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) , 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 previously unknown virus infections of cell cultures. Transcriptome sequencing (RNA-Seq), whole exome sequencing (WES), and whole genome sequencing (WGS) data enabled us to screen cell lines for a multitude of viruses by analyzing 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 . 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. The NGS analysis showed that the human breast cancer cell line SK-BR-3 contains bovine polyoma virus sequences. Sequences of this virus can be found in fetal bovine serum. However, it is currently unclear, how this virus was able to infect the human cells, whereas no other cell line was shown to be infected.
The approach was also applied for the RNA-Seq data generated from a set of 100 leukemia and lymphoma cell lines (LL100) from the DSMZ cell culture collection . All virus infections previously detected by PCR were confirmed by NGS analysis and no further virus species were found.
The analysis of publicly available RNA-Seq data sets of further 640 human cell lines applying the Taxonomer tool showed previously unknown virus infections with bovine viral diarrhea virus, feline sarcoma virus, human papilloma virus type 18, Mason-Pfizer monkey virus, parainfluenza virus type 5, squirrel monkey retrovirus, and xenotropic murine leukemia virus.
During the corona virus pandemic, we screened a number of cell lines from the cell lines bank expressing the corona virus cellular entry proteins ACE2 and TMPRSS2 with respect to their permissiveness to SARS-CoV-2. CL-14, CL-40 (both colon carcinoma cell lines), and CAL-51 (breast carcinoma cell line) were newly shown to be highly permissive, whereas a few other cell lines proved to be low permissive. The cell lines can be useful tools for the study of SARS-CoV-2 cell infections .
Pommerenke C, Rand U, Uphoff CC, Nagel S, Zaborski M, Hauer V, Kaufmann M, Meyer C, Denkmann SA, Riese P, Eschke K, Kim Y, Macak Safranko Z, Kurolt I-C, Markotic A, Cicin-Sain L, Steenpaß L (2021) Identification of cell lines CL-14, CL-40 and CAL-51 as suitable models for SARS-CoV-2 infection studies. PLoS One (under review).
Quentmeier H, Pommerenke C, Dirks WG, Eberth S, Koeppel M, MacLeod RAF, Nagel S, Steube K, Uphoff CC, Drexler HG (2019) The LL-100 panel: 100 cell lines for blood cancer studies. Sci Rep 9 (1): 8218.
Uphoff,C.C., Pommerenke,C., Denkmann,S.A., Drexler,H.G. (2019) Screening human cell lines for viral infections applying RNA-Seq data analysis. PLoS One 14 (1): e0210404.
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).
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).