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

Abstract

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.

Sequencing of Historical Isolates, K-mer Mining and High Serological Cross-Reactivity with Ross River Virus Argue against the Presence of Getah Virus in Australia

“…Aside from biosample contamination, infection of cell lines with adventitious viruses is also a well-documented phenomenon [ 69 , 70 , 71 ]. For instance, Phasi Charoen-like virus and Cell fusing agent virus can be found in the Aag2 mosquito cell line [ 72 ], a rhabdovirus infects Sf9 cells [ 73 ], and a bovine polyomavirus infection was identified in the breast cancer cell line, SK-BR-3 [ 74 ]. Contamination of MARC-145 cultures with GETV was recently reported [ 22 ]; and herein we illustrate GETV contamination of NIH 3T3 cultures ( Table 1 , Bioproject PRJNA561663), although it is unlikely that all MARC-145 and NIH 3T3 cell cultures globally are similarly contaminated.…”

Single-Cell Heterogeneity of Cutaneous T-Cell Lymphomas Revealed Using RNA-Seq Technologies

“…Because of unbiased analysis of the entire transcriptome, RNA sequencing enables us to identify previously undescribed transcripts, such as lncRNAs, gene isoforms, or pathways of gene expression regulated by enhancer RNAs. Another advantage of the RNA-seq method is the ability to identify non-human transcripts, for example, those of viral origin, that can confirm or exclude a potential infectious aetiology of human diseases [ 16 , 17 ].…”

The LL-100 Cell Lines Panel: Tool for Molecular Leukemia–Lymphoma Research

“…It is particulary noteworthy to stress their detailed clinical annotation and their comprehensive profiling (encompassing morphology, immunophenotyping, cytogenetics, molecular analysis, cell culturing). The strengths of the panel include, furthermore, the intensive identity and quality control to which the cell lines have been subjected (domains like authentication and exclusion of cross-contamination; documentation of freedom from inadvertent mycoplasm and viral contamination; references [40][41][42][43]). Panel development was absolutely contingent upon the ability to exclude cross-contaminated and non-representative cell lines.…”