Skip to content
1887

Journal of General Virology header logo Journal of General Virology

Volume 106, Issue 11

Defective but promising: evaluating the utility of currently available bioinformatic pipelines for detecting defective viral genomes in RNA-Seq data Open Access

Abstract

Defective viral genomes (DVGs) affect viral dynamics, pathogenicity and evolution, have been found in many viral infections, and in theory can be detected from sequencing data. We explored the utility of the currently available bioinformatic programs ViReMa, DI-tector, DVGfinder, DG-Seq and VODKA2 for identifying junction points in plant virus high-throughput sequencing data, looking at whether the outputs from these bioinformatic tools generally agree and exploring the possibility of using these tools to help us understand whether DVGs are consistently generated and maintained in a specific virus-host combination. We conducted a meta-analysis of eight previously published RNA sequencing datasets utilizing all five programs and compared the degree of output overlap, the most common junctions present in each output and whether these junctions match previously reported junctions for that virus. Our results demonstrate a low degree of agreement regarding identified junctions between programs, including the most frequently identified one, although the most frequently identified junctions typically corresponded to large, disruptive deletions. We found preliminary support for our prevalence hypothesis, although we ultimately conclude that a more robust dataset generated expressly for testing this hypothesis will be required for a convincing answer. Finally, we suggest that when using bioinformatic programs to search for DVGs, it is best to run the same dataset through multiple programs and look at the overlap to inform decisions on downstream characterization.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): bioinformatics , defective interfering RNA , defective interfering virus and plant virus
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.002176
2025-11-17
2025-12-16

Metrics

Loading full text...

Full text loading...

/deliver/fulltext/jgv/106/11/jgv002176.html?itemId=/content/journal/jgv/10.1099/jgv.0.002176&mimeType=html&fmt=ahah

