Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids

Abstract

The use of high-throughput sequencing (HTS) to identify viruses in biologicals differs from current molecular approaches, since its use enables an unbiased approach to detection without the need to design specific primers to preamplify target sequences. Its broad range of detection and analytical sensitivity make it an important tool to ensure that biologicals are free from adventitious viruses. Similar to other molecular methods, however, identification of viral sequences in cells by HTS does not prove viral infection, since this could reflect carryover of inert viral sequences from reagents or other sources or the presence of transcriptionally inactive cellular sequences. Due to the broad range of detection associated with HTS, the above can potentially be perceived as a drawback for the testing of pharmaceutical biological products using this method. In order to avoid the identification of inert viral sequences, we present a methodology based on metabolic RNA labeling and sequencing, which enables the specific identification of newly synthesized viral RNAs in infected cells, resulting in the ability to unambiguously distinguish active infection by DNA or RNA viruses from inert nucleic acids. In the present study, we report the ability to differentiate Vero cells acutely infected by a single-stranded positive-sense RNA virus (tick-borne encephalitis virus) from cells which have been in contact with nonreplicating virus particles. Additionally, we also found a laboratory contamination by the squirrel monkey retrovirus of our Vero cell line, which was derived from an Old World (African green) monkey, a type of contamination which until now has been identified only in cells derived from primates from the New World. IMPORTANCE The use of high-throughput sequencing (HTS) to identify viral contamination of biological products is extremely sensitive and provides a broad range of detection. Nevertheless, viral sequences identified can also be inert. Examples include contamination resulting from reagents or the presence of inactivated viruses in animal-derived components of the cell culture medium. We therefore developed a method that relies on the sequencing of newly synthesized RNAs, an unequivocal sign of the presence of a transcriptionally active virus. This improvement in the specificity of viral testing increases the acceptability of HTS as a standard test for cells used in manufacturing biologicals and in biotherapies.

Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection

“…Possible laboratory follow-ups include confirmation of intact full-length viral genomes 27,28 , expression analysis 29,30 and infectivity assays 4,31 . It is also essential to validate the HTS test under Good Manufacturing Practice in order to apply the method to new vaccine products as release specification tests, as a substitution of in vivo tests to detect adventitious virus and to potentially replace/substitute specific tests such as PCRs used in routine testing.…”

Report of the second international conference on next generation sequencing for adventitious virus detection in biologics for humans and animals

“… The development of five live, reference virus stocks for NGS evaluation and standardization and completion of 24-month stability studies [ 6 ]. The use of NGS for the detection of active viral RNAs by transcriptomics analysis [ 14 , 15 ]. …”

“…Dr. Marc Eloit (Institut Pasteur and PathoQuest) described newly developed processes for specifically identifying cell infection by live viruses, either through the analysis of the cell transcriptome by stranded RNA analysis or by metabolic labelling of RNAs [ 14 , 15 ]. Tested cells can be master cell lines or cell therapy drug substances, or cells used in in vitro assays to amplify very small amounts of virus that are present in for instance working or master cell lines, in raw materials, or in seeds.…”

“…The limit of detection using EBV-infected B95-8 cells in a background of non-infected lymphoid cells has been evaluated [ 15 ]. In terms of the range of detection of RNA-Seq, adventitious virus has been identified in a human lymphoid cell line [ 15 ], and contamination by squirrel monkey retrovirus (SMRV) in a Vero cell line has also been identified [ 14 ]. This technique has also been demonstrated to enable the differentiation between active and inert sequences in persistent viral infections [ 14 ].…”

“…In terms of the range of detection of RNA-Seq, adventitious virus has been identified in a human lymphoid cell line [ 15 ], and contamination by squirrel monkey retrovirus (SMRV) in a Vero cell line has also been identified [ 14 ]. This technique has also been demonstrated to enable the differentiation between active and inert sequences in persistent viral infections [ 14 ]. Regarding analytical sensitivity of RNA-Seq, acute infection at very low doses is detected early after infection [ 15 ].…”