Evaluation of high-throughput sequencing for replacing the conventional adventitious virus detection assays used for biologics

1. US Food and Drug Adminstration. Guidance for Industry: Characterizatoin and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications. http://www.fda.gov/downloads/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/vaccines/ucm202439.pdf (2010).

2. European Pharmacopoeia. Chapter 5.2.3. Cell substrates for the production of vaccines for human use. 5.0 (Council of Europe, 2005).

3. European Pharmacopoeia. Chapter 2.6.16. Tests for extraneous agents in viral vaccines for human use. 6.0 (Council of Europe, 2008).

4. WHO Expert Committee on Biological Standardization. in WHO Technical Report Series Vol. No. 978 Annex 3 (ed) 79–187 (World Health Organization, 2013).

5. Khan, A. S. et al. Emerging Methods for Virus Detection (U.S. Food and Drug Administration (FDA), 2015).

6. Lambert, C. et al. Considerations for optimization of high-throughput sequencing bioinformatics pipelines for virus detection. Viruses 10, 528, https://doi.org/10.3390/v10100528 (2018).

   Google Scholar 

7. Ng, S. H. et al. Current Perspectives on High-Throughput Sequencing (HTS) for adventitious virus detection: upstream sample processing and library preparation. Viruses 10, 566, https://doi.org/10.3390/v10100566 (2018).

   Google Scholar 

8. International Council for Hamonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). Q5a(r2): Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin (Food and Drug Administration, 2023).

9. Victoria, J. G. et al. Viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus. J. Virol. 84, 6033–6040, https://doi.org/10.1128/JVI.02690-09 (2010).

   Google Scholar 

10. Ma, H., Galvin, T. A., Glasner, D. R., Shaheduzzaman, S. & Khan, A. S. Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines. J. Virol. 88, 6576–6585, https://doi.org/10.1128/JVI.00780-14 (2014).

    Google Scholar 

11. Cox, M. M. & Hollister, J. R. FluBlok, a next-generation influenza vaccine manufactured in insect cells. Biologicals 37, 182–189, https://doi.org/10.1016/j.biologicals.2009.02.014 (2009).

    Google Scholar 

12. Dubin, G. et al. Investigation of a regulatory agency enquiry into potential porcine circovirus type 1 contamination of the human rotavirus vaccine, Rotarix: approach and outcome. Hum. Vaccin. Immunother. 9, 2398–2408, https://doi.org/10.4161/hv.25973 (2013).

    Google Scholar 

13. Walsh, G. Biopharmaceutical benchmarks 2014. Nat. Biotechnol. 32, 992–1000, https://doi.org/10.1038/nbt.3040 (2014).

    Google Scholar 

14. Sanchez-Martinez, Z. V., Alpuche-Lazcano, S. P., Stuible, M. & Durocher, Y. CHO cells for virus-like particle and subunit vaccine manufacturing. Vaccine 42, 2530–2542, https://doi.org/10.1016/j.vaccine.2024.03.034 (2024).

    Google Scholar 

15. Chin, P. J. et al. Virus detection by short read high throughput sequencing in a high virus low cellular background. NPJ Vaccines 10, 61. https://doi.org/10.1038/s41541-025-01104-1 (2025).

    Google Scholar 

16. Council of Europe. European Convention for the protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. EU Directive 2010/63/EU. (Council of Europe, 2010).

17. Russell, W. M. S. & Burch, R. L. The Principles of Humane Experimental Technique (Charles C. Thomas Publishing, 1959).

18. Khan, A. S. et al. A multicenter study to evaluate the performance of high-throughput sequencing for virus detection. mSphere 2, e00307–e00317, https://doi.org/10.1128/mSphere.00307-17 (2017).

    Google Scholar 

19. BEI Resources, N., NIH. NR-59622 Center for Biologics Evaluation and Research Next Generation Sequencing Virus Reagents, <https://www.beiresources.org/Catalog/AssaysPanels/NR-59622.aspx> (2024).

20. Goodacre, N., Aljanahi, A., Nandakumar, S., Mikailov, M. & Khan, A. S. A reference viral database (RVDB) to enhance bioinformatics analysis of high-throughput sequencing for novel virus detection. mSphere 3, e00069–18, https://doi.org/10.1128/mSphereDirect.00069-18 (2018).

    Google Scholar 

21. Chin, P. J. et al. Refinement of the reference viral database (RVDB) for improving bioinformatics analysis of virus detection by high-throughput sequencing (HTS). mSphere 10, e0028625. https://doi.org/10.1128/msphere.00286-25 (2025).