References

  1. Olmo-Uceda MJ, Muñoz-Sánchez JC, Lasso-Giraldo W, Arnau V, Díaz-Villanueva W et al. DVGfinder: a metasearch tool for identifying defective viral genomes in rna-seq data. Viruses 2022; 14:1–18 [View Article] [PubMed]
     [Google Scholar]
  2. Rand U, Kupke SY, Shkarlet H, Hein MD, Hirsch T et al. Antiviral activity of influenza A virus defective interfering particles against sars-cov-2 replication in vitro through stimulation of innate immunity. Cells 2021; 10:1756 [View Article] [PubMed]
     [Google Scholar]
  3. Szittya G, Molnár A, Silhavy D, Hornyik C, Burgyán J. Short defective interfering RNAs of tombusviruses are not targeted but trigger post-transcriptional gene silencing against their helper virus. Plant Cell 2002; 14:359–372 [View Article] [PubMed]
     [Google Scholar]
  4. Leeks A, Young PG, Turner PE, Wild G, West SA. Cheating leads to the evolution of multipartite viruses. PLoS Biol 2023; 21:e3002092 [View Article]
     [Google Scholar]
  5. Hillman BI, Carrington JC, Morris TJ. A defective interfering RNA that contains a mosaic of a plant virus genome. Cell 1987; 51:427–433 [View Article] [PubMed]
     [Google Scholar]
  6. Scholthof KBG, Scholthof HB, Jackson AO. The effect of defective interfering rnas on the accumulation of tomato bushy stunt virus proteins and implications for disease attenuation. Virology 1995; 211:324–328 [View Article]
     [Google Scholar]
  7. Van Magnus P. Incomplete forms of influenza virus. Adv Virus Res 1954; 2:59–79 [View Article]
     [Google Scholar]
  8. Budzyńska D, Zwart MP, Hasiów-Jaroszewska B. Defective RNA particles of plant viruses-origin, structure and role in pathogenesis. Viruses 2022; 14:2814 [View Article] [PubMed]
     [Google Scholar]
  9. Celix A, Rodriguez-Cerezo E, Garcia-Arenal F. New satellite RNAs, but no DI RNAs, are found in natural populations of tomato bushy stunt tombusvirus. Virology 1997; 239:277–284 [View Article] [PubMed]
     [Google Scholar]
  10. Knorr DA, Mullin RH, Hearne PQ, Morris TJ. De novo generation of defective interfering RNAs of tomato bushy stunt virus by high multiplicity passage. Virology 1991; 181:193–202 [View Article] [PubMed]
     [Google Scholar]
  11. Law MD, Morris TJ. De novo generation and accumulation of tomato bushy stunt virus defective interfering RNAs without serial host passage. Virology 1994; 198:377–380 [View Article] [PubMed]
     [Google Scholar]
  12. Budzyńska D, Minicka J, Hasiów‐Jaroszewska B, Elena SF. Molecular evolution of tomato black ring virus and de novo generation of a new type of defective RNAs during long‐term passaging in different hosts. Plant Pathology 2020; 69:1767–1776 [View Article]
     [Google Scholar]
  13. Mawassi M, Gafny R, Gagliardi D, Bar-Joseph M. Populations of citrus tristeza virus contain smaller-than-full-length particles which encapsidate sub-genomic RNA molecules. J Gen Virol 1995; 76 (Pt 3):651–659 [View Article] [PubMed]
     [Google Scholar]
  14. Mawassi M, Karasev AV, Mietkiewska E, Gafny R, Lee RF et al. Defective RNA molecules associated with citrus tristeza virus. Virology 1995; 208:383–387 [View Article] [PubMed]
     [Google Scholar]
  15. Romero J, Huang Q, Pogany J, Bujarski JJ. Characterization of defective interfering RNA components that increase symptom severity of broad bean mottle virus infections. Virology 1993; 194:576–584 [View Article] [PubMed]
     [Google Scholar]
  16. Stenger DC. Genotypic variability and the occurrence of less than genome-length viral dna forms in a field population of beet curly top geminivirus. Phytopathology 1996; 85:1316 [View Article]
     [Google Scholar]
  17. Albiach-Martí MR, Guerri J, de Mendoza AH, Laigret F, Ballester-Olmos JF et al. Aphid transmission alters the genomic and defective rna populations of citrus tristeza virus isolates. Phytopathology 2000; 90:134–138 [View Article] [PubMed]
     [Google Scholar]
  18. Visser PB, Brown DJF, Brederode FT, Bol JF. Nematode transmission of tobacco rattle virus serves as a bottleneck to clear the virus population from defective interfering RNAs. Virology 1999; 263:155–165 [View Article] [PubMed]
     [Google Scholar]
