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Detection of human pathogenic virusesDetection of human pathogenic viruses

Detection of Human Pathogenic Viruses


Contamination of cultured cells with human pathogenic viruses may pose health risks. Cell cultures of unknown origin or from non-approved sources should be regarded as potentially harmful until their infection status is known. The DSMZ tests all primate cell lines for a range of human pathogenic viruses by PCR/RT-PCR and other methods.

As known from transfusion and transplantation medicine, human cells can harbor several human pathogens putting sufficiently contagious to warrant testing. In particular, human pathogenic viruses, like human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus type I and II (HTLV-I and -II), and hepatitis viruses, e.g. hepatitis B virus (HBV) and hepatitis C virus (HCV), may be found in human donor material. Human tumor cell lines may be infected with these viruses or others linked to their tumors of origin, e.g. human herpesvirus type 8 (HHV-8) or human papilloma viruses (HPV). Indeed, some cell lines are known to harbor human pathogenic viruses, notably the classic widely distributed HeLa cell line which carries HPV integrated within its genome. Besides the donor-infected primary material, contaminations of cell cultures can also be introduced secondarily by laboratory personnel, or from other infected cells present in the lab.

Figure 1: EBV episomes attached to the chromosomes of the RAJI cell line.

In all human and other primate cell lines we perform polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR to detect specific DNA or RNA sequences of the human pathogenic viruses HIV-1, HTLV-I and -II, EBV (Epstein-Barr virus), HBV, and HCV. Additionally, select cell lines are tested for both ubiquitous viruses, e.g. squirrel monkey retrovirus (SMRV) and those targeting specific cell types, e.g. the Kaposi's sarcoma-associated herpes virus HHV-8, HPV, or xenotropic murine leukemia-virus related virus (XMRV). Many human and animal cell lines were also tested for reverse transcriptase activity to detect unspecified retroviruses (1).

EBV has the unique capability of immortalizing resting B cells and transforming these into permanently growing B-lymphoblastoid cell lines (B-LCL). As EBV is ubiquitously distributed in all human populations and more than 90% of the individuals are so infected, the detection of EBV is a priori not a safety issue. However, for quality control reasons and moreover due to the potential of EBV to transform B cells, we routinely determine the EBV infection status of cell lines. This helps distinguish B-LCLs from tumor cell lines. Figure 1 shows episomes of EBV attached to the chromosomes of the RAJI cell line established from a tumor (Burkitt lymphoma) in which EBV actually plays a pathologic role.

Figure 2: Western blot analysis with antibodies raised against the immediate early protein ZEBRA of EBV.

Whenever virus specific sequences are detected, we attempt to determine whether active viruses are produced by the infected cell line. Hence, we detect viral proteins linked to the replication of the viruses or harvest the suspected viruses by ultracentrifugation, resuspend the pellet, digest the free DNA of the eukaryotic cells which might stick to particles, and extract the remaining DNA for further PCR analyses. Figure 2 shows the expression of the immediate-early protein ZEBRA which initiates the EBV replication. For more detailed information, please consult our publications (2, 3).

 

 

References:
1. Eberle J, Seibl R: A new method for measuring reverse transcriptase activity by ELISA. J Virol Meth 40: 347-356 (1992). PubMedID 1282132
2. Uphoff CC, Habig S, Carbone A, Gaidano G, Drexler HG: HHV-8 infection is specific for cell lines derived from primary effusion (body cavity-based) lymphomas. Leukemia 12: 1806-1809 (1998). PubMedID 9823957
3. Uphoff CC, Denkmann SA, Steube KG, Drexler HG: Detection of EBV, HBV, HCV, HIV-1, HTLV-I and -II and SMRV in human and other primate cell lines. J Biomed Biotechnol, Vol. 2010: Article ID 904767, 23 pages (2010). PubMedID 20454443