Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Influenza A virus isolation, culture and identification

Nature Protocols volume 9, pages 2663–2681 (2014)Cite this article

Subjects

Abstract

Influenza A viruses (IAVs) cause epidemics and pandemics that result in considerable financial burden and loss of human life. To manage annual IAV epidemics and prepare for future pandemics, an improved understanding of how IAVs emerge, transmit, cause disease and acquire pandemic potential is urgently needed. Fundamental techniques essential for procuring such knowledge are IAV isolation and culture from experimental and surveillance samples. Here we present a detailed protocol for IAV sample collection and processing, amplification in chicken eggs or mammalian cells, and identification from samples containing unknown pathogens. This protocol is robust, and it allows for the generation of virus cultures that can be used for downstream analyses. Once experimental or surveillance samples are obtained, virus cultures can be generated and the presence of IAVs can be verified in 3–5 d via reverse-transcription (RT)-PCR or hemagglutination assay. Increased time frames may be required for less experienced laboratory personnel, or when large numbers of samples will be processed.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: IAV isolation, culture and identification protocol flowchart.
Figure 2: Embryonated egg inoculation and allantoic fluid collection.
Figure 3: IAV-induced cytopathic effects (CPEs) in MDCK cells.
Figure 4: Hemagglutination assay.
Figure 5: RT-PCR analysis.

Similar content being viewed by others

References

  1. World Health Organization. Influenza (Seasonal) Fact Sheet No. 211 http://www.who.int/mediacentre/factsheets/fs211/en/index.html (2013).

  2. Taubenberger, J.K. & Morens, D.M. 1918 influenza: the mother of all pandemics. Emerg. Infect. Dis. 12, 15–22 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Tong, S. et al. A distinct lineage of influenza A virus from bats. Proc. Natl. Acad. Sci. USA 109, 4269–4274 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. World Health Organization. Influenza at the Human-Animal Interface–Summary and Assessment as of 27 June 2014. http://www.who.int/influenza/human_animal_interface/Influenza_Summary_IRA_HA_interface_27June14.pdf?ua=1 (2014).

  5. Imai, M. et al. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486, 420–428 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Herfst, S. et al. Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336, 1534–1541 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chen, L.M. et al. In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity. Virology 422, 105–113 (2012).

    Article  CAS  PubMed  Google Scholar 

  8. Gao, R. et al. Human infection with a novel avian-origin influenza A (H7N9) virus. N. Engl. J. Med. 368, 1888–1897 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. World Health Organization. WHO Risk Assessment, Human Infections with Avian Influenza A(H7N9) Virus, Summary of Surveillance and Investigation Findings as of 27 June 2014. http://www.who.int/influenza/human_animal_interface/influenza_h7n9/riskassessment_h7n9_27june14.pdf?ua=1 (2014).

  10. Qi, X. et al. Probable person-to-person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation. BMJ 347, f4752–4759 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Watanabe, T. et al. Characterization of H7N9 influenza A viruses isolated from humans. Nature 501, 551–555 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Belser, J.A. et al. Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice. Nature 501, 556–559 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhu, H. et al. Infectivity, transmission, and pathology of human-isolated H7N9 influenza virus in ferrets and pigs. Science 341, 183–186 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Zhang, Q. et al. H7N9 influenza viruses are transmissible in ferrets by respiratory droplet. Science 341, 410–414 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. World Health Organization. Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza. http://whqlibdoc.who.int/publications/2011/9789241548090_eng.pdf (2011).

  16. World Health Organization. Manual on Animal Influenza Diagnosis and Surveillance. http://www.who.int/csr/resources/publications/influenza/en/whocdscsrncs20025rev.pdf (2002).

  17. World Health Organization. WHO Information for Molecular Diagnosis of Influenza Virus in Humans. http://www.who.int/influenza/gisrs_laboratory/molecular_diagnosis_influenza_virus_humans_update_201211.pdf (2012).

