Virus Diagnostics

Virus infections still represent a challenge regarding their detection, evaluation and handling in cell culture technology and particularly in pharmacological and medical applications. Accurate determination is impeded by structural heterogeneity of virus particles and their diverse life cycles in eukaryotic cells and higher organisms. The lack of knowledge of which viruses do possess the potential to infect different cultured cells and, in particular, which viruses are able to reproduce within the cells are further difficulties in this matter. Thus, until now there is no general and practical method for a comprehensive detection of viruses in cell cultures (which is, of course, similarly true for patients suffering from unspecified diseases). Usually, cell culture viruses (1) originate from an infection of a patient or donor, (2) are deliberately introduced into the cell culture (e.g. for immortalization), (3) might be transmitted secondarily during cell culture manipulation, e.g. xenotransplantation for tumorigenicity testing, by cross contamination from an infected culture, (4) by contaminated cell culture media supplements (e.g. fetal bovine serum; FBS) [2], or (5) from laboratory staff (e.g. adenovirus) due to poor aseptic practice or failure of microbiological safety cabinets.

We have shown that xeno- and polytropic murine leukemia viruses (X/P-MLV) are able to infect cells from numerous species and various tissues in vitro and to replicate in cell cultures. All human cell lines from the cell culture collection were investigated for the presence of murine leukemia viruses (MLV). A number of cell lines were identified to be contaminated with MLV and sequence analyses of the MLV PCR products and the complete MLV genomes revealed at least three groups of related MLV genotypes. Most of the cell lines were shown to produce active retroviruses. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma entities. A contamination of primary human cells from healthy volunteers could not be substantiated. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection.

Next generation sequencing (NGS) and the new bioinformatics tools promised to be useful for the detection of already known and potentially new virus infections of cell cultures. RNA-Seq, whole exome sequencing, and whole genome sequencing data enabled us to screen cell lines for a multitude of viruses by screening of nucleotide sequences not mapping to the human genome. The results were compared and cross-evaluated with data based on PCR analyses. In this study, we first investigated the NGS data sets of cell lines already tested by conventional PCR for the same set of viruses by aligning all reads of a data set to the genome sequences of the individual virus genomes. Next, we used the publicly available metagenomics analysis software Taxonomer to screen the data sets for all known viral nucleotide and protein sequences [13]. This approach provided the opportunity to evaluate the usefulness of the NGS data and of the analysis tools for the determination of virus contamination. It also enables a thorough validation of the currently applied panel of PCR assays for the characterization and risk assessment of virus infections in cell lines.

Selected References

  1. Uphoff CC, Pommerenke C, Denkmann SA, Drexler HG. Screening human cell lines for viral infections applying RNA-Seq data analysis. PLoS One 14: e0210404 (2019).
  2. Nagel S, Uphoff CC, Dirks WG, Pommerenke C, Meyer C, Drexler HG. Epstein Barr virus (EBV) activates NKL homeobox gene HLX in DLBCL. PLoS One 14: e0216898 (2019).
  3. Raymond BBA, Madhkoor R, Schleicher I, Uphoff CC, Turnbull L, Whitchurch CB, Rohde M, Padula MP, Djordjevic SP: Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae. Front Cell Infect Microbiol 8 :54( 2018 ).
  4. Quentmeier H, Pommerenke C, Bernhart SH, Dirks WG, Hauer V, Hoffmann S, Nagel S, Siebert R, Uphoff CC, Zaborski M, Drexler HG, Consortium IM-S. RBFOX2 and alternative splicing in B-cell lymphoma. Blood Cancer J 8: 77 (2018).
  5. Raymond BBA, Turnbull L, Jenkins C, Madhkoor R, Schleicher I, Uphoff CC, Whitchurch CB, Rohde M, Djordjevic SP. Mycoplasma hyopneumoniae resides intracellularly within porcine epithelial cells. Sci Rep8: 17697 (2018).
  6. Drexler HG, Dirks WG, MacLeod RA, Uphoff CC. False and mycoplasma-contaminated leukemia-lymphoma cell lines: time for a reappraisal. Int J Cancer 140(5): 1209-1214 (2017). 
  7. Quentmeier H, Pommerenke C, Ammerpohl O, Geffers R, Hauer V, MacLeod RA, Nagel S, Romani J, Rosati E, Rosen A, Uphoff CC, Zaborski M, Drexler HG. Subclones in B-lymphoma cell lines: isogenic models for the study of gene regulation. Oncotarget 7: 63456-63465 (2016).
  8. Quentmeier H, Drexler HG, Hauer V, MacLeod RA, Pommerenke C, Uphoff CC, Zaborski M, Berglund M, Enblad G, Amini RM. Diffuse Large B Cell Lymphoma Cell Line U-2946: Model for MCL1 Inhibitor Testing. PLoS One 11: e0167599 (2016).
  9. Uphoff CC, Lange S, Denkmann SA, Garritsen HS, Drexler HG. Prevalence and characterization of murine leukemia virus contamination in human cell lines. PLoS One10(4): e0125622 (2015). 
  10. Dirks WG, Uphoff CC. Quality control essentials in human cell culture: Cell line cross-contamination and microbiological infections. Animal Cell Biotechnology in Biologics Production: 102-113 (2014). 
  11. Uphoff CC, Drexler HG. Detection of Mycoplasma contamination in cell cultures. Curr Protoc Mol Biol106: 28/4/1-28/4/14 (2014).
  12. Uphoff CC, Drexler HG. Eradication of Mycoplasma contaminations from cell cultures. Curr Protoc Mol Biol106: 28/5/1-28/5/12 (2014).