Publications

Use of a new RNA next-generation sequencing approach for the specific detection of virus infection in cells

Audrey Brussela; Kerstin Brackb; Erika Muthc; Rudolf Zirwesc; Justine Chevalc; Charles Hebertc; Jean-Marie Charpinc; Alice Marinacib; Benoit Flana; Horst Ruppachb; Pascale Beurdeleyc; Marc Eloitcde a LFB, Courtaboeuf, France b Charles River Laboratories Germany GmbH, Erkrath, Germany c PathoQuest, Paris, France d National Veterinary School of Alfort, Paris-Est University, Maisons-Alfort, France e Pathogen Discovery Laboratory, Biology of Infection Unit, Institut Pasteur, Paris, France

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.