Skip to main content

Propagation and Titration of Influenza Viruses

  • Protocol
  • First Online:
Influenza Virus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1836))

Abstract

Influenza viruses are constantly circulating among humans, in which they cause seasonal epidemics of severe respiratory disease. Additionally, these zoonotic viruses infect different mammals and birds, from which new antigenic variants are occasionally transmitted to humans leading to devastating global pandemics. Surveillance programs, in which viruses from the main reservoir (waterfowl), intermediate hosts (like pigs and other farm animals), and other affected species are isolated and characterized, are crucial for the global influenza prevention strategy. This chapter gives an overview of the most commonly used methods for the propagation and titration of influenza viruses, which are key steps in surveillance procedures, as well as in vaccine development and basic research. Depending on the host and the viral strain, primary isolates are obtained from biological samples of different origin and subsequently amplified in embryonated chicken eggs or cell cultures. These propagation procedures are the focus of the first part of this chapter. Once the initial isolates have been amplified, virus titration methods based on particular characteristics of influenza viruses, such as their ability to agglutinate red blood cells (RBCs) or to induce cytopathic effects (CPE) in cell monolayers, are used to estimate the amount of viral particles. Such approaches, like the hemagglutination assay (HA assay), 50% tissue culture infectious dose (TCID50), or plaque assay, are included in the second part of this chapter. Although they are simple and cost-effective, some of these techniques have been partially replaced by faster and more sensitive methods based on the quantification of viral genomes, such as the quantitative real-time reverse transcription PCR (RT-qPCR), which is presented at the end of this section. The different protocols are explained in detail in order to facilitate the preparation and quantification of infectious virus stocks.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 249.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. World Health Organization (November 2016) Influenza fact sheet. http://www.who.int/mediacentre/factsheets/fs211/en/

  2. Parrish CR, Murcia PR, Holmes EC (2015) Influenza virus reservoirs and intermediate hosts: dogs, horses, and new possibilities for influenza virus exposure of humans. J Virol 89(6):2990–2994. https://doi.org/10.1128/jvi.03146-14

    Article  PubMed  CAS  Google Scholar 

  3. Yoon SW, Webby RJ, Webster RG (2014) Evolution and ecology of influenza A viruses. Curr Top Microbiol Immunol 385:359–375. https://doi.org/10.1007/82_2014_396

    Article  PubMed  CAS  Google Scholar 

  4. Neumann G, Kawaoka Y (2015) Transmission of influenza A viruses. Virology 479-480:234–246. https://doi.org/10.1016/j.virol.2015.03.009

    Article  PubMed  CAS  Google Scholar 

  5. Taubenberger JK, Kash JC (2010) Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe 7(6):440–451. https://doi.org/10.1016/j.chom.2010.05.009

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Neumann G, Noda T, Kawaoka Y (2009) Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 459(7249):931–939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Smith GJD, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus OG, Ma SK, Cheung CL, Raghwani J, Bhatt S, Peiris JSM, Guan Y, Rambaut A (2009) Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459(7250):1122–1125. https://doi.org/10.1038/nature08182

    Article  PubMed  CAS  Google Scholar 

  8. Lai S, Qin Y, Cowling BJ, Ren X, Wardrop NA, Gilbert M, Tsang TK, Wu P, Feng L, Jiang H, Peng Z, Zheng J, Liao Q, Li S, Horby PW, Farrar JJ, Gao GF, Tatem AJ, Yu H (2016) Global epidemiology of avian influenza A H5N1 virus infection in humans, 1997-2015: a systematic review of individual case data. Lancet Infect Dis 16(7):e108–e118. https://doi.org/10.1016/S1473-3099(16)00153-5

    Article  PubMed  PubMed Central  Google Scholar 

  9. Qi X, Qian YH, Bao CJ, Guo XL, Cui LB, Tang FY, Ji H, Huang Y, Cai PQ, Lu B, Xu K, Shi C, Zhu FC, Zhou MH, Wang H (2013) Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation. BMJ 347:f4752. https://doi.org/10.1136/bmj.f4752

    Article  PubMed  PubMed Central  Google Scholar 

  10. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z, Yang Y, Zhao X, Zhou L, Li X, Zou S, Zhang Y, Li X, Yang L, Guo J, Dong J, Li Q, Dong L, Zhu Y, Bai T, Wang S, Hao P, Yang W, Zhang Y, Han J, Yu H, Li D, Gao GF, Wu G, Wang Y, Yuan Z, Shu Y (2013) Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med 368(20):1888–1897. https://doi.org/10.1056/NEJMoa1304459