  19. Bora M, Manu M, Karki M. Exploring the mechanisms of interference, persistence and antiviral potential of defective interfering particles. Discov Viruses 2025; 2:1 [View Article]
     [Google Scholar]
  20. Levi LI, Rezelj VV, Henrion-Lacritick A, Erazo D, Boussier J et al. Defective viral genomes from chikungunya virus are broad-spectrum antivirals and prevent virus dissemination in mosquitoes. PLoS Pathog 2021; 17:e1009110 [View Article] [PubMed]
     [Google Scholar]
  21. Lin M-H, Li D, Tang B, Li L, Suhrbier A et al. Defective interfering particles with broad-acting antiviral activity for dengue, zika, yellow fever, respiratory syncytial and SARS-CoV-2 virus infection. Microbiol Spectr 2022; 10:e0394922 [View Article] [PubMed]
     [Google Scholar]
  22. Wasik MA, Eichwald L, Genzel Y, Reichl U. Cell culture-based production of defective interfering particles for influenza antiviral therapy. Appl Microbiol Biotechnol 2018; 102:1167–1177 [View Article] [PubMed]
     [Google Scholar]
  23. Wu M, Zhou E, Sheng R, Fu X, Li J et al. Defective interfering particles of influenza virus and their characteristics, impacts, and use in vaccines and antiviral strategies: a systematic review. Viruses 2022; 14:2773 [View Article] [PubMed]
     [Google Scholar]
  24. Yang Y, Lyu T, Zhou R, He X, Ye K et al. The antiviral and antitumor effects of defective interfering particles/genomes and their mechanisms. Front Microbiol 2019; 10:1–14 [View Article]
     [Google Scholar]
  25. Burgyan J, Grieco F, Russo M. A Defective interfering RNA Molecule in Cymbidium ringspot virus infections. J Gen Virol 1989; 70:235–239 [View Article]
     [Google Scholar]
  26. de Oliveira Resende R, de Haan P, de Avila AC, Kitajima EW, Kormelink R et al. Generation of envelope and defective interfering RNA mutants of tomato spotted wilt virus by mechanical passage. J Gen Virol 1991; 72:2375–2383 [View Article]
     [Google Scholar]
  27. de Oliveira Resende R, de Haan P, van de Vossen E, de avila AC, Goldbach R et al. Defective interfering L RNA segments of tomato spotted wilt virus retain both virus genome termini and have extensive internal deletions. J Gen Virol 1992; 73:2509–2516 [View Article]
     [Google Scholar]
  28. Inoue-Nagata AK, Kormelink R, Nagata T, Kitajima EW, Goldbach R et al. Effects of temperature and host on the generation of tomato spotted wilt virus defective interfering RNAs. Virology 1997; 87:1168–1173 [View Article]
     [Google Scholar]
  29. Li XH, Heaton LA, Morris TJ, Simon AE. Turnip crinkle virus defective interfering RNAs intensify viral symptoms and are generated de novo. Proc Natl Acad Sci USA 1989; 86:9173–9177 [View Article] [PubMed]
     [Google Scholar]
  30. White KA, Brancroft JB, Mackie GA. Defective RNAs of clover yellow mosaic virus encode nonstructural/coat protein fusion products. Virology 1991; 183:479–486 [View Article]
     [Google Scholar]
  31. González Aparicio LJ, López CB, Felt SA. A virus is a community: diversity within negative-sense RNA virus populations. Microbiol Mol Biol Rev 2022; 86:e0008621 [View Article] [PubMed]
     [Google Scholar]
  32. Routh A, Johnson JE. Discovery of functional genomic motifs in viruses with ViReMa-a Virus Recombination Mapper-for analysis of next-generation sequencing data. Nucleic Acids Res 2014; 42:1–10 [View Article] [PubMed]
     [Google Scholar]
  33. Beauclair G, Mura M, Combredet C, Tangy F, Jouvenet N et al. DI-tector: defective interfering viral genomes’ detector for next-generation sequencing data. RNA 2018; 24:1285–1296 [View Article] [PubMed]
     [Google Scholar]
  34. Boussier J, Munier S, Achouri E, Meyer B, Crescenzo-Chaigne B et al. RNA-seq accuracy and reproducibility for the mapping and quantification of influenza defective viral genomes. RNA 2020; 26:1905–1918 [View Article] [PubMed]
     [Google Scholar]
  35. Bosma TJ, Karagiannis K, Santana-Quintero L, Ilyushina N, Zagorodnyaya T et al. Identification and quantification of defective virus genomes in high throughput sequencing data using DVG-profiler, a novel post-sequence alignment processing algorithm. PLoS One 2019; 14:e0216944 [View Article] [PubMed]
     [Google Scholar]