    Google Scholar 

22. Gauvin, L. et al. Respiratory infection of mice with mammalian reoviruses causes systemic infection with age and strain dependent pneumonia and encephalitis. Virol J 10, 67, https://doi.org/10.1186/1743-422X-10-67 (2013).

    Google Scholar 

23. van Erp, E. A. et al. Pathogenesis of respiratory syncytial virus infection in BALB/c mice differs between intratracheal and intranasal inoculation. Viruses 11, 508, https://doi.org/10.3390/v11060508 (2019).

    Google Scholar 

24. Gombold, J. et al. Systematic evaluation of in vitro and in vivo adventitious virus assays for the detection of viral contamination of cell banks and biological products. Vaccine 32, 2916–2926, https://doi.org/10.1016/j.vaccine.2014.02.021 (2014).

    Google Scholar 

25. Khan, A. S. & Study Group. Proposed 1st international virus reference standards for adventitious virus detection in biological products by next-generation sequencing (NGS) technologies (CBER-5) (WHO, 2020).

26. Charlebois, R. L. et al. Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection. J Vaccines 5, 61 (2020).

    Google Scholar 

27. Lerner, A. M., Cherry, J. D. & Finland, M. Hemagglutination with reoviruses. Virology 19, 58–65, https://doi.org/10.1016/0042-6822(63)90024-2 (1963).

    Google Scholar 

28. Smith, S. C., Krystofiak, E. & Ogden, K. M. Mammalian orthoreovirus can exit cells in extracellular vesicles. PLoS Pathog. 20, e1011637, https://doi.org/10.1371/journal.ppat.1011637 (2024).

    Google Scholar 

29. Barone, P. W. et al. Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next-generation sequencing (NGS). Biologicals 81, 101661. https://doi.org/10.1016/j.biologicals.2022.11.003 (2023).

    Google Scholar 

30. Graham, B. S., Perkins, M. D., Wright, P. F. & Karzon, D. T. Primary respiratory syncytial virus infection in mice. J. Med. Virol. 26, 153–162, https://doi.org/10.1002/jmv.1890260207 (1988).

    Google Scholar 

31. Khan, A. S., Chin, P.-J., Fuentes, S. M. & Tsou, J.-H. Study Group. A collaborative study to evaluate the proposed first WHO international reference panel for adventitious virus detection in biological products by high-throughput sequencing (HTS) technologies. https://cdn.who.int/media/docs/default-source/biologicals/call-for-comments/who_bs_2024.2471_1st-inter-ref-panel-for-adventitious-virus-detection_cber_khan–arifa.pdf?sfvrsn=e410a551_2 (2024).

32. European Pharmacopoeia. Chapter 2.6.1 Sterility (Council of Europe, 2005).

33. European Pharmacopoeia. EP 2.6.7 Mycoplasmas, 6th ed. Strasbourg, France (Council of Europe, 2010).

34. Japanese Pharmacopoeia. Chapter 4.06. Sterility test. Eighteenth Edition (Pharmaceuticals and Medical Devices Agency, 2021).

35. United States Pharmacopeia. USP 33/NF 28 <63> Mycoplasma Tests, Rockville, MD (United States Pharmacopeial Convention (USP), 2009).

36. U. S. Pharmacopeia. USP <71> Sterility tests, In: 2018 United States Pharmacopeia and National Formulary. USP 41-NF 36. U.S. Pharmacopeial Convention, Rockville MD. 5984–5991 (2018).

37. U. S. Food and Drug Administration. US FDA Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals. http://www.fda.gov/downloads/BiologicsBloodVaccines/SafetyAvailability/UCM162863.pdf (1993).

38. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J Mol Biol 215, 403–410, https://doi.org/10.1016/S0022-2836(05)80360-2 (1990).

    Google Scholar 

39. Bushnell, B. BBMap: sourceforge.net/projects/bbmap/. Accessed 7 March 2025.

40. Andrews, S. FastQC: a quality control tool for high throughput sequence data, 2010).

41. Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 37, 907–915 (2019).

    Google Scholar 

42. Li, W. & Godzik, A. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 1658–1659, https://doi.org/10.1093/bioinformatics/btl158 (2006).

    Google Scholar 

43. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120, https://doi.org/10.1093/bioinformatics/btu170 (2014).

    Google Scholar 

44. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760, https://doi.org/10.1093/bioinformatics/btp324 (2009).

    Google Scholar 

45. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079, https://doi.org/10.1093/bioinformatics/btp352 (2009).

