title: A flow cytometry-based assay for quantifying non-plaque forming strains of yellow fever virus.
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.
authors.affiliation: Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon, United States of America.
authors.forename: Erika
authors.initials: E
authors.lastname: Hammarlund
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Amanna
authors.forename: Melissa E
authors.initials: ME
authors.lastname: Dubois
authors.forename: Alex
authors.initials: A
authors.lastname: Barron
authors.forename: Flora
authors.initials: F
authors.lastname: Engelmann
authors.forename: Ilhem
authors.initials: I
authors.lastname: Messaoudi
authors.forename: Mark K
authors.initials: MK
authors.lastname: Slifka
mesh_terms: D000330:Aedes; D000818:Animals; D000914:Antibodies, Viral; D002460:Cell Line; D005260:Female; D005434:Flow Cytometry; D008253:Macaca mulatta; D008297:Male; D051379:Mice; D019562:Viral Load / Q000379:methods*; D010948:Viral Plaque Assay; D015005:Yellow fever virus / Q000254:growth & development* / Q000276:immunology
chemical_list: D000914:Antibodies, Viral