1. Field of the Invention
The present invention relates to a method for replication of hepatitis C virus (HCV) genome, cells for said replication and the use of said cells. The present invention also relates to methods for distinguishing a virus having high infectivity and virus having less infectivity. The methods are useful, for example, in a clinical test relating to HCV.
2. Related Art
The genome of HCV, the main cause of transfusion-related non-A, non-B hepatitis, has been cloned (Choo, W. L. et al., Science, 244, 359-362, 1989). Synthesis of cDNA by reverse transcription of viral RNA and amplification by PCR of the nucleotide sequence of the HCV cDNA has been useful for detecting HCV genome (Weiner, A. J. et al., Lancet, 335, 1-3, 1990). Immunodiagnostic assays for antibodies against HCV proteins have also been developed (Kuo G. et al., Science, 244, 362-364, 1989). Studies with chimpanzees have provided evidence that the virus is probably enveloped (Feinstone, S. M. et al., Infect. Immun, 41, 816-821, 1983), and is approximately 30-60 nm in diameter (He, L. F. et al., Infect Dis., 156, 636-640, 1987). Additional research, however, has been hampered because of the limited availability of chimpanzees and the relatively low titer of the virus in clinical samples.
Hillings et al. reported on the transmission of non-A, non-B hepatitis (presumably hepatitis C) to chimpanzees by inoculation with leucocytes, most probably T leucocytes, derived from either acutely or chronically infected patents or chimpanzees (Hellings, J. A. et al., J. Virol. Methods, 1985). In addition, it was also recently found that lactose dehydrogenase (LDH)-elevating virus, another unclassified enveloped RNA virus, replicates more efficiently in mouse cells when they are superinfected with murine leukemia virus (Inada, T. et al., Gen. Virol., 72, 2437-2444, 1991). EPC Unexamined Patent Publication No. 0,414,475A describes the replication of HCV in a cell line.
On the basis of cloning an HCV gene, methods for detecting the HCV using hybridization with a DNA probe have been developed. However, with the probe hybridization methods, it is impossible to distinguish a virus with high infectivity and a virus with less infectivity. Accordingly, to distinguish same it is necessary to inoculate a chimpanzee with a virus to be tested and test the infection thereof in the chimpanzee. However, since chimpanzees are expensive and the number of chimpanzees available is limited, an in-vitro method to distinguish a highly infective virus and a less infective virus is desirable.
Data on physical properties of HCV are being accumulated. Miyamoto et al. (J. Gen. Viral. 73: 713-718, 1992) estimated the density of HCV virions (as measured by RT/PCR) to be 1.08 g/ml by SDG centrifugation. In potassium bromide, a slightly higher density of 1.10 g/ml has been reported (Takahashi. K. et al., J. Gen. Virol. 73: 667-672, 1992). Bradly et al. (J. Med. Virol. 34: 206-206, 1991) found the density of infectious HCV (as measured by inoculation of chimpanzees) to be 1.09-1.11 g/ml in sucrose.