    Google Scholar 

46. Danecek, P. et al. Twelve years of SAMtools and BCFtools. Gigascience 10, giab008. https://doi.org/10.1093/gigascience/giab008 (2021).

    Google Scholar 

47. Jiang, H., An, L., Lin, S. M., Feng, G. & Qiu, Y. A statistical framework for accurate taxonomic assignment of metagenomic sequencing reads. PLoS One 7, e46450, https://doi.org/10.1371/journal.pone.0046450 (2012).

    Google Scholar 

48. Shen, W. et al. KMCP: accurate metagenomic profiling of both prokaryotic and viral populations by pseudo-mapping. Bioinformatics 39, btac845, https://doi.org/10.1093/bioinformatics/btac845 (2023).

    Google Scholar 

49. Illumina. https://emea.support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html.

50. Institute, J. G. https://jgi.doe.gov/data-and-tools/software-tools/bbtools/.

51. Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890, https://doi.org/10.1093/bioinformatics/bty560 (2018).

    Google Scholar 

52. Shen, W., Le, S., Li, Y. & Hu, F. SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation. PLoS One 11, e0163962, https://doi.org/10.1371/journal.pone.0163962 (2016).

    Google Scholar 

53. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842, https://doi.org/10.1093/bioinformatics/btq033 (2010).

    Google Scholar 

54. Rice, P. M. EMBOSS User’s Guide: Practical Bioinformatics (Cambridge University Press, 2011).

55. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357–359, https://doi.org/10.1038/nmeth.1923 (2012).

    Google Scholar 

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Author notes

1. Jen-Hui Tsou

   Present address: Office of the Director, Facility of Biotechnology Resources, CBER USA FDA, Silver Spring, MD, USA

2. Manuel Noll

   Present address: Integrative Biological Sciences (InBioS) Bât. B22 Integrative Biological Sciences, Liège 1, Belgium

3. These authors contributed equally: Pei-Ju Chin, Jen-Hui Tsou, Alison Armstrong, Noémie Deneyer, Valeria Zanda.

Authors and Affiliations

1. Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA

   Pei-Ju Chin, Jen-Hui Tsou, Sandra M. Fuentes & Arifa S. Khan

2. Millipore Sigma, Rockville, MD, USA

   Alison Armstrong, Nikolay Korokhov, Alfonso Lavorgna, Afshin Sohrabi & Mary Whiteman

3. GSK, Wavre, Wavre, Belgium

   Noémie Deneyer

4. EMD Serono RBM S.p.A. Istituto di Ricerche Biomediche A. Marxer, Via Ribes 1 10010 Colleretto Giacosa, Italy, an affiliate of Merck KGaA, Darmstadt, Germany

   Valeria Zanda & Simone Olgiati

5. Akkodis Belgium c/o GSK, Evere, 1140, Belgium

   Sophie Ayama-Canden & Manuel Noll

6. GSK, Rixensart, Rixensart, Belgium

   Anne-Sophie Colinet, Christophe Lambert & Michel Protz

7. GSK Marburg, Marburg, Germany

   Shahjahan Shaid

Contributions

A.S.K. designed and coordinated the study, provided oversight for the data analysis, P-J. C. performed bioinformatics analysis and data interpretation, J-H.T. performed the lab work, S.F. prepared and distributed the test samples and provided support for the lab work; N.D., C.L., M.P. and S.S. were involved in the conception and design of the study. N.D., C.L., A.S.C., and M.P. selected and/or developed methods to carry out the study. M.N., S.A-C. and M.P. acquired the data. M.N., S.A-C., M.P., C.L., A.S.C. and N.D. analyzed and interpreted the results. A.A., A.S., S.O. and V.Z. were involved in the conception and design of the study. A.A., A.L., N.K., S.O., V.Z. and M.W. selected and/or developed methods to carry out the study. A.A., A.L., N.K., S.O., V.Z. and M.W. analyzed and interpreted the results. All authors were involved in drafting the manuscript or revising it critically for important intellectual content. All authors had full access to the data and approved the final manuscript before it was submitted by the corresponding author.

Corresponding author

Correspondence to Arifa S. Khan.

Competing interests

C.L., M.P., A-S.C., S.S. and N.D. are employees of the GSK group of companies and may hold shares in GSK; A.A., A.S., N.K., A.L., and M.W. are employees of the MilliporeSigma organization and may hold shares in MilliporeSigma. V.Z. and S.O. are employees of EMD Serono RBM S.p.A, and affiliate of Merck KGaA, Darmstadt, Germany, and may hold shared in Merck KGaA. All other authors declare no competing interests.

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