title: A high-throughput drop microfluidic system for virus culture and analysis.
abstract: High mutation rates and short replication times lead to rapid evolution in RNA viruses. New tools for high-throughput culture and analysis of viral phenotypes will enable more effective studies of viral evolutionary processes. A water-in-oil drop microfluidic system to study virus-cell interactions at the single event level on a massively parallel scale is described here. Murine norovirus (MNV-1) particles were co-encapsulated with individual RAW 264.7 cells in 65 pL aqueous drops formed by flow focusing in 50 μm microchannels. At low multiplicity of infection (MOI), viral titers increased greatly, reaching a maximum 18 h post-encapsulation. This system was employed to evaluate MNV-1 escape from a neutralizing monoclonal antibody (clone A6.2). Further, the system was validated as a means for testing escape from antibody neutralization using a series of viral point mutants. Finally, the replicative capacity of single viral particles in drops under antibody stress was tested. Under standard conditions, many RNA virus stocks harbor minority populations of genotypic and phenotypic variants, resulting in quasispecies. These data show that when single cells are encapsulated with single viral particles under antibody stress without competition from other virions, the number of resulting infectious particles is nearly equivalent to the number of viral genomes present. These findings suggest that lower fitness virions can infect cells successfully and replicate, indicating that the microfluidics system may serve as an effective tool for isolating mutants that escape evolutionary stressors.
authors.affiliation: The Johns Hopkins University Applied Physics Laboratory, 11100 Johns Hopkins Road, Laurel, MD 20723, USA.
authors.forename: Audrey E
authors.initials: AE
authors.lastname: Fischer
authors.affiliation: The Johns Hopkins University Applied Physics Laboratory, 11100 Johns Hopkins Road, Laurel, MD 20723, USA.
authors.forename: Susan K
authors.initials: SK
authors.lastname: Wu
authors.affiliation: The Johns Hopkins University Applied Physics Laboratory, 11100 Johns Hopkins Road, Laurel, MD 20723, USA.
authors.forename: Jody B G
authors.initials: JB
authors.lastname: Proescher
authors.affiliation: Harvard School of Engineering and Applied Sciences, 9 Oxford Street, Cambridge, MA 02138, USA.
authors.forename: Assaf
authors.initials: A
authors.lastname: Rotem
authors.affiliation: Harvard School of Engineering and Applied Sciences, 9 Oxford Street, Cambridge, MA 02138, USA.
authors.forename: Connie B
authors.initials: CB
authors.lastname: Chang
authors.affiliation: Harvard School of Engineering and Applied Sciences, 9 Oxford Street, Cambridge, MA 02138, USA.
authors.forename: Huidan
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Harvard School of Engineering and Applied Sciences, 9 Oxford Street, Cambridge, MA 02138, USA.
authors.forename: Ye
authors.initials: Y
authors.lastname: Tao
authors.affiliation: The Johns Hopkins University Applied Physics Laboratory, 11100 Johns Hopkins Road, Laurel, MD 20723, USA.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Mehoke
authors.affiliation: The Johns Hopkins University Applied Physics Laboratory, 11100 Johns Hopkins Road, Laurel, MD 20723, USA.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Thielen
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA.
authors.forename: Abimbola O
authors.initials: AO
authors.lastname: Kolawole
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, TX 77555, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Smith
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA.
authors.forename: Christiane E
authors.initials: CE
authors.lastname: Wobus
authors.affiliation: Harvard School of Engineering and Applied Sciences, 9 Oxford Street, Cambridge, MA 02138, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Weitz
authors.affiliation: The Johns Hopkins University Applied Physics Laboratory, 11100 Johns Hopkins Road, Laurel, MD 20723, USA.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Lin
authors.affiliation: The Johns Hopkins University Applied Physics Laboratory, 11100 Johns Hopkins Road, Laurel, MD 20723, USA.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Feldman
authors.affiliation: The Johns Hopkins University Applied Physics Laboratory, 11100 Johns Hopkins Road, Laurel, MD 20723, USA. Electronic address: joshua.wolfe@jhuapl.edu.
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Wolfe
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D002460:Cell Line; D057166:High-Throughput Screening Assays / Q000379:methods*; D054884:Host-Pathogen Interactions; D057131:Immune Evasion; D008264:Macrophages / Q000821:virology; D051379:Mice; D044085:Microfluidics / Q000379:methods*; D029322:Norovirus / Q000502:physiology; D019562:Viral Load; D014773:Virology / Q000379:methods*; D014776:Virus Cultivation / Q000379:methods; D014779:Virus Replication
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral
keywords: Antibody neutralization; Evolution; Microfluidics; Murine norovirus