Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1997-09-12
1998-11-03
Woodward, Michael P.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435237, 4352351, 435325, 435236, 4242241, A61K 39208, C12N 700, C12N 708
Patent
active
058306382
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a permanent cell line and a process for replicating infectious rabies virus and to its quantitative detection by means of the cytopathic effect (CPE) using this cell line, and to a process for detecting inhibitors of rabies virus replication.
Living cells are required in order to produce proteins and viral antigens. In the state of the art, diploid cell strains or permanent cell lines are used for this purpose provided they are suitable for producing the desired proteins or viral antigens. As compared with diploid cell strains, permanent cell lines enjoy the advantage that they exhibit unlimited growth, i.e. they are immortalized. Over broad passage ranges, such permanent cell lines exhibit constant properties as regards cell and antigen replication since, in their case, no cell differentiation takes place as it does in diploid cell strains. Apart from the limited lifespan (passage number) of diploid cell strains, a further, serious disadvantage of these strains is that the organs, tissues, and also chick embryos, which are required as starting material for diploid cell strains are not available in adequate quantity and at all times. Furthermore, the starting material can be latently and/or inapparently contaminated (viruses, mycoplasmas and bacteria), as a result of which optimally reproducible antigen production is not guaranteed.
While it is known that rabies virus can be replicated in cell cultures derived from a multiplicity of species, there are only a few reports in the literature of a cytopathic effect being obtained (Egert et al., Acta Virol. 33 (1989), 353-358, Consales et al., J. Virol. Meth. 27 (1990), 227-286, Campbell and Charlton, in: Development in veterinary virology: Rabies Kluwer Academic Publishers, Boston, Dordrecht, London, 1988). The cytopathic effect (CPE) is a virally-determined, specific cell destruction (lysis) which can readily be detected under the light microscope.
In general, however, replication of rabies virus in tissue cultures either proceeds without any CPE (Campbell and Charlton (1988)) or else virally-determined cytopathic changes are only irregular (Kaplan and Koprowski, Laboratory techniques in rabies, III. Edit. Wld. Hlth. Org. Monograph Series 23, Geneva (1973)) or only occur transiently (Smith et al., Intervirol. 8 (1977), 92-99), so that rabies viruses cannot be detected reliably by evaluating the CPE when these cell lines are used.
The present invention was therefore based on the technical problem of providing a permanent cell line and processes which do not exhibit these disadvantages, i.e. which generally enable infectious rabies viruses to be replicated with an improved yield and with the appearance of the cytopathic effect (CPE), which can be used to detect them quantitatively in a sensitive manner.
This technical problem is solved by the provision of the embodiments which are characterized in the claims.
It has been found, surprisingly, that rabies viruses having a cytopathic effect (CPE) replicate in high yields in the novel permanent cell line PH-2. Other virus species from different virus families, such as picornaviruses, herpesviruses, paramyxoviruses, reoviruses and togaviruses, can likewise be replicated in this cell line. This cell line is suitable both for the production of virus antigens and for virus and antibody detection. It is particularly surprising that rabies virus, which belongs to the rhabdoviruses, always produces a cytopathic effect (CPE) when it is replicated in this cell line, as a result of which this cell line is particularly suitable for the simple detection and for the quantitative determination of rabies virus and (neutralizing) rabies virus antibodies.
Consequently, the invention relates to the permanent cell line PH-2 and to cell lines which are derived therefrom. The cell line PH-2 has been deposited in the DSM Deutsche Sammlung fur Mikroorganismen (German Collection of Microorganisms) in Braunschweig under number DSM ACC 2165.
In a preferred embodiment, the present invention relates to a process for re
REFERENCES:
patent: 4664912 (1987-05-01), Wiktor et al.
Cleide A. Consales et al., "Cytopathic effect induced by rabies virus in McCoy cells", Journal of Virological Methods, 27, pp. 277-286 (1990).
J. Egert et al., "Properties of Rabies Strain (,, Pasteur Potsdam) Adapted to Primary Dog Kidney Cells", Acta virol. 33:553-558 (1989).
Abigail L. Smith et al., "Isolation and Assay of Rabies Serogroup Viruses in CER Cells", Intervirology 8:92-99 (1977).
Bernhardt Dieter
Groner Albrecht
Behringwerke Aktiengesellschaft
Fuldner Rebecca
Woodward Michael P.
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