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AIM To study persistence and replication ofhepatitis C virus(HCV)in patients’ peripheralblood mononuclear cells(PBMC)cultured invitro.METHODS Epstein-Barr virus(EBV)was usedto transform the hepatitis C virus from a HCVpositive patient to permanent lymphoblastoidceil lines(LCL).Positive and negative HCV RNAstrands of the cultured cells and growth mediawere detected by reverse transcriptase-polymerase chain reaction(RT-PCR)eachmonth.Core and NS5 proteins of HCV werefurther tested using immunohistochemical SPmethod and in situ RT-PCR.RESULTS HCV RNA positive strands wereconsistently detected the cultured cells for oneyear.The negative-strand RNA in LCL cells andthe positive-strand RNA in supernatants wereobserved intermittently.Immunohistochemicalresults medicated expression of HCV NS3 and Cproteins in LCL cytoplasm mostly.The positivesignal of PCR product was dark blue and mainlylocalized to the LCL cytoplasm.The RT-PCRsignal was eliminated by overnight RNasedigestion but not DNase digestion.CONCLUSION HCV may exist and remainfunctional in a cultured cell line for a longperiod.
AIM To study persistence and replication of hepatitis C virus (HCV) in patients’ peripheral blood mononuclear cells (PBMC) cultured invitro.METHODS Epstein-Barr virus (EBV) was used to transform the hepatitis C virus from a HCVpositive patient to permanent lymphoblastoid lines (LCL) . Positive and negative HCV RNAstrands of the cultured cells and growth media detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV werefurther tested using immunohistochemical SPmethod and in situ RT-PCR .RESULTS HCV RNA positive strands wereconsistently detected the cultured cells for oneyear. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants wereobserved intermittently. Immunohistochemical results of the medicated expression of HCV NS3 and Cproteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCRsignal was eliminated by overnight RNasedigestion but not DNase diges tion.CONCLUSION HCV may exist and remain functional in a cultured cell line for a long period.