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Isolation of Swine Influenza A Virus in Cell Cultures and Embryonated Chicken Eggs

  • Jianqiang ZhangEmail author
  • Phillip C. Gauger
Protocol
  • 115 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2123)

Abstract

Influenza virus isolation is a procedure to obtain a live and infectious virus that can be used for antigenic characterization, pathogenesis investigation, vaccine production, and so on. Embryonated chicken egg inoculation is traditionally considered the “gold standard” method for influenza virus isolation and propagation. However, many primary cells and continuous cell lines have also been examined or developed for influenza virus isolation and replication. Specifically, influenza A virus in swine (IAV-S) isolation and propagation has been attempted and compared in embryonated chicken eggs, some primary porcine cells, and a number of continuous cell lines. Currently, Madin-Darby canine kidney (MDCK) cells remain the most commonly used cell line for the isolation, propagation, and titration of IAV-S. Virus isolation in embryonated chicken eggs or in different cell lines offers alternative approaches when IAV-S isolation in MDCK cells is unsuccessful. Optimal specimens for IAV-S isolation includes nasal swabs, nasopharyngeal swabs, oral fluids, bronchoalveolar lavage, lung tissues, and so on. In this chapter, we describe the procedures of sample processing, IAV-S isolation in MDCK cells and in embryonated chicken eggs, as well as the methods used for confirming the virus isolation results.