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

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

  20. ECDC. European Influenza Surveillance Network (EISN) 〈http://ecdc.europa.eu/en/activities/surveillance/EISN/Pages/index.aspx〉 (2013).

  21. World Health Organization. WHO Collaborating Centres for Influenza and Essential Regulatory Laboratories. http://www.who.int/influenza/gisrs_laboratory/collaborating_centres/list/en/ (2013).

  22. Fouchier, R.A. et al. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J. Clin. Microbiol. 38, 4096–4101 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. van Elden, L.J., Nijhuis, M., Schipper, P., Schuurman, R. & van Loon, A.M. Simultaneous detection of influenza viruses A and B using real-time quantitative PCR. J. Clin. Microbiol. 39, 196–200 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Goodpasture, E.W., Woodruff, A.M. & Buddingh, G.J. The cultivation of vaccine and other viruses in the chorioallantoic membrane of chick embryos. Science 74, 371–372 (1931).

    Article  CAS  PubMed  Google Scholar 

  25. Ito, T. et al. 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, 3357–3362 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lu, B., Zhou, H., Chan, W., Kemble, G. & Jin, H. Single amino acid substitutions in the hemagglutinin of influenza A/Singapore/21/04 (H3N2) increase virus growth in embryonated chicken eggs. Vaccine 24, 6691–6693 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Lu, B., Zhou, H., Ye, D., Kemble, G. & Jin, H. Improvement of influenza A/Fujian/411/02 (H3N2) virus growth in embryonated chicken eggs by balancing the hemagglutinin and neuraminidase activities, using reverse genetics. J. Virol. 79, 6763–6771 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Stevens, J. et al. Receptor specificity of influenza A H3N2 viruses isolated in mammalian cells and embryonated chicken eggs. J. Virol. 84, 8287–8299 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Katz, J.M. & Webster, R.G. Efficacy of inactivated influenza A virus (H3N2) vaccines grown in mammalian cells or embryonated eggs. J. Infect. Dis. 160, 191–198 (1989).

    Article  CAS  PubMed  Google Scholar 

  30. Kodihalli, S., Justewicz, D.M., Gubareva, L.V. & Webster, R.G. Selection of a single amino acid substitution in the hemagglutinin molecule by chicken eggs can render influenza A virus (H3) candidate vaccine ineffective. J. Virol. 69, 4888–4897 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Gaush, C.R., Hard, W.L. & Smith, T.F. Characterization of an established line of canine kidney cells (MDCK). Proc. Soc. Exp. Biol. Med. 122, 931–935 (1966).

    Article  CAS  PubMed  Google Scholar 

  32. Gaush, C.R. & Smith, T.F. Replication and plaque assay of influenza virus in an established line of canine kidney cells. Appl. Microbiol. 16, 588–594 (1968).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tobita, K., Sugiura, A., Enomote, C. & Furuyama, M. 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, 9–14 (1975).

    Article  CAS  PubMed  Google Scholar 

  34. Davies, H.W., Appleyard, G., Cunningham, P. & Pereira, M.S. The use of a continuous cell line for the isolation of influenza viruses. Bull. World Health Organ. 56, 991–993 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Meguro, H., Bryant, J.D., Torrence, A.E. & Wright, P.F. Canine kidney cell line for isolation of respiratory viruses. J. Clin. Microbiol. 9, 175–179 (1979).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lazarowitz, S.G. & Choppin, P.W. Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology 68, 440–454 (1975).

    Article  CAS  PubMed  Google Scholar 

  37. Klenk, H.D., Rott, R., Orlich, M. & Blodorn, J. Activation of influenza A viruses by trypsin treatment. Virology 68, 426–439 (1975).

    Article  CAS  PubMed  Google Scholar 

  38. Skehel, J.J. & Wiley, D.C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu. Rev. Biochem. 69, 531–569 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Hatakeyama, S. et al. Enhanced expression of an α2,6-linked sialic acid on MDCK cells improves isolation of human influenza viruses and evaluation of their sensitivity to a neuraminidase inhibitor. J. Clin. Microbiol. 43, 4139–4146 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Hirst, G.K. The agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus. Science 94, 22–23 (1941).

