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. 2001 Jun;7(3):370-5.
doi: 10.3748/wjg.v7.i3.370.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

J L Cheng  1 , B L Liu, Y Zhang, W B Tong, Z Yan, B F Feng
Affiliations

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

J L Cheng et al. World J Gastroenterol. 2001 Jun.

Abstract

Aim: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro.

Methods: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR.

Results: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion.

Conclusion: HCV may exist and remain functional in a cultured cell line for a long period.

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Figures

Figure 1
Figure 1
There was increasing development of cell aggregates of proliferative lymphoblast cells.
Figure 2
Figure 2
The immortal cell line of human peripheral blood mononuclear cells transformed by Epstein-Barr virus. The cell bodies are larger and sphereical. There are the false foot-like hairs or thorns on their surface. The cell nuclei are large and varied. The cells multiply and grow as cell regiments or clusters.
Figure 3
Figure 3
Identification of HCV RNA with RT-PCR in serum and PBMC with the chronic hepatitis C patient. Lane 1: negative control; Lane 2: the last wash; Lane 3: HCV RNA positive serum; Lane 4: PBMC from hepatitis C patient; Lane 5: positive control. Lane M: pGEM-72f(+)/Hae III markers
Figure 4
Figure 4
Identification of HCV RNA plus-strand with RT-PCR in the EBV transformed PBMC and the growth medium. Lane 1: Negative control; Lane 2: LCL cells; Lane 3: the last wash of LCL; Lane 4: growth medium; Lane 5: positive control; Lane M: pGEM-72f(+)/Hae III markers
Figure 5
Figure 5
HCV RNA minus strand with RT-PCR in the EBV transformed PBMC and the growth medium. Lane 1: Negative control; Lane 2: growth medium; Lane 3: In cells; Lane 4: Positive control; Lane M: pGEM-72f(+)/Hae III markers
Figure 6
Figure 6
Immunohistochemical SP method, DAB staining and hematoxylin counter staining of cell nuclei. NS3 antibody positive granules seen as a brown lump in the cytoplasm of LCL. × 400
Figure 7
Figure 7
Immunohistochemical SP method, DAB staining and hematoxylin counter-staining cell nuclei. Core antibody positive granules are seen as a brown lump with in the cytoplasm of LCL. × 400
Figure 8
Figure 8
Immunohistochemical negative control (anti-HBs replaced anti-HCV), a positive brown granule is not seen in the cytoplasm. × 400
Figure 9
Figure 9
The blue-black positive signals of HCV RNA are exhibited in the LCL cell plasma but not in the nucleolus. × 400
Figure 10
Figure 10
The blue-black signals are not exhibited in negative control cells in situ RT-PCR. × 400
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