    Article  PubMed  CAS  Google Scholar 

  11. World Health Organization (2013) Global Influenza Surveillance and Response System (GISRS). http://www.who.int/influenza/gisrs_laboratory/en/

  12. US Center for Disease Control (CDC) (2013) Seasonal Influenza Activity and Surveillance US. http://www.cdc.gov/flu/weekly/fluactivitysurv.htm

  13. European Centre for Disease Prevention and Control (ECDC) (2013) European Influenza Surveillance Network (EISN). http://ecdc.europa.eu/en/activities/surveillance/EISN/Pages/index.aspx

  14. Goodpasture EW, Woodruff AM, Buddingh GJ (1931) The cultivation of vaccine and other viruses in the chorioallantoic membrane of chick embryos. Science 74(1919):371–372. https://doi.org/10.1126/science.74.1919.371

    Article  PubMed  CAS  Google Scholar 

  15. Tobita K, Sugiura A, Enomote C, Furuyama M (1975) Plaque assay and primary isolation of influenza A viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin. Med Microbiol Immunol 162(1):9–14

    Article  CAS  PubMed  Google Scholar 

  16. Hirst GK (1941) The agglutination of red cells by amniotic fluid of chick embryos infected with influenza virus. Science 94(2427):22–23. https://doi.org/10.1126/science.94.2427.22

    Article  PubMed  CAS  Google Scholar 

  17. Miller GL (1965) Improved measurement of influenza virus hemagglutinin titer. J Immunol 95(2):336–344

    PubMed  CAS  Google Scholar 

  18. Miller GL, Stanley WM (1944) Quantitative aspects of the red blood cells agglutination test for influenza virus. J Exp Med 79(2):185–195

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Reed LJ, Muench H (1938) A simple method of estimating fifty percent endpoints. Am J Hyg 27:493–497

    Google Scholar 

  20. Fouchier RA, Bestebroer TM, Herfst S, Van Der Kemp L, Rimmelzwaan GF, Osterhaus AD (2000) Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J Clin Microbiol 38(11):4096–4101

    PubMed  PubMed Central  CAS  Google Scholar 

  21. McGinnis J, Laplante J, Shudt M, George KS (2016) Next generation sequencing for whole genome analysis and surveillance of influenza A viruses. J Clin Virol 79:44–50. https://doi.org/10.1016/j.jcv.2016.03.005

    Article  PubMed  CAS  Google Scholar 

  22. Seong M-W, Cho SI, Park H, Seo SH, Lee SJ, Kim E-C, Park SS (2016) Genotyping influenza virus by next-generation deep sequencing in clinical specimens. Ann Lab Med 36(3):255–258. https://doi.org/10.3343/alm.2016.36.3.255

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. World Health Organization (2011) Manual for the laboratory diagnosis and virological surveillance of influenza. http://whqlibdoc.who.int/publications/2011/9789241548090_eng.pdf

  24. World Health Organization (2002) Manual on animal influenza diagnosis and surveillance. http://www.who.int/csr/resources/publications/influenza/en/whocdscsrncs20025rev.pdf

  25. World Health Organization (2012) Information for molecular diagnosis of influenza virus in humans. http://www.who.int/influenza/gisrs_laboratory/molecular_diagnosis_influenza_virus_humans_update_201211.pdf

  26. World Health Organization (2015) WHO information for molecular diagnosis of influenza virus (H1N1). http://www.who.int/influenza/gisrs_laboratory/molecular_diagnosis_influenza_virus_humans_update_201403rev201505.pdf

  27. Ito T, Suzuki Y, Takada A, Kawamoto A, Otsuki K, Masuda H, Yamada M, Suzuki T, Kida H, Kawaoka Y (1997) Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection. J Virol 71(4):3357–3362

    PubMed  PubMed Central  CAS  Google Scholar 

  28. Gaush CR, Hard WL, Smith TF (1966) Characterization of an established line of canine kidney cells (MDCK). Proc Soc Exp Biol and Med 122(3):931–935

    Article  CAS  Google Scholar 

  29. Gaush CR, Smith TF (1968) Replication and plaque assay of influenza virus in an established line of canine kidney cells. Appl Microbiol 16(4):588–594

