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Visualizing Virus-Derived dsRNA Using Antibody-Independent and -Dependent Methods

  • Sarah J. Poynter
  • Stephanie J. DeWitte-OrrEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1656)

Abstract

Long double-stranded (ds) RNA molecules are produced as a byproduct of viral replication. Studying virus-derived dsRNA is important for understanding virus replication, understanding host responses to virus infections, and as a diagnostic tool for virus presence and replication. Here, we describe four different techniques for visualizing dsRNA; two antibody-dependent methods (immunoblotting and immunocytochemistry), as well as two antibody-independent methods (differential digestion and acridine orange staining). The benefits and disadvantages of each technique are also discussed.

Key words

dsRNA Immunoblot Differential digestion Viral dsRNA Acridine orange Immunocytochemistry 

References

  1. 1.
    Jacobs BL, Langland JO (1996) When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA. Virology 219(2):339–349CrossRefGoogle Scholar
  2. 2.
    Ablasser A, Bauernfeind F, Hartmann G, Latz E, Fitzgerald KA, Hornung V (2009) RIG-I-dependent sensing of poly (dA: dT) through the induction of an RNA polymerase III–transcribed RNA intermediate. Nat Immunol 10(10):1065–1072CrossRefGoogle Scholar
  3. 3.
    DeWitte-Orr SJ, Mossman KL (2010) dsRNA and the innate antiviral immune response. Future Virology 5(3):325–341CrossRefGoogle Scholar
  4. 4.
    Alcami A, Koszinowski UH (2000) Viral mechanisms of immune evasion. Immunol Today 21(9):447–455CrossRefGoogle Scholar
  5. 5.
    Edy VG, Szekely M, Loviny T, Dreyer C (1976) Action of nucleases on double-stranded RNA. Eur J Biochem 61(2):563–572CrossRefGoogle Scholar
  6. 6.
    Weber F, Wagner V, Rasmussen SB, Hartmann R, Paludan SR (2006) Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol 80(10):5059–5064CrossRefGoogle Scholar
  7. 7.
    Aloni Y (1972) Extensive symmetrical transcription of simian virus 40 DNA in virus-yielding cells. PNAS 69(9):2404–2409CrossRefGoogle Scholar
  8. 8.
    Schonborn J, Oberstraβ J, Breyel E, Tittgen J, Schumacher J, Lukacs N (1991) Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res 19(11):2993–3000CrossRefGoogle Scholar
  9. 9.
    Doherty L, Poynter SJ, Aloufi A, DeWitte-Orr SJ (2016) Fish viruses make dsRNA in fish cells: characterization of dsRNA production in rainbow trout (Oncorhynchus mykiss) cells infected with viral haemorrhagic septicaemia virus, chum salmon reovirus and frog virus 3. J Fish Dis 39:1133–1137CrossRefGoogle Scholar
  10. 10.
    Lukács N (1994) Detection of virus infection in plants and differentiation between coexisting viruses by monoclonal antibodies to double-stranded RNA. J Virol Methods 47(3):255–272CrossRefGoogle Scholar
  11. 11.
    Pichlmair A, Schulz O, Tan CP, Rehwinkel J, Kato H, Takeuchi O, Akira S, Way M, Schiavo G, Reis e Sousa C (2009) Activation of MDA5 requires higher-order RNA structures generated during virus infection. J Virol 83(20):10761–10769CrossRefGoogle Scholar
  12. 12.
    Borst P, Weissmann C (1965) Replication of viral RNA, 8. Studies on the enzymatic mechanism of replication of ms2 RNA. PNAS 54(3):982–987CrossRefGoogle Scholar
  13. 13.
    Wolf K, Quimby MC (1962) Established Eurythermic line of fish cells in vitro. Science 23(3508):1065–1066CrossRefGoogle Scholar
  14. 14.
    Lannan CN, Winton JR, Fryer JL (1984) Fish cell lines: establishment and characterization of nine cell lines from salmonids. In Vitro 20(9):671–676CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of BiologyWilfrid Laurier UniversityWaterlooCanada
  2. 2.Departments of Health Sciences and BiologyWilfrid Laurier UniversityWaterlooCanada

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