Generation of an H3N2 Influenza A Reporter Virus to Visualize Modern Infection Dynamics

Simple item page
dc.contributor.committeeMemberPekosz, Andrew S.
dc.contributor.committeeMemberKlein, Sabra L.
dc.creatorThompson, Emily
dc.date.accessioned2018-10-03T02:17:00Z
dc.date.available2018-10-03T02:17:00Z
dc.date.created2018-05
dc.date.issued2018-04-25
dc.date.submittedMay 2018
dc.date.updated2018-10-03T02:17:00Z
dc.description.abstractThe public health burden of influenza is exacerbated by the continuous emergence of new strains and unanswered questions about viral spread within the host. Previously, groups have utilized reporter viruses encoding fluorescent proteins or luciferases to visualize real-time infection dynamics and elucidate complex interactions between viruses and host cells. In 2013, Tran et. al. developed a replication-competent influenza A reporter virus expressing nanoluciferase in the PA segment of a 1933 strain of H1N1, A/WSN/33, that closely mimicked wild-type virus behavior and replication dynamics. However, currently circulating strains of influenza are predominantly H3N2 viruses, urging development of a reporter influenza A virus that more closely represents the behavior of these modern strains. To address this need, we aimed to create two influenza A reporter viruses expressing nanoluciferase and the fluorescent protein mPlum in the PA segment of the A/Victoria/361/2011 strain of H3N2. Through various cloning techniques, the reporter genes were inserted at the end of the PA segment, and a reverse genetics approach was used to generate the two reporter viruses. Characterization of infectious titers and replication kinetics of the reporter viruses in cell culture (through TCID50 and plaque assays) demonstrated that the mPlum reporter virus, rA/Vic-mPlum, was slightly attenuated compared to the nanoluciferase reporter virus, rA/Vic-NLuc, and the wild-type virus, rA/Vic-WT. In addition, expression of mPlum fluorescent protein in the reporter virus could not be validated. However, rA/Vic-NLuc stably maintained luciferase activity and grew to higher titers and with similar kinetics as rA/Vic-WT. The results suggest rA/Vic-NLuc is replication competent and can be used in future applications to quickly assess the behavior of emerging H3N2 influenza viruses and further investigate influenza A virus replication and pathogenesis.
dc.format.mimetypeapplication/pdf
dc.identifier.urihttp://jhir.library.jhu.edu/handle/1774.2/59248
dc.language.isoen_US
dc.publisherJohns Hopkins University
dc.publisher.countryUSA
dc.subjectinfluenza
dc.subjectreporter
dc.subjectvirus
dc.subjectH3N2
dc.subjectluciferase
dc.subjectfluorescent
dc.subjectmPlum
dc.subjectfar-red
dc.subjectreporter virus
dc.subjectfluorescence microscopy
dc.subjectinfluenza A
dc.subjectnanoluciferase
dc.subjectPA
dc.subjectpolymerase
dc.titleGeneration of an H3N2 Influenza A Reporter Virus to Visualize Modern Infection Dynamics
dc.typeThesis
dc.type.materialtext
thesis.degree.departmentMolecular Microbiology and Immunology
thesis.degree.disciplinenot listed
thesis.degree.grantorJohns Hopkins University
thesis.degree.grantorBloomberg School of Public Health
thesis.degree.levelMasters
thesis.degree.nameSc.M.
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
THOMPSON-THESIS-2018.pdf
Size:
2.87 MB
Format:
Adobe Portable Document Format
Download
License bundle
Now showing 1 - 2 of 2
No Thumbnail Available
Name:
PROQUEST_LICENSE.txt
Size:
5.84 KB
Format:
Plain Text
Description:
Download
No Thumbnail Available
Name:
LICENSE.txt
Size:
2.68 KB
Format:
Plain Text
Description:
Download
Collections
ETD -- Graduate theses