# US 6,566,117 B2 · Immortal cell line derived from grouper Epinephelus coioides and its applications therein

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- Number: US 6,566,117 B2
- Document ID: US6566117B2
- Status: Granted
- Kind: B2
- Application number: 09/998,212
- Filed: 2001-12-03
- Published: 2002-11-07
- Granted: 2003-05-20
- Earliest claimed: 1998-12-03
- Assignees: National Science Council
- Inventors: Shau-Chi Chi
- Web page: https://patentgrep.com/patent/US6566117B2

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61P 31/12: Antivirals
- C07K 16/08: against material from viruses
- C12Q 1/02: involving viable microorganisms
- C12Q 1/701: {Specific hybridization probes}
- G01N 33/56983: {Viruses}
- A61K 2039/5252: {inactivated (killed)}
- A61K 2039/542: {oral/gastrointestinal}
- C12N 2770/30051: Methods of production or purification of viral material
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention describes (1) an immortal cell line derived from grouper and a method for establishing the cell line; (2) methods for mass producing and purifying aquatic viruses using the immortal cell line from grouper; (3) an anti-NNV antibody and a method for producing the anti-NNV antibody; and (4) a vaccine of NNV and a method for protecting fish against NNV infection. The present immortal cell line is derived from the grouper and is susceptible to the viral families of Birnaviridae such as Infectious Pancreatic Necrosis Virus (IPNV); Herpesviridae such as Eel Herpes Virus Formosa (EHVF); Reoviridae such as Hard Clam Reovirus (HCRV); and Nodaviridae such as Nervous Necrosis Virus (NNV).

## Claims

### Claim 1

A method for growing a virus comprising:
inoculating said virus into an established cell culture comprising an immortal cell line which is ATCC deposit no. PTA-859; and
incubating said cell culture in a nutrient medium suitable for growth and replication of said virus.

### Claim 2 (depends on 1)

The method for growing a virus according to claim 1, wherein said virus is selected from the group consisting of Birnaviridae, Herpesviridae, Reoviridae, and Nodaviridae.

### Claim 3 (depends on 2)

The method for growing a virus according to claim 2, wherein said Birnaviridae is Infectious Pancreatic Necrosis Virus (IPNV).

### Claim 4 (depends on 2)

The method for growing a virus according to claim 2, wherein said Herpesviridae is Eel Herpes Virus Formosa (EHVF).

### Claim 5 (depends on 2)

The method for growing a virus according to claim 2, wherein said Reoviridae is Hard Clam Reovirus (HCRV).

### Claim 6 (depends on 2)

The method for growing a virus according to claim 2, wherein said Nodaviridae is a fish nodavirus which is selected from the group consisting of Nervous Necrosis Virus (NNV), Fish Encephalitis Virus (EFV), Piscine Neuropathy Nodavirus (PNN), Grouper Nervous Necrosis Virus (GNNV), Stripped Jack Nervous Necrosis Virus (SJNNV), Tiger Puffer Nervous Necrosis Virus (TPNNV), Berfin Flounder Nervous Necrosis Virus (BFNNV) and Red Spotted Grouper Nervous Necrosis Virus (RGNNV).

### Claim 7

A method for mass producing a virus comprising:
inoculating the virus in the established cell culture comprising an immortal cell line which is ATCC deposit no. PTA-859;
incubating said cell culture in a nutrient medium suitable for growth and replication of said virus until an appearance of cytopathic effects (CPE); and
harvesting said virus from said cell line.

### Claim 8

A method for purifying a virus comprising:
inoculating the virus into an established cell culture comprising an immortal cell line which is ATCC deposit no. PTA-859;
incubating said cell culture in a nutrient medium suitable for growth and replication of said virus until an appearance of cytopathic effects (CPE);
harvesting said virus from said cell line; and
purifying said virus using a density gradient centrifugation.

### Claim 9 (depends on 8)

The method for purifying a virus according to claim 8, wherein said density gradient centrifugation is a CsCl density gradient centrifugation.

### Claim 10 (depends on 8)

The method for purifying a virus according to claim 8, wherein said virus is NNV, and wherein said NNV has a buoyant density of approximately 1.34 g/ml in CsCl.