Key words

Influenza A virus Swine Virus isolation Cell culture Embryonated chicken eggs 

References

  1. 1.
    Yeolekar LR, Dhere RM (2012) Development and validation of an egg-based potency assay for a trivalent live attenuated influenza vaccine. Biologicals 40:146–150CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    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 227:493–499CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Katz JM, Naeve CW, Webster RG (1987) Host cell-mediated variation in H3N2 influenza viruses. Virology 156:386–395CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Robertson JS, Naeve CW, Webster RG, Bootman JS, Newman R, Schild GC (1985) Alterations in the hemagglutinin associated with adaptation of influenza B virus to growth in eggs. Virology 143:166–174CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Schild GC, Oxford JS, de Jong JC, Webster RG (1983) Evidence for host-cell selection of influenza virus antigenic variants. Nature 303:706–709CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Stevens J, Chen LM, Carney PJ, Garten R, Foust A, Le J, Pokorny BA, Manojkumar R, Silverman J, Devis R, Rhea K, Xu X, Bucher DJ, Paulson JC, Cox NJ, Klimov A, Donis RO (2010) Receptor specificity of influenza A H3N2 viruses isolated in mammalian cells and embryonated chicken eggs. J Virol 84:8287–8299CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Katz JM, Wang M, Webster RG (1990) Direct sequencing of the HA gene of influenza (H3N2) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus. J Virol 64:1808–1811CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Robertson JS, Bootman JS, Nicolson C, Major D, Robertson EW, Wood JM (1990) The hemagglutinin of influenza B virus present in clinical material is a single species identical to that of mammalian cell-grown virus. Virology 179:35–40CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    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 71:3357–3362CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Sriwilaijaroen N, Kondo S, Yagi H, Wilairat P, Hiramatsu H, Ito M, Ito Y, Kato K, Suzuki Y (2009) Analysis of N-glycans in embryonated chicken egg chorioallantoic and amniotic cells responsible for binding and adaptation of human and avian influenza viruses. Glycoconj J 26:433–443CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Frank AL, Couch RB, Griffis CA, Baxter BD (1979) Comparison of different tissue cultures for isolation and quantitation of influenza and parainfluenza viruses. J Clin Microbiol 10:32–36CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Meguro H, Bryant JD, Torrence AE, Wright PF (1979) Canine kidney cell line for isolation of respiratory viruses. J Clin Microbiol 9:175–179PubMedPubMedCentralGoogle Scholar
  13. 13.
    Tobita K (1975) Permanent canine kidney (MDCK) cells for isolation and plaque assay of influenza B viruses. Med Microbiol Immunol 162:23–27CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    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 162:9–14CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Govorkova EA, Kaverin NV, Gubareva LV, Meignier B, Webster RG (1995) Replication of influenza A viruses in a green monkey kidney continuous cell line (Vero). J Infect Dis 172:250–253CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Govorkova EA, Murti G, Meignier B, de Taisne C, Webster RG (1996) African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses. J Virol 70:5519–5524CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    de Ona M, Melon S, de la Iglesia P, Hidalgo F, Verdugo AF (1995) Isolation of influenza virus in human lung embryonated fibroblast cells (MRC-5) from clinical samples. J Clin Microbiol 33:1948–1949CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Schultz-Cherry S, Dybdahl-Sissoko N, McGregor M, Hinshaw VS (1998) Mink lung epithelial cells: unique cell line that supports influenza A and B virus replication. J Clin Microbiol 36:3718–3720CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Huang YT, Turchek BM (2000) Mink lung cells and mixed mink lung and A549 cells for rapid detection of influenza virus and other respiratory viruses. J Clin Microbiol 38:422–423PubMedPubMedCentralGoogle Scholar
  20. 20.
    Zhirnov OP, Vorobjeva IV, Saphonova OA, Malyshev NA, Ovcharenko AV, Klenk HD (2007) Specific biochemical features of replication of clinical influenza viruses in human intestinal cell culture. Biochemistry (Mosc) 72:398–408CrossRefGoogle Scholar
  21. 21.
    Reina J, Fernandez-Baca V, Blanco I, Munar M (1997) Comparison of Madin-Darby canine kidney cells (MDCK) with a green monkey continuous cell line (Vero) and human lung embryonated cells (MRC-5) in the isolation of influenza A virus from nasopharyngeal aspirates by shell vial culture. J Clin Microbiol 35:1900–1901CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Hamilton SB, Wyatt DE, Wahlgren BT, O'Dowd MK, Morrissey JM, Daniels DE, Lednicky JA (2011) Higher titers of some H5N1 and recent human H1N1 and H3N2 influenza viruses in Mv1 Lu vs. MDCK cells. Virol J 8:66CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Landolt GA, Karasin AI, Hofer C, Mahaney J, Svaren J, Olsen CW (2005) Use of real-time reverse transcriptase polymerase chain reaction assay and cell culture methods for detection of swine influenza A viruses. Am J Vet Res 66:119–124CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Li IW, Chan KH, To KW, Wong SS, Ho PL, Lau SK, Woo PC, Tsoi HW, Chan JF, Cheng VC, Zheng BJ, Chen H, Yuen KY (2009) Differential susceptibility of different cell lines to swine-origin influenza A H1N1, seasonal human influenza A H1N1, and avian influenza A H5N1 viruses. J Clin Virol 46:325–330CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Moresco KA, Stallknecht DE, Swayne DE (2012) Evaluation of different embryonating bird eggs and cell cultures for isolation efficiency of avian influenza A virus and avian paramyxovirus serotype 1 from real-time reverse transcription polymerase chain reaction-positive wild bird surveillance samples. J Vet Diagn Investig 24:563–567CrossRefGoogle Scholar
