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

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.

Figure 1

There was increasing development of cell aggregates of proliferative lymphoblast cells.

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

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

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

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

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

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

Immunohistochemical negative control (anti-HBs replaced anti-HCV), a positive brown granule is not seen in the cytoplasm. × 400

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

The blue-black signals are not exhibited in negative control cells in situ RT-PCR. × 400