Skip to main content
SpringerLink
Log in

Isolation of Exosomes or Extracellular Vesicles from West Nile Virus-Infected N2a Cells, Primary Cortical Neurons, and Brain Tissues

  • Protocol
  • First Online:
West Nile Virus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2585))

Abstract

Several flaviviruses compromise the blood-brain barrier integrity, infect the central nervous system, and elicit neuroinvasion to successfully cause neuropathogenesis in the vertebrate host. Therefore, understanding the pathway(s) and mechanism(s) to block the transmission and/or dissemination of flaviviruses and perhaps other neuroinvasive viruses is considered as an important area of research. Moreover, studies that address mechanism(s) of neuroinvasion by flaviviruses are limited. In this chapter, we discuss detailed methods to isolate exosomes or extracellular vesicles (EVs) from mouse and human N2a cells, primary cultures of murine cortical neurons, and mouse brain tissue. Two different methods including differential ultracentrifugation and density gradient exosome (DG-Exo) isolation are described for the preparation of exosomes/EVs from N2a cells and cortical neurons. In addition, we discuss the detailed DG-Exo method for the isolation of exosomes from murine brain tissue. Studies on neuronal exosomes will perhaps enhance our understanding of the mechanism of neuroinvasion by these deadly viruses.

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

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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

Similar content being viewed by others

References

  1. Kimura T, Sasaki M, Okumura E, Sawa H (2010) Flavivirus encephalitis: pathological aspects of mouse and other animal models. Vet Pathol 47(5):806–818

    Article  CAS  PubMed  Google Scholar 

  2. Blahove MR, Carter JR (2021) Flavivirus persistence in wildlife populations. Viruses 13(10):2099

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Chala B, Hamde F (2021) Emerging and re-emerging vector-borne infectious diseases and the challenges for control: a review. Front Public Health 9:715759

    Article  PubMed  PubMed Central  Google Scholar 

  4. Chambers TJ, Diamond MS (2003) Pathogenesis of flavivirus encephalitis. Adv Virus Res 60:273–342

    Article  CAS  PubMed  Google Scholar 

  5. Ferraris P, Wichit S, Cordel N, Misse D (2021) Human host genetics and susceptibility to ZIKV infection. Infect Genet Evol 95:105066

    Article  CAS  PubMed  Google Scholar 

  6. Kemenesi G, Banyai K (2019) Tick-borne flaviviruses, with a focus on Powassan virus. Clin Microbiol Rev 32(1). https://doi.org/10.1128/CMR.00106-17

  7. Kuhn RJ, Dowd KA, Beth Post C, Pierson TC (2015) Shake, rattle, and roll: impact of the dynamics of flavivirus particles on their interactions with the host. Virology 479-480:508–517

    Article  CAS  PubMed  Google Scholar 

  8. Mukhopadhyay S, Kuhn RJ, Rossmann MG (2005) A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 3(1):13–22

    Article  CAS  PubMed  Google Scholar 

  9. Pan Y, Cai W, Cheng A, Wang M, Yin Z, Jia R (2022) Flaviviruses: innate immunity, inflammasome activation, inflammatory cell death, and cytokines. Front Immunol 13:829433

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Schneider WM, Hoffmann HH (2022) Flavivirus-host interactions: an expanding network of proviral and antiviral factors. Curr Opin Virol 52:71–77

    Article  CAS  PubMed  Google Scholar 

  11. Vasilakis N, Weaver SC (2017) Flavivirus transmission focusing on Zika. Curr Opin Virol 22:30–35

    Article  PubMed  Google Scholar 

  12. Xie X, Zeng J (2021) Neuroimmune evasion of Zika virus to facilitate viral pathogenesis. Front Cell Infect Microbiol 11:662447

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhao R, Wnag M, Cao J, Shen J, Zhou X, Wang D et al (2021) Flavivirus: from structure to therapeutics development. Life (Basel) 11(7):615

    CAS  Google Scholar 

  14. Neal JW (2014) Flaviviruses are neurotropic, but how do they invade the CNS? J Infect 69(3):203–215

    Article  CAS  PubMed  Google Scholar 

  15. Sips GJ, Wilschut J, Smit JM (2012) Neuroinvasive flavivirus infections. Rev Med Virol 22(2):69–87

    Article  CAS  PubMed  Google Scholar 

  16. Suen WW, Prow NA, Hall RA, Bielefeldt-Ohmann H (2014) Mechanism of West Nile virus neuroinvasion: a critical appraisal. Viruses 6(7):2796–2825

    Article  PubMed  PubMed Central  Google Scholar 

  17. Anwar MN, Akhtar R, Abid M, Khan SA, Rehman ZU, Tayyub M et al (2022) The interactions of flaviviruses with cellular receptors: implications for virus entry. Virology 568:77–85