  26. 26.
    Zeng H, Goldsmith CS, Maines TR, Belser JA, Gustin KM, Pekosz A, Zaki SR, Katz JM, Tumpey TM (2013) Tropism and infectivity of influenza virus, including highly pathogenic avian H5N1 virus, in ferret tracheal differentiated primary epithelial cell cultures. J Virol 87:2597–2607CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Swenson SL, Vincent LL, Lute BM, Janke BH, Lechtenberg KE, Landgraf JG, Schmitt BJ, Kinker DR, McMillen JK (2001) A comparison of diagnostic assays for the detection of type A swine influenza virus from nasal swabs and lungs. J Vet Diagn Investig 13:36–42CrossRefGoogle Scholar
  28. 28.
    Lombardo T, Dotti S, Renzi S, Ferrari M (2012) Susceptibility of different cell lines to avian and swine influenza viruses. J Virol Methods 185:82–88CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Clavijo A, Tresnan DB, Jolie R, Zhou EM (2002) Comparison of embryonated chicken eggs with MDCK cell culture for the isolation of swine influenza virus. Can J Vet Res 66:117–121PubMedPubMedCentralGoogle Scholar
  30. 30.
    Chiapponi C, Zanni I, Garbarino C, Barigazzi G, Foni E (2010) Comparison of the usefulness of the CACO-2 cell line with standard substrates for isolation of swine influenza A viruses. J Virol Methods 163:162–165CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Bowman AS, Nelson SW, Edwards JL, Hofer CC, Nolting JM, Davis IC, Slemons RD (2013) Comparative effectiveness of isolation techniques for contemporary influenza A virus strains circulating in exhibition swine. J Vet Diagn Investig 25:82–90CrossRefGoogle Scholar
  32. 32.
    Punyadarsaniya D, Liang CH, Winter C, Petersen H, Rautenschlein S, Hennig-Pauka I, Schwegmann-Wessels C, Wu CY, Wong CH, Herrler G (2011) Infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses. PLoS One 6:e28429CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Ferrari M, Scalvini A, Losio MN, Corradi A, Soncini M, Bignotti E, Milanesi E, Ajmone-Marsan P, Barlati S, Bellotti D, Tonelli M (2003) Establishment and characterization of two new pig cell lines for use in virological diagnostic laboratories. J Virol Methods 107:205–212CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Sun Z, Huber VC, McCormick K, Kaushik RS, Boon AC, Zhu L, Hause B, Webby RJ, Fang Y (2012) Characterization of a porcine intestinal epithelial cell line for influenza virus production. J Gen Virol 93:2008–2016CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Bateman AC, Karasin AI, Olsen CW (2013) Differentiated swine airway epithelial cell cultures for the investigation of influenza A virus infection and replication. Influenza Other Respir Viruses 7:139–150CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Khatri M, Saif YM (2011) Epithelial cells derived from swine bone marrow express stem cell markers and support influenza virus replication in vitro. PLoS One 6:e29567CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Londt BZ, Brookes SM, Nash BJ, Nunez A, Stagg DA, Brown IH (2013) The infectivity of pandemic 2009 H1N1 and avian influenza viruses for pigs: an assessment by ex vivo respiratory tract organ culture. Influenza Other Respir Viruses 7:393–402CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    OIE (2013) Chapter 2.8.8 Swine influenza, OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2013Google Scholar
  39. 39.
    Detmer S, Gramer M, Goyal S, Torremorell M, Torrison J (2013) Diagnostics and surveillance for swine influenza. Curr Top Microbiol Immunol 370:85–112PubMedPubMedCentralGoogle Scholar
  40. 40.
    Romagosa A, Gramer M, Joo HS, Torremorell M (2012) Sensitivity of oral fluids for detecting influenza A virus in populations of vaccinated and non-vaccinated pigs. Influenza Other Respir Viruses 6:110–118CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Detmer SE, Patnayak DP, Jiang Y, Gramer MR, Goyal SM (2011) Detection of influenza A virus in porcine oral fluid samples. J Vet Diagn Investig 23:241–247CrossRefGoogle Scholar
  42. 42.
    Ramirez A, Wang C, Prickett JR, Pogranichniy R, Yoon KJ, Main R, Johnson JK, Rademacher C, Hoogland M, Hoffmann P, Kurtz A, Kurtz E, Zimmerman J (2012) Efficient surveillance of pig populations using oral fluids. Prev Vet Med 104:292–300CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Goodell CK, Prickett J, Kittawornrat A, Zhou F, Rauh R, Nelson W, O'Connell C, Burrell A, Wang C, Yoon KJ, Zimmerman JJ (2013) Probability of detecting influenza A virus subtypes H1N1 and H3N2 in individual pig nasal swabs and pen-based oral fluid specimens over time. Vet Microbiol 166:450–460CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Lazarowitz SG, Choppin PW (1975) Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology 68:440–454CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Zhirnov O, Klenk HD (2003) Human influenza A viruses are proteolytically activated and do not induce apoptosis in CACO-2 cells. Virology 313:198–212CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Krauss S, Walker D, Webster RG (2012) Influenza virus isolation. In Kawaoka Y and Neumann G (ed) Influenza virus: methods and protocols. Methods Mol Biol 865:11–24CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply. 2020

Authors and Affiliations

  1. 1.Department of Veterinary Diagnostic and Production Animal MedicineCollege of Veterinary Medicine, Iowa State UniversityAmesUSA

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