Detection of Murine Leukemia Virus in the Epstein-Barr Virus-Positive Human B-Cell Line JY, Using a Computational RNA-Seq-Based Exogenous Agent Detection Pipeline, PARSES

Abstract

bMany cell lines commonly used for biological studies have been found to harbor exogenous agents such as the human tumor viruses Epstein-Barr virus (EBV) and human papillomavirus. Nevertheless, broad-based, unbiased approaches to globally assess the presence of ectopic organisms within cell model systems have not previously been available. We reasoned that highthroughput sequencing should provide unparalleled insights into the microbiomes of tissue culture cell systems. Here we have used our RNA-seq analysis pipeline, PARSES (Pipeline for Analysis of RNA-Seq Exogenous Sequences), to investigate the presence of ectopic organisms within two EBV-positive B-cell lines commonly used by EBV researchers. Sequencing data sets from both the Akata and JY B-cell lines were found to contain reads for EBV, and the JY data set was found to also contain reads from the murine leukemia virus (MuLV). Further investigation revealed that MuLV transcription in JY cells is highly active. We also identified a number of MuLV alternative splicing events, and we uncovered evidence of APOBEC3G (apolipoprotein B mRNAediting enzyme, catalytic polypeptide-like 3G)-dependent DNA editing. Finally, reverse transcription-PCR analysis showed the presence of MuLV in three other human B-cell lines (DG75, Ramos, and P3HR1 Cl.13) commonly used by investigators in the Epstein-Barr virus field. We believe that a thorough examination of tissue culture microbiomes using RNA-seq/PARSES-like approaches is critical for the appropriate utilization of these systems in biological studies.

Latent Expression of the Epstein-Barr Virus (EBV)-Encoded Major Histocompatibility Complex Class I TAP Inhibitor, BNLF2a , in EBV-Positive Gastric Carcinomas

“…Notably, very high murine leukemia virus (MuLV) read levels (1.5% of human mapped reads) were detected in the LMSU cell line ( Fig. 1B; see also Table S3 in the supplemental material), which likely reflects incidental laboratory infection of this cell line, similar to previous MuLV virome findings from our lab for other cell lines (9,13).…”

Epstein-Barr Virus and Human Herpesvirus 6 Detection in a Non-Hodgkin's Diffuse Large B-Cell Lymphoma Cohort by Using RNA Sequencing

“…Although IHC and PCR approaches have been important for the detection of infectious agents in cancers, they have also led to false discovery and/or controversy. Several groups, including ours, have utilized RNA sequencing (RNA-seq) for the discovery and investigation of infectious agents; for example, Merkel cell virus was linked to Merkel cell carcinoma (2), Fusobacterium was associated with colorectal carcinoma (3,4), EBV was studied with gastric carcinoma samples (5), murine leukemia virus (MuLV) was detected in human B-cell lines (6), and large sequencing databases were screened for oncoviruses (7). Next-generation sequencing (NGS) approaches have several advantages over previous detection methods for this type of study.…”

High-Throughput RNA Sequencing-Based Virome Analysis of 50 Lymphoma Cell Lines from the Cancer Cell Line Encyclopedia Project

“…The average number of reads per cell line was approximately 164 million, ranging from 74.6 million to 209.8 million. Based on our previous investigations of microbial agents in biological specimens and cell lines (13)(14)(15), these read depths were deemed to be well above the minimum levels required to detect meaningful pathological infections.…”