    Article  CAS  PubMed  Google Scholar 

  18. Huang YJ, Higgs S, Horne KM, Vanlandingham DL (2014) Flavivirus-mosquito interactions. Viruses 6(11):4703–4730

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jordan TX, Randall G (2016) Flavivirus modulation of cellular metabolism. Curr Opin Virol 19:7–10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Klein RS, Diamond MS (2008) Immunological headgear: antiviral immune responses protect against neuroinvasive West Nile virus. Trends Mol Med 14(7):286–294

    Article  CAS  PubMed  Google Scholar 

  21. Sultana H, Foellmer HG, Neelakanta G, Oliphant T, Engle M, Ledizet M et al (2009) Fusion loop peptide of the West Nile virus envelope protein is essential for pathogenesis and is recognized by a therapeutic cross-reactive human monoclonal antibody. J Immunol 183(1):650–660

    Article  CAS  PubMed  Google Scholar 

  22. Sultana H, Neelakanta G, Foellmmer HG, Montgomery RR, Anderson JF, Koski RA et al (2012) Semaphorin 7A contributes to West Nile virus pathogenesis through TGF-beta1/Smad6 signaling. J Immunol 189(6):3150–3158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Paterson R (2005) How West Nile virus crosses the blood-brain barrier. Lancet Neurol 4(1):18

    Article  PubMed  Google Scholar 

  24. Kumar M, Nerurkar VR (2016) In vitro and in vivo blood-brain barrier models to study West Nile virus pathogenesis. Methods Mol Biol 1435:103–113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Morrey JD, Olsen AL, Siddhartan V, Motter NE, Wang H, Taro BS et al (2008) Increased blood-brain barrier permeability is not a primary determinant for lethality of West Nile virus infection in rodents. J Gen Virol 89(Pt 2):467–473

    Article  CAS  PubMed  Google Scholar 

  26. Kobiler D, Lustig S, Gozes Y, Ben-Nathan D, Akov Y (1989) Sodium dodecylsulphate induces a breach in the blood-brain barrier and enables a West Nile virus variant to penetrate into mouse brain. Brain Res 496(1–2):314–316

    Article  CAS  PubMed  Google Scholar 

  27. Verma S, Lo Y, Chapagain M, Lum S, Kumar M, Gurjav U et al (2009) West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: transmigration across the in vitro blood-brain barrier. Virology 385(2):425–433

    Article  CAS  PubMed  Google Scholar 

  28. Roe K, Orillo B, Verma S (2014) West Nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model. PLoS One 9(7):e102598

    Article  PubMed  PubMed Central  Google Scholar 

  29. Diamond MS, Klein RS (2004) West Nile virus: crossing the blood-brain barrier. Nat Med 10(12):1294–1295

    Article  CAS  PubMed  Google Scholar 

  30. Zhou W, Woodson M, Sherman MB, Neelakanta G, Sultana H (2019) Exosomes mediate Zika virus transmission through SMPD3 neutral sphingomyelinase in cortical neurons. Emerg Microbes Infect 8(1):307–326

    Article  PubMed  PubMed Central  Google Scholar 

  31. Zhou W, Woodson M, Neupane B, Bai F, Sherman MB, Choi KH et al (2018) Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells. PLoS Pathog 14(1):e1006764

    Article  PubMed  PubMed Central  Google Scholar 

  32. Zhou W, Tahir F, Wang JC, Woodson M, Sherman MB, Karim S et al (2020) Discovery of exosomes from tick saliva and salivary glands reveals therapeutic roles for CXCL12 and IL-8 in wound healing at the tick-human skin interface. Front Cell Dev Biol 8:554

    Article  PubMed  PubMed Central  Google Scholar 

  33. Vora A, Zhou W, Londodno-Renteria B, Woodson M, Sherman MB, Colpitts TM et al (2018) Arthropod EVs mediate dengue virus transmission through interaction with a tetraspanin domain containing glycoprotein Tsp29Fb. Proc Natl Acad Sci U S A 115(28):E6604–E6613

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sultana H, Neelakanta G (2020) Arthropod exosomes as bubbles with message(s) to transmit vector-borne diseases. Curr Opin Insect Sci 40:39–47

    Article  PubMed  Google Scholar 

  35. Sultana H (2016) Examination of West Nile virus neuroinvasion and neuropathogenesis in the central nervous system of a murine model. Methods Mol Biol 1435:83–101

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by start-up funds from the University of Tennessee, Knoxville, to HS and GN.

Author information

Authors and Affiliations

  1. Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA

    Hameeda Sultana & Girish Neelakanta

Authors
  1. Hameeda Sultana
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Girish Neelakanta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hameeda Sultana .

Editor information

Editors and Affiliations

  1. Department of Cell and Molecular Biology, Center for Molecular and Cellular Biosciences The University of Southern Mississippi, Hattiesburg, MS, USA

    Fengwei Bai

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sultana, H., Neelakanta, G. (2023). Isolation of Exosomes or Extracellular Vesicles from West Nile Virus-Infected N2a Cells, Primary Cortical Neurons, and Brain Tissues. In: Bai, F. (eds) West Nile Virus. Methods in Molecular Biology, vol 2585. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2760-0_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2760-0_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2759-4

  • Online ISBN: 978-1-0716-2760-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation