A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus

Erika Hammarlund, Ian J. Amanna, Melissa E. Dubois, Alex Barron, Flora Engelmann, Ilhem Messaoudi, Mark K. Slifka

    Research output: Contribution to journalArticle

    5 Scopus citations

    Abstract

    Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.

    Original languageEnglish (US)
    Article numbere41707
    JournalPloS one
    Volume7
    Issue number9
    DOIs
    StatePublished - Sep 19 2012

    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology(all)
    • Agricultural and Biological Sciences(all)
    • General

    Fingerprint Dive into the research topics of 'A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus'. Together they form a unique fingerprint.

  • Cite this

    Hammarlund, E., Amanna, I. J., Dubois, M. E., Barron, A., Engelmann, F., Messaoudi, I., & Slifka, M. K. (2012). A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus. PloS one, 7(9), [e41707]. https://doi.org/10.1371/journal.pone.0041707