1994 Fiscal Year Final Research Report Summary
HEPATITIS C VIRUS PARTICLE DETECTED BY IMMUNOELECTRON MICROSCOPIC STUDY
| Project/Area Number |
05807046
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Gastroenterology
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| Research Institution | MIE UNIVERSITY |
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Principal Investigator |
KAITO Masahiko MIE UNIVERSITY,FACULTY OF MEDICINE,ASSISTANT, 医学部, 助手 (70214244)
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| Co-Investigator(Kenkyū-buntansha) |
WATANABE Shozo MIE UNIVERSITY,CENTER FOR HEALTH MAINTENANCE,PROFESSOR, 保健管理センター, 教授 (20134934)
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| Project Period (FY) |
1993 – 1994
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| Keywords | HEPATITIS C VIRUS / VIRUS PARTICLE / ELECTRON MICROSCOPE / COLLOIDAL GOLD / PCR METHOD / MONOCLONAL ANTIBODY / PLASMA / ALANITE AMINOTRANSFERASE |
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| Research Abstract |
To clarify the morphology of hepatitis C virus (HCV), an indirect immunogold electron microscopic study was carried out on two plasma samples with high HCV-RNA titer using polyclonal and monoclonal antibodies specific to the putative HCV envelope protein. Quantification of HCV-RNA was accomplished by competitive PCR and twe plasma samples (subtype 1b) with more than 4x10^7 copies/ml were chosen. These plasma samples were ulrtacentrifuged. An approximately 1000-fold concentrated suspension was layred onto a continuous sucrose gradient. HCV-RNA titers in each sucrose fraction were assayd by competitive PCR.High HCV-RNA titiers more than 4x10^8 copies/ml were found at 1.14 to 1.16g/ml in samples.Spherical virus-like particles 55 to 65 nm in diameter with spike-like projections were found in these fractions. The morphological features of these particles were similar to those of flavivirus and found only in HCV-infected blood donors. The indirect immunogold electron microscopic procedure followed the method of Murti and Webster with a minor modification, using the polyclonal and monoclonal anti-HCV putative envelope antibodies (subtype 1b). These particles specifically reacted with the polyclonal and monoclonal antibodies and their antibody haloes were identified by specific gold lebelling. In conclusion, HCV particles are 55 to 65 nm diameter spherical particles with fine surface splike-like projections, 6 nm in length, and have a 30 to 35 nm inner core. HCV particles were obseved to be similar to flavivirus virions. This is the first known report in which the morphology of the HCV particle is clearly shown. Later, to clarify the localization of HCV envelope 2/NS1 protein on the surface of HCV particles, an indirect immunogold electron microscopic study was carried out on these samples using polyclonal and monoclonal antibodies specific to the putative HCV envelope 2/NSI protein. HCV envelope 2/NS1 protein was observed on the surface of the HCV particle.
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