Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1999-11-30
2002-08-20
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S235100, C435S239000, C424S204100
Reexamination Certificate
active
06436702
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an immortal cell line (GF-1) derived from the fin tissue of grouper
Epinephelus coioides
and the method of establishing the GF-1 cell line. The GF-1 cell line is susceptible to a number of aquatic viruses, including, but not limited to, Infectious Pancreatic Necrosis Virus (IPNV), Eel Herpes Virus Formosa (EHVF), and Nervous Necrosis Virus (NNV). This invention also relates to the method of mass producing and purifying the aquatic viruses using an immortal cell line from grouper such as the GF-1 cell line as a host. Additionally, this invention relates to an anti-NNV antibody and the method of producing the anti-NNV antibody. Finally, this invention relates to a vaccine of NNV and the method for protecting fish against NNV infection.
BACKGROUND OF THE INVENTION
Nervous necrosis virus (NNV), a pathogen found in many varieties of hatchery-reared marine fish, has caused mass mortality of such fish at their larval or juvenile stages. NNV belongs to the family Nodaviridae. Fish nodaviruses isolated from different species (such as SJNNV, BFNNV, JFNNV, TPNNV, RGNNV, GNNV etc.) are closely related to each other owing to the high similarity of the conserved region of their coat protein genes. NNV, also named as fish encephalitis virus (FEV) and piscine neuropathy nodavirus (PNN), is an unenveloped spherical virus with particles sized between 25 and 34 nm. The virus is characterized by vacuolation of the nerve tissues. Viral Nervous Necrosis (VNN) disease has been found in many countries under various names such as viral fish encephalitis, fish encephalomyelitis, cardiac myopathy syndrome. The hosts of NNV include many species of marine fish, for example: parrotfish, sea bass, turbot, grouper, stripped jack, tiger puffer, berfin flounder, halibut, barramundi, and spotted wolffish.
According to the statistics shown in 1993, approximately 159 fish cell lines have been established which have demonstrated a capacity for growing fish viruses (Fryer and Lannan,
J. Tissue Culture Method
(1994), 10:57-94). Most of these cell lines are derived from the tissues of freshwater fish. There are only thirty-four cell lines which are originated from marine fish. Although some of the fish cell lines, which include RTG-2, CHSE-214, BF2, SBL, FHM, EPC, have been tested for the susceptibility of fish nodavirus, none of these cells lines has shown cytopathic effects (CPE) after viral inoculations.
In 1996, SSN-1 cell line, a cell line derived from striped snakehead
Channa Striatus
, has been successfully used for isolating sea bass nodavirus (Frerichs et al.,
J. General Virology
(1996)77:20672071). However, SSN-1 cell line has been known to be persistently contaminated with C-type retrovirus (Frerichs et al.,
J. General Virology
(1991) 72:2537-2539). Therefore, it is not suitable for the production of fish nodavirus.
Viral diseases cannot be cured by therapeutic reagents. The best ways to contain viral diseases include prevention through early detection and the development of vaccines. In either way, the understanding of the biological, biochemical, and serological characteristics of the virus is fundamentally required, which in turn relies on the industry to have the capacity of mass producing the pure form of viruses, preferably through an in vitro cell culture system. Therefore, the development of a new cell line which can be susceptible to fish nodavirus is desperately in demand in order to control the wide spread of fish viral diseases due to fish nodavirus infection.
Grouper is an important hatchery fish in Taiwan. In recent years, there have been several reports regarding the establishment of cell lines derived from grouper. For example, Chen et. al. (
Japan Scientific Society Press
(Tokyo) (1988) 218-227) have reported their establishment of several cell lines from the fin and kidney tissues of grouper
Epinephelus awoara
. Lee (Master Thesis from the Department of Zoology at the National Taiwan University, 1993) also has reported his establishment of the cell lines derived from the eye pigment cells and brain tissue of grouper
Epinephelus amblycephalus
. However, Chen et al. do not provide sufficient data in support of the claim for immortality in their cell lines and Lee expressly indicates in his thesis that his grouper cell lines are not immortal. Moreover, neither Chen et al.'s nor Lee's cell lines are susceptible to fish nodavirus.
Recently, severe mortality among groupers has repeatedly occurred which is caused primarily by nodavirus. As present, fish nodavirus has been discovered in grouper and can be isolated from moribund grouper which possess symptoms of VNN disease (Chi et al.,
J. Fish Disease
(1997) 20:185-193). Electron microscopic examination of the tissues from grouper shows that, in addition to nodavirus infection, grouper is susceptible to other viral infections (Chi,
COA Fisheries Series No.
61
, Reports on Fish Disease Research
(1997) 18:59-69). The fact that some viruses have host specificity makes a cell line derived from grouper more appropriate for investigating the specific viruses isolated from grouper.
In the invention to be presented below, an immortal cell line derived from the fin tissue of grouper
Epinephelus coioides
(Hamilton) will be introduced: The cell line of the present invention is susceptible to various viruses, particularly fish nodavirus such as GNNV. Using the present cell line, various aquatic viruses can be mass produced and purified. The purified viruses are useful for antibody and vaccine production to protect fish from viral infections.
SUMMARY OF THE INVENTION
A first embodiment of the present invention provides for an immortal cell line derived from grouper, preferably, an immortal cell line (GF-1) which is derived from the fin tissue of grouper
Epinephelus coioides
. GF-1 is susceptible to, and can mass produce viruses which include, but are not limited to, viruses from the 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 grouper nervous necrosis virus [GNNV]).
The first embodiment also provides for a method of establishing an immortal cell line. The method comprises the steps of : (1) establishing a primary cell culture by placing cells released from the fin tissue of grouper
Epinephelus coioides
in a tissue culture flask to form a monolayer of cells; (2) subculturing and maintaining the monolayer of cells in a media suitable for cell subculturing; and (3) monitoring a transformation of cells which is characterized by a change in chromosome number distribution, plating efficiency, fetal bovine serum (FBS) requirement , and susceptibility to aquatic viruses, particularly fish nodavirus such as GNNV.
A second embodiment of the invention provides for a method for growing a virus using the immortal cell line derived from grouper, preferably the GF-1 cell line. The method comprises the steps of. (1) inoculating the virus into the cell line; and (2) incubating the cell line in a nutrient medium suitable for growth and replication of the virus. The viruses which are susceptible to and can be replicated in the immortal cell line include viruses from the families of Birnaviridae, Herpesviridae, Reoviridae, and Nodaviridae, and, in particular, lPNV of Birnaviridae, EHVF of Herpesviridae, HCRV of Reoviridae, and GNNV of Nodaviridae.
The second embodiment also provides for methods of mass producing the viruses using the immortal grouper cell line, purifying the viruses, and detecting the viruses in the cell line. The method for mass producing the virus comprises: (1) inoculating the virus into the grouper cell line; (2) incubating the cell line in a nutrient medium suitable for growth and replication of the virus; and (3) harvesting the virus from the cell line.
The method for purifying a virus comprises: (1) inoculating the virus into the grouper cell line; (2) incubating the
Chao Fei-Fei
National Science Council
Scheiner Laurie
Venable Baetjer Howard & Civiletti LLP
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