Deneyer, N.
, Chin, P.
, Bayon-Vicente, G.
, Beurdeley, P.
, Cleveland, M.
, Colinet, A.
, Cruveiller, S.
, Eloit, M.
, Gilchrist, S.
, Hashiba, N.
, Lambert, C.
, Lembo, A.
, Leong, K.
, Michaels, B.
, Moreira, S.
, Nakashima, R.
, Olgiati, S.
, Salehi, N.
, Sun, Q.
, Uchida, K.
, Vandeputte, O.
, Yuan, Y.
, Yusa, K.
, Zanda, V.
and Khan, A.
(2026),
A collaborative study evaluating the detection of virus-infected cells by transcriptomic high-throughput sequencing, Microbiology Spectrum, [online], https://doi.org/10.1128/spectrum.00066-26, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=960518 (Accessed September 24, 2026)
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A collaborative study evaluating the detection of virus-infected cells by transcriptomic high-throughput sequencing
Published
Author(s)
Noémie Deneyer, Pei-Ju Chin, Guillaume Bayon-Vicente, Pascale Beurdeley, , Anne-Sophie Colinet, Stephane Cruveiller, Marc Eloit, Shanaz Gilchrist, Noriko Hashiba, Christophe Lambert, Antonio Lembo, Ka-Wai Leong, Brandye Michaels, Sandrine Moreira, Reiko Nakashima, Simone Olgiati, Nasrin Salehi, Qinyu Sun, Kazuhisa Uchida, Oliver Vandeputte, Yuzhe Yuan, Keisuke Yusa, Valeria Maria Zanda, Arifa Khan
Abstract
High-throughput sequencing (HTS) is an agnostic virus detection technology that can replace or supplement the conventional adventitious virus assays used for safety testing of biologics. Transcriptomic HTS is particularly suitable for the detection of replicating viruses. We have evaluated the sensitivity of virus detection by transcriptomic HTS using targeted and non-targeted bioinformatics analysis of infected cells spiked into a background of uninfected cells, mimicking material representing a test sample from product manufacturing. Eight laboratories tested a mix of Raji cells (expressing Epstein-Barr virus [EBV] RNAs without virus production) and EBV-negative Ramos cells at ratios ranging from 0.5 to 0.00005. Seven laboratories performed the analysis using different HTS protocols, while the eighth laboratory quantified EBV RNA recovery in all extracted samples by quantifying EBNA-1 by RT-ddPCR assay. All participants detected EBV transcripts at a 0.001 ratio with the targeted analysis and a 0.01 ratio with the non-targeted analysis (i.e., one infected cell in 100 uninfected cells), with four laboratories achieving detection at the 0.0001 ratio for both analysis. The results of the spiking studies demonstrated the capabilities of transcriptomic HTS for adventitious virus detection in cell lines used for the production of biologics, and highlight that optimization in the workflow can improve HTS virus detection.Citation
Microbiology Spectrum
Pub Type
Journals
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Keywords
biomanufacturing, NGS, digital PCR, dPCR
Citation
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Created September 22, 2026