Generation of an H3N2 Influenza A Reporter Virus to Visualize Modern Infection Dynamics
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Date
2018-04-25
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Johns Hopkins University
Abstract
The 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.
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influenza, reporter, virus, H3N2, luciferase, fluorescent, mPlum, far-red, reporter virus, fluorescence microscopy, influenza A, nanoluciferase, PA, polymerase