  36. Achouri E, Felt SA, Hackbart M, Rivera-Espinal NS, López CB. VODKA2: a fast and accurate method to detect non-standard viral genomes from large RNA-seq data sets. RNA 2023; 30:16–25 [View Article] [PubMed]
     [Google Scholar]
  37. Sun Y, Kim EJ, Felt SA, Taylor LJ, Agarwal D et al. A specific sequence in the genome of respiratory syncytial virus regulates the generation of copy-back defective viral genomes. PLoS Pathog 2019; 15:e1007707 [View Article]
     [Google Scholar]
  38. Wu J, Bisaro DM. Cell-cell communication and initial population composition shape the structure of potato spindle tuber viroid quasispecies. Plant Cell 2024a; 36:1036–1055 [View Article]
     [Google Scholar]
  39. Wu J, Bisaro DM. Potato spindle tuber viroid (PSTVd) loop 27 mutants promote cell-to-cell movement and phloem unloading of the wild type: Insights into RNA-based viroid interactions. Virology 2024b; 597:110137 [View Article]
     [Google Scholar]
  40. Wu J, Zhang Y, Nie Y, Yan F, Zirbel CL et al. RNA three-dimensional structure drives the sequence organization of potato spindle tuber viroid quasispecies. PLoS Pathog 2024; 20:e1012142 [View Article] [PubMed]
     [Google Scholar]
  41. Chang YC, Borja M, Scholthof HB, Jackson AO, Morris TJ. Host effects and sequences essential for accumulation of defective interfering RNAs of cucumber necrosis and tomato bushy stunt tombusviruses. Virology 1995; 210:41–53 [View Article] [PubMed]
     [Google Scholar]
  42. Inoue-Nagata AK, Kormelink R, Sgro J-Y, Nagata T, Kitajima EW et al. Molecular characterization of tomato spotted Wilt virus defective interfering RNAs and detection of truncated L proteins. Virology 1998; 248:342–356 [View Article] [PubMed]
     [Google Scholar]
  43. Nagata T, Inoue-Nagata AK, Prins M, Goldbach R, Peters D. Impeded thrips transmission of defective tomato spotted wilt virus isolates. Phytopathology 2000; 90:454–459 [View Article] [PubMed]
     [Google Scholar]
  44. Dexheimer S, Shrestha N, Chapagain BS, Bujarski JJ, Yin Y. Characterization of variant RNAs encapsidated during bromovirus infection by high-throughput sequencing. Pathogens 2024; 13:96 [View Article] [PubMed]
     [Google Scholar]
  45. Damayanti TA, Nagano H, Mise K, Furusawa I, Okuno T. Positional effect of deletions on viability, especially on encapsidation, of Brome mosaic virus D-RNA in barley protoplasts. Virology 2002; 293:314–319 [View Article] [PubMed]
     [Google Scholar]
  46. Damayanti TA, Nagano H, Mise K, Furusawa I, Okuno T. Brome mosaic virus defective RNAs generated during infection of barley plants. J Gen Virol 1999; 80 (Pt 9):2511–2518 [View Article] [PubMed]
     [Google Scholar]
  47. Graves MV, Roossinck MJ. Characterization of defective RNAs derived from RNA 3 of the Fny strain of cucumber mosaic cucumovirus. J Virol 1995; 69:4746–4751 [View Article] [PubMed]
     [Google Scholar]
  48. Szittya G, Moxon S, Pantaleo V, Toth G, Rusholme Pilcher RL et al. Structural and functional analysis of viral siRNAs. PLoS Pathog 2010; 6:e1000838 [View Article] [PubMed]
     [Google Scholar]
  49. Ghasemazadeh A, Haar M, Shams-bakhsh M, Pirovano W, Pantaleo V. Shannon entropy to evaluate substitution rate variation among viral nucleotide positions in datasets of viral siRNAs. In Pantaleo V, Chiumenti M. eds Viral Metagenomics: Methods and Protocols Humana Press/Springer; 2018 pp 187–197
     [Google Scholar]
  50. Lv J, Deng M, Li Z, Zhu H, Wang Z et al. Integrative analysis of the transcriptome and metabolome reveals the response mechanism to tomato spotted wilt virus. Horticultural Plant J 2023; 9:958–970 [View Article]
     [Google Scholar]
/content/journal/jgv/10.1099/jgv.0.002176
Loading
Defective but promising: evaluating the utility of currently available bioinformatic pipelines for detecting defective viral genomes in RNA-Seq data
J. Gen. Virol. 106, 002176 (2025); https://doi.org/10.1099/jgv.0.002176
/content/journal/jgv/10.1099/jgv.0.002176
/content/journal/jgv/10.1099/jgv.0.002176
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An error occurred
Approval was partially successful, following selected items could not be processed due to error