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 anal...

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Autores principales:	Justine Cheval, Erika Muth, Gaëlle Gonzalez, Muriel Coulpier, Pascale Beurdeley, Stéphane Cruveiller, Marc Eloit
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	4-thiouridine RNA-seq Vero biologicals high-throughput sequencing metagenomics Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9917511c9eb944cb851a21b7b3b67334
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spelling	oai:doaj.org-article:9917511c9eb944cb851a21b7b3b673342021-11-15T15:22:20ZAdventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids10.1128/mSphere.00298-192379-5042https://doaj.org/article/9917511c9eb944cb851a21b7b3b673342019-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00298-19https://doaj.org/toc/2379-5042ABSTRACT 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.Justine ChevalErika MuthGaëlle GonzalezMuriel CoulpierPascale BeurdeleyStéphane CruveillerMarc EloitAmerican Society for Microbiologyarticle4-thiouridineRNA-seqVerobiologicalshigh-throughput sequencingmetagenomicsMicrobiologyQR1-502ENmSphere, Vol 4, Iss 3 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	4-thiouridine RNA-seq Vero biologicals high-throughput sequencing metagenomics Microbiology QR1-502
spellingShingle	4-thiouridine RNA-seq Vero biologicals high-throughput sequencing metagenomics Microbiology QR1-502 Justine Cheval Erika Muth Gaëlle Gonzalez Muriel Coulpier Pascale Beurdeley Stéphane Cruveiller Marc Eloit Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids
description	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.
format	article
author	Justine Cheval Erika Muth Gaëlle Gonzalez Muriel Coulpier Pascale Beurdeley Stéphane Cruveiller Marc Eloit
author_facet	Justine Cheval Erika Muth Gaëlle Gonzalez Muriel Coulpier Pascale Beurdeley Stéphane Cruveiller Marc Eloit
author_sort	Justine Cheval
title	Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids
title_short	Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids
title_full	Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids
title_fullStr	Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids
title_full_unstemmed	Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids
title_sort	adventitious virus detection in cells by high-throughput sequencing of newly synthesized rnas: unambiguous differentiation of cell infection from carryover of viral nucleic acids
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/9917511c9eb944cb851a21b7b3b67334
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Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids

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