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Infection of eukaryotic cells with viruses in vitro

A technology for eukaryotic cells and infecting cells, applied to viruses, antiviral agents, cells modified by introducing foreign genetic material, etc. low cost effect

Inactive Publication Date: 2003-05-28
达尼埃尔·法夫尔
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the 2.2.15 cell line has several disadvantages: 1) The 2.2.15 cell line produces infectious granules (Danne granules) at a very low rate
[0014] In summary, despite many attempts, the prior art has not described an efficient and reliable in vitro infection system for HBV and HCV (Robinson, 1996, pp. 1149, 1155, 1161; Koff, 1993, pp. 498-499; Hollinger, 1996, p. 2742; Ganem, 1996, p. 2709; RobinSon, 1996, p. 1189; Lohmann et al., 1999, its reference 6; Schinazi, 1999)

Method used

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  • Infection of eukaryotic cells with viruses in vitro
  • Infection of eukaryotic cells with viruses in vitro
  • Infection of eukaryotic cells with viruses in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] This example demonstrates the effective and stable infection of HepG2 cell line by HBV. The proof of HBV infection is supported by the following tests: the presence of HbsAg in the supernatant (1) and bound to the cells (2), (3), and (4); the transformation effect of HBV (5); dextran Overgrowth in the combined treatment of methasone and insulin (6); long-term insulation (7). (1) The presence of HBsAg in the cell culture supernatant

[0045]In order to detect HBsAg in the supernatant or on the cell surface, the AXsYM (Abbott Laboratories) routine test is usually used to detect the HBV surface antigen. In order to maintain a convenient concentration of protein in the sample to obtain accurate results, the following procedure is usually considered: usually the cell culture supernatant from the mock infection culture and HBV infection culture is mixed with AUSAB-negative, HBV-negative human serum. A final concentration of 50-90% serum is reached in the analyzed sample. The resu...

Embodiment 2

[0055] This example demonstrates the effective and stable infection of Huh-7 cell line by HBV. Proof of HBV infection is supported by long-term insulation.

[0056] According to the method of the present invention, Huh-7 cells are infected with low-titer HBV. The cells can then be kept as a monolayer for up to 35 days (Table 4a). During the entire incubation period, only about 75-80% of the volume of the complete medium is replaced every 5-7 days.

[0057] Table 4a

Embodiment 3

[0059] This example demonstrates the effective and stable infection of HepG2 cell line by HCV. The proof of HCV infection is supported by RNA detection (COBAS AmpliGor) in the supernatant and cytopathic effects.

[0060] To detect HCV, Roche's COBAS Amplicor HCV Monitor test is routinely used. In order to maintain a convenient concentration of protein in the sample to obtain accurate results, the following procedure is usually considered: usually the cell culture supernatant from the simulant infection culture and the HCV infection culture is mixed with HCV-negative human serum, the sample that will usually be analyzed In the final concentration of 50-90% serum. HCV negative serum was used as a negative control; as for the positive control, human plasma or human plasma from HCV infected patients with known HCV viremia was treated in a similar manner to samples from mock-infected cells and HCV-infected cell culture medium serum.

[0061] The results are given in the form of an inde...

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PUM

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Abstract

The invention related to a process for infecting eukaryotic cells with one or more virus species, preferentially hepatitis B or hepatitis C virus, as well as cell cultures infected by the same. To achieve this goal, plasma or serum obtained from individuals infected by viruses, preferentially hepadnaviridae and flaviviridae, is used an inoculum to infect established eukaryotic cell lines or primary cells, preferentially hepatocytes, which in turn produce viral particles. This process, the resulting infected cells and cell culture supernatant can be used in many situations, notably the screening of drug candidates, the detection of antibodies, the generation of a diagnostic kit and the production of vaccines.

Description

Invention field [0001] The present invention relates to a method for infecting eukaryotic cell lines or primary cells with viruses in vitro, and the resulting infected cell culture. To achieve this goal, plasma or serum obtained from individuals infected with viruses (preferably hepatoviridae and flaviviridae) is used as an inoculum to infect established eukaryotic cell lines or primary cells (preferably hepatocytes), Then virus particles are generated. This method, the resulting infected cells, and cell culture supernatants can be used in many situations, especially for screening drug candidates, detecting antibodies, generating diagnostic kits, and producing vaccines. [0002]The following references are either cited in the text or related to the prior art: Acs et al., Proc. Natl. Acad. Sci. USA, 84: 4641-4644, 1987 Agnello et al., Proc. Natl. Acad. Sci. USA , 96: 12766-12771, 1999 Aoki et al., Virology, 250: 140-150, 1998 Bchini et al., J. Virol., 64: 3125-3032, 1990 Bertolin...

Claims

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Application Information

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IPC IPC(8): G01N33/50A61K39/12A61K39/29A61P31/12C12N5/02C12N5/07C12N5/071C12N7/00C12N7/01C12Q1/70G01N33/15
CPCA61K39/292A61K39/29C12N7/00C12N2730/10134A61K39/12A61P31/12Y02A50/30
Inventor 达尼埃尔·法夫尔
Owner 达尼埃尔·法夫尔
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