    PubMed  PubMed Central  CAS  Google Scholar 

  30. Meguro H, Bryant JD, Torrence AE, Wright PF (1979) Canine kidney cell line for isolation of respiratory viruses. J Clin Microbiol 9(2):175–179

    PubMed  PubMed Central  CAS  Google Scholar 

  31. Davies HW, Appleyard G, Cunningham P, Pereira MS (1978) The use of a continuous cell line for the isolation of influenza viruses. Bull World Health Organ 56(6):991–993

    PubMed  PubMed Central  CAS  Google Scholar 

  32. Lazarowitz SG, Choppin PW (1975) Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology 68(2):440–454

    Article  CAS  PubMed  Google Scholar 

  33. Klenk HD, Rott R, Orlich M, Blodorn J (1975) Activation of influenza A viruses by trypsin treatment. Virology 68(2):426–439

    Article  CAS  PubMed  Google Scholar 

  34. Skehel JJ, Wiley DC (2000) Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69:531–569. https://doi.org/10.1146/annurev.biochem.69.1.531

    Article  PubMed  CAS  Google Scholar 

  35. Hartenstein V (2006) Blood cells and blood cell development in the animal kingdom. Annu Rev Cell Dev Biol 22:677–712. https://doi.org/10.1146/annurev.cellbio.22.010605.093317

    Article  PubMed  CAS  Google Scholar 

  36. Tobita K (1975) Permanent canine kidney (MDCK) cells for isolation and plaque assay of influenza B viruses. Med Microbiol Immunol 162(1):23–27

    Article  CAS  PubMed  Google Scholar 

  37. Spearman C (1908) The method of ‘Right and Wrong Cases’ (‘Constant Stimuli’) without Gauss’s formulae. Br J Psychol 2:227–242

    Google Scholar 

  38. Karber G (1931) Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Archiv für Experimentelle Pathologie und Pharmakologie 162:480–483

    Google Scholar 

  39. van Elden LJ, Nijhuis M, Schipper P, Schuurman R, van Loon AM (2001) Simultaneous detection of influenza viruses A and B using real-time quantitative PCR. J Clin Microbiol 39(1):196–200. https://doi.org/10.1128/jcm.39.1.196-200.2001

    Article  PubMed  PubMed Central  Google Scholar 

  40. Piralla A, Daleno C, Pariani E, Conaldi P, Esposito S, Zanetti A, Baldanti F (2013) Virtual quantification of influenza a virus load by real-time RT-PCR. J Clin Virol 56(1):65–68. https://doi.org/10.1016/j.jcv.2012.09.011

    Article  PubMed  CAS  Google Scholar 

  41. Scholtissek C, Muller K (1988) Effect of dimethylsulfoxide (DMSO) on virus replication and maturation. Arch Virol 100(1–2):27–35

    Article  CAS  PubMed  Google Scholar 

  42. Medeiros R, Escriou N, Naffakh N, Manuguerra JC, van der Werf S (2001) Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes. Virology 289(1):74–85. https://doi.org/10.1006/viro.2001.1121

    Article  PubMed  CAS  Google Scholar 

  43. Ito T, Suzuki Y, Mitnaul L, Vines A, Kida H, Kawaoka Y (1997) Receptor specificity of influenza A viruses correlates with the agglutination of erythrocytes from different animal species. Virology 227(2):493–499. https://doi.org/10.1006/viro.1996.8323

    Article  PubMed  CAS  Google Scholar 

  44. Matrosovich M, Matrosovich T, Garten W, Klenk HD (2006) New low-viscosity overlay medium for viral plaque assays. Virol J 3:63. https://doi.org/10.1186/1743-422x-3-63

    Article  PubMed  PubMed Central  Google Scholar 

  45. World Health Organization (2014) Information for molecular diagnosis of influenza virus (Update). http://www.who.int/influenza/gisrs_laboratory/molecular_diagnosis_influenza_virus_humans_update_201403.pdf

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Emilio Yángüez .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Karakus, U., Crameri, M., Lanz, C., Yángüez, E. (2018). Propagation and Titration of Influenza Viruses. In: Yamauchi, Y. (eds) Influenza Virus. Methods in Molecular Biology, vol 1836. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8678-1_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8678-1_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8677-4

  • Online ISBN: 978-1-4939-8678-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics