anguilla japonica-K cell culture (ECACC accession number 16062701) in terms of the virus titre at 4, 5, 6, 7, 9 and 11 days post-harvest, infected at an MOI of 0.1, 0.5 and 1.
• Figure 1 shows the Amplification products for COl gene (left hand set, lanes 1-4) and 16S gene (right hand set, lanes 7-10) of pre master Anguilla japonica-K cell seed pi 18 (1&2 and 7&8 and master cell pl21 (3&4 and 9&10). Lanes 5, 6, 11 and 12 are negative controls.
• the cell culture was stored in 50/50 by volume of 10% DMSO in Minimal Essential Medium (MEM) and Foetal Bovine Serum (FBS) undiluted, at a density of 2 x 10 6 cells/ml.
• MEM Minimal Essential Medium
• FBS Foetal Bovine Serum
• Anguilla japonica-K cells were generally maintained at 28°C with L-15 medium (Invitrogen) supplemented with 10% L-glutamine and 1 ⁇ 7 ⁇ of gentamicin, and 7.5% Foetal Bovine Serum (FBS).
• L-15 medium Invitrogen
• FBS Foetal Bovine Serum
• the Anguilla japonica-K cells were maintained at between 15 and 28°C in Minimal Essential Medium (MEM), 7.5% Foetal Bovine Serum (FBS), 0.5% sodium bicarbonate, and 1% L-alanyl-L-Glutamine dipeptide (Glutamax).
• MEM Minimal Essential Medium
• FBS Foetal Bovine Serum
• Glutamax 1% L-alanyl-L-Glutamine dipeptide
• Anguilla japonica-K cells infected with IPN were maintained at 15°C.
• Anguilla japonica-K cells infected with nodavirus were maintained at 28°C.
• T e Anguilla japonica-K cell culture deposited with the ECACC relates to a sample of the pre- master cell seed at passage (p) 124. Analysis showed the cell culture to be mainly epithelial, with fibroblasts present.
• IPN Infectious Pancreatic Necrosis
• SPDV Salmon Pancreas Disease Virus
• Nodavirus Nodavirus
• the cells were inoculated at a MOI of 1.0 for IPN and SPDV, and MOI's of 0.1, 0.5 and 1.0 for Nodavirus.
• the growth curves for the viruses are shown in Figure 2 (Infectious Pancreatic Necrosis Virus), Figure 3 (Salmon Pancreatic Disease Virus) and Figure 4 (Nodavirus).
• the virus was harvested following full CPE by centrifugation of the suspension at 2500xg followed by recovery of the supernatant.
• the average titres of the viruses are shown in Table 2 below. Table 2
• Anguilla japonica-K (TCIDso/ml) at batches (cell harvest time in cells optimal harvest time factories) days postover several research infection (days) scale batches (cell

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