    Article  CAS  PubMed  Google Scholar 

  41. Francis, T., Pearson, H.E., Salk, J.E. & Brown, P.N. Immunity in human subjects artificially infected with influenza virus, type B. Am. J. Public Health Nations Health 34, 317–334 (1944).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ito, T. et al. Receptor specificity of influenza A viruses correlates with the agglutination of erythrocytes from different animal species. Virology 227, 493–499 (1997).

    Article  CAS  PubMed  Google Scholar 

  43. Medeiros, R., Escriou, N., Naffakh, N., Manuguerra, J.C. & van der Werf, S. Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes. Virology 289, 74–85 (2001).

    Article  CAS  PubMed  Google Scholar 

  44. Wiriyarat, W. et al. Erythrocyte binding preference of 16 subtypes of low-pathogenic avian influenza and 2009 pandemic influenza A (H1N1) viruses. Vet. Microbiol. 146, 346–349 (2010).

    Article  PubMed  Google Scholar 

  45. Gavin, P.J. & Thomson, R.B. Jr. Review of rapid diagnostic tests for influenza. Clin. Appl. Immunol. Rev. 4, 151–172 (2003).

    Article  Google Scholar 

  46. Hurt, A.C., Alexander, R., Hibbert, J., Deed, N. & Barr, I.G. Performance of six influenza rapid tests in detecting human influenza in clinical specimens. J. Clin. Virol. 39, 132–135 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Nutter, S. et al. Evaluation of indirect fluorescent antibody assays compared to rapid influenza diagnostic tests for the detection of pandemic influenza A (H1N1) pdm09. PLoS ONE 7, e33097 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Al Johani, S.M., Al Balawi, M., Al Alwan, B., Al Hefdhi, R. & Hajeer, A. Validity of two rapid point-of-care influenza tests and direct fluorescence assay in comparison of real-time PCR for swine of origin influenza virus. J. Infect. Public Health 4, 7–11 (2011).

    Article  CAS  PubMed  Google Scholar 

  49. Rahman, M. et al. Performance of Directigen flu A+B enzyme immunoassay and direct fluorescent assay for detection of influenza infection during the 2004–2005 season. Diagn. Microbiol. Infect. Dis. 58, 413–418 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Hirst, G.K. The quantitative determination of influenza virus and antibodies by means of red cell agglutination. J. Exp. Med. 75, 49–64 (1942).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rowe, T. et al. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J. Clin. Microbiol. 37, 937–943 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. World Health Organization. Serological Diagnosis of Influenza by Microneutralization Assay. http://www.who.int/influenza/gisrs_laboratory/2010_12_06_serological_diagnosis_of_influenza_by_microneutralization_assay.pdf (2006).

  53. St. George, K. Diagnosis of influenza virus. In Influenza Virus: Methods and Protocols Methods in Molecular Biology (eds. Kawaoka, Y. & Neumann, G.) Ch. 4, (Humana Press, 2012).

  54. Hoffmann, E., Stech, J., Guan, Y., Webster, R.G. & Perez, D.R. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146, 2275–2289 (2001).

    Article  CAS  PubMed  Google Scholar 

  55. Lee, C.W., Senne, D.A. & Suarez, D.L. Development and application of reference antisera against 15 hemagglutinin subtypes of influenza virus by DNA vaccination of chickens. Clin. Vaccine Immunol. 13, 395–402 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. US Centers for Disease Control. Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th edn, http://www.cdc.gov/biosafety/publications/bmbl5/ (2009).

  57. Kostka, V. & Carpenter, F.H. Inhibition of chymotrypsin activity in crystalline trypsin preparations. J. Biol. Chem. 239, 1799–1803 (1964).

