Detection of EBV, HBV, HCV, HIV-1, HTLV-I and -II, and SMRV in Human and Other Primate Cell Lines

The high prevalence of contaminated cell cultures suggests that viral contaminations might be distributed among cultures. We investigated more than 460 primate cell lines for Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus I and II (HTLV-I/-II), and squirrel monkey retrovirus (SMRV) infections for risk assessment. None of the cell lines were infected with HCV, HIV-1, or HTLV-I/-II. However, one cell line displayed reverse transcriptase activity. Thirty-nine cell lines harbored EBV DNA sequences. Studies on the lytic phase of EBV revealed that five cell lines produce EBV particles and six further cell lines produced EBV upon stimulation. One cell line contained an integrated HBV genome fragment but showed no virus production. Six cell lines were SMRV-infected. Newly established cell lines should be tested for EBV infections to detect B-lymphoblastoid cell lines (B-LCL). B-LCLs established with EBV from cell line B95-8 should be tested for SMRV infections

Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin

A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant

Screening human cell lines for viral infections applying RNA-Seq data analysis.

Monitoring viral infections of cell cultures is largely neglected although the viruses may have an impact on the physiology of cells and may constitute a biohazard regarding laboratory safety and safety of bioactive agents produced by cell cultures. PCR, immunological assays, and enzyme activity tests represent common methods to detect virus infections. We have screened more than 300 Cancer Cell Line Encyclopedia RNA sequencing and 60 whole exome sequencing human cell lines data sets for specific viral sequences and general viral nucleotide and protein sequence assessment applying the Taxonomer bioinformatics tool developed by IDbyDNA. The results were compared with our previous findings from virus specific PCR analyses. Both, the results obtained from the direct alignment method and the Taxonomer alignment method revealed a complete concordance with the PCR results: twenty cell lines were found to be infected with five virus species. Taxonomer further uncovered a bovine polyomavirus infection in the breast cancer cell line SK-BR-3 most likely introduced by contaminated fetal bovine serum. RNA-Seq data sets were more sensitive for virus detection although a significant proportion of cell lines revealed low numbers of virus specific alignments attributable to low level nucleotide contamination during RNA preparation or sequencing procedure. Low quality reads leading to Taxonomer false positive results can be eliminated by trimming the sequence data before analysis. One further important result is that no viruses were detected that had never been shown to occur in cell cultures. The results prove that the currently applied testing of cell cultures is adequate for the detection of contamination and for the risk assessment of cell cultures. The results emphasize that next generation sequencing is an efficient tool to determine the viral infection status of human cells

Prevalence and characterization of murine leukemia virus contamination in human cell lines.

Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. 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. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%

Nucleotide sequence alignment of MLV infected cell lines.

<p>The multiple sequence alignment was performed using Vector NTI. The multiple sequence file was then imported to showalign of the EMBOSS program collection. The numbers determine the position from the forward primer. Missing nucleotides are represented by a dash and nucleotides different to the consensus sequence are represented by the respective character. * Cell line sequences originating from the NCBI database.</p

PCR-Assays for the detection of MLV sequences in cell lines infected with MLV from contaminated cell lines.

<p>The cell lines NCI-H82 and VERO-B4 were infected with cell culture supernatant of the cell lines DEL, KELLY, KYSE-70, LCL-HO, NAMALWA.CSN/70, or NCEB-1. DNA of the infected cell lines was prepared after 44 or 45 days (dpi, days post infection). NCI-H82 and VERO-B4 were previously shown to be MLV-negative. Only the 604–642 bp bands and the 150–174 bp bands are specific for MLV contaminations determined by the outer and inner primers, respectively.</p

Primer Sequences.

<p>* <b>Primers used for preparing 5´-half of the MLV genome for sequencing</b></p><p>^<b>#</b><b>Primers used for preparing 3´-half of the MLV genome for sequencing</b></p><p>Primer Sequences.</p

Detection of RT activity in human and mouse (ELM-I-1 and PSI-2) cell culture supernatants by PERT assay.

<p>The assay was carried out applying two different buffers containing MgCl₂ or MnCl₂, respectively. The Mn²⁺-buffer was more sensitive in most cases, indicating that the contaminating viruses presumably belong to the Mn²⁺-dependent C-type retroviruses. The CML-T1 cell line was MLV-PCR positive due to contamination of the genomic DNA with mouse derived DNA. The size of the MS2 PCR product is 112 bp.</p

PCR-Assays for the sensitive and reliable detection of MLV sequences in cell line DNA.

<p>The upper two panels show the ethidium bromide stained gels of the MLV-specific PCR assays performed with the outer (first panel) and inner primers (second panel). The sizes of the MLV-specific bands are 604–642 bp and 150–174 bp, respectively, depending on the MLV genotype. Some cell lines produce an unspecific band with the outer primers (also seen in some MLV-negative cell lines). The inner primers usually also produce several unspecific bands seen only in MLV-positive cell lines. The cell lines KYSE-30 and NAMALWA are MLV-negative in the assay, whereas the cell lines from the same series or subclones are MLV-positive, respectively. The third panel demonstrates the integrity of the DNAs used for the analyses by amplification of the human ABL gene. The lower panel shows contamination of the genomic DNA with mouse DNA by amplification of mouse specific IAP coding sequences. The PCR produces a double band of approximately 300 bp. The cell line NCEB-1 harbors several mouse-derived chromosomes.</p