    Article  CAS  PubMed  Google Scholar 

  58. World Health Organization. WHO Collaborating Centres. http://www.who.int/collaboratingcentres/en/ (2013).

  59. Daley, P., Castriciano, S., Chernesky, M. & Smieja, M. Comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients. J. Clin. Microbiol. 44, 2265–2267 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Walsh, P. et al. Comparison of respiratory virus detection rates for infants and toddlers by use of flocked swabs, saline aspirates, and saline aspirates mixed in universal transport medium for room temperature storage and shipping. J. Clin. Microbiol. 46, 2374–2376 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Munywoki, P.K. et al. Improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time PCR using nasopharyngeal flocked swabs. J. Clin. Microbiol 49, 3365–3367 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Hernes, S.S. et al. Swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon flocked swabs. Eur. J. Clin. Microbiol. Infect. Dis. 30, 159–165 (2011).

    Article  CAS  PubMed  Google Scholar 

  63. Wadowsky, R.M., Laus, S., Libert, T., States, S.J. & Ehrlich, G.D. Inhibition of PCR-based assay for Bordetella pertussis by using calcium alginate fiber and aluminum shaft components of a nasopharyngeal swab. J. Clin. Microbiol. 32, 1054–1057 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Wiebe, J.P. & Dinsdale, C.J. Inhibition of cell proliferation by glycerol. Life Sci. 48, 1511–1517 (1991).

    Article  CAS  PubMed  Google Scholar 

  65. Miller, G.L. Influence of pH and of certain other conditions on the stability of the infectivity and red cell agglutinating activity of influenza virus. J. Exp. Med. 80, 507–520 (1944).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Wang, X., Zoueva, O., Zhao, J., Ye, Z. & Hewlett, I. Stability and infectivity of novel pandemic influenza A (H1N1) virus in blood-derived matrices under different storage conditions. BMC Infect. Dis. 11, 354 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Beato, M.S. et al. Infectivity of H7 LP and HP influenza viruses at different temperatures and pH and persistence of H7 HP virus in poultry meat at refrigeration temperature. Virology 433, 522–527 (2012).

    Article  CAS  PubMed  Google Scholar 

  68. Greiff, D., Blumenthal, H., Chiga, M. & Pinkerton, H. The effects on biological materials of freezing and drying by vacuum sublimation. II. Effect on influenza virus. J. Exp. Med. 100, 89–101 (1954).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. World Health Organization. Collecting, Preserving and Shipping Specimens for the Diagnosis of Avian Influenza A(H5N1) Virus Infection. http://www.who.int/csr/resources/publications/surveillance/CDS_EPR_ARO_2006_1.pdf (2006).

Download references

Acknowledgements

The authors are grateful to M. Hatta, S. Fan, J. Ping, Z. Najacht and A. Karasin for helpful discussions, to P. Jester and Z. Najacht for assistance with hemagglutination assay and RT-PCR figure components and to S. Watson for scientific editing of the manuscript. This work was supported by grants-in-aid from the Ministry of Health, Labour and Welfare, Japan, by ERATO (Japan Science and Technology Agency), by US National Institute of Allergy and Infectious Diseases (NIAID) Public Health Service research grants and by a NIAID-funded Center for Research on Influenza Pathogenesis grant (CRIP, HHSN266200700010C).

Author information

Authors and Affiliations

  1. Department of Pathobiological Sciences, Influenza Research Institute, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA

    Amie J Eisfeld, Gabriele Neumann & Yoshihiro Kawaoka

  2. Exploratory Research for Advanced Technology (ERATO) Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan

    Yoshihiro Kawaoka

  3. Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan

    Yoshihiro Kawaoka

  4. Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan

    Yoshihiro Kawaoka

Authors
  1. Amie J Eisfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gabriele Neumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yoshihiro Kawaoka
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.J.E., G.N. and Y.K. wrote the manuscript.

Corresponding author

Correspondence to Yoshihiro Kawaoka.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Eisfeld, A., Neumann, G. & Kawaoka, Y. Influenza A virus isolation, culture and identification. Nat Protoc 9, 2663–2681 (2014). https://doi.org/10.1038/nprot.2014.180

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2014.180

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology