Dataset Information

Real-time tracking of bioluminescent influenza A virus infection in mice.

ABSTRACT: Despite the availability of vaccines and antiviral therapies, seasonal influenza infections cause 400,000 human deaths on average per year. Low vaccine coverage and the occurrence of drug-resistant viral strains highlight the need for new and improved countermeasures. While influenza A virus (IAV) engineered to express a reporter gene may serve as a valuable tool for real-time tracking of viral infection, reporter gene insertion into IAV typically attenuates viral pathogenicity, hindering its application to research. Here, we demonstrate that lethal or even sublethal doses of bioluminescent IAV carrying the NanoLuc gene in the C-terminus of PB2 can be tracked in real-time in live mice without compromising pathogenicity. Real-time tracking of this bioluminescent IAV enables spatiotemporal viral replication tracking in animals that will facilitate the development of countermeasures by enhancing the interpretation of clinical signs and prognosis while also allowing less animal usage.

SUBMITTER: Kim JH

PROVIDER: S-EPMC8873407 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Real-time tracking of bioluminescent influenza A virus infection in mice.

Kim Jin H JH Bryant Hannah H Fiedler Edward E Cao TuAnh T Rayner Jonathan O JO

Scientific reports 20220224 1

Despite the availability of vaccines and antiviral therapies, seasonal influenza infections cause 400,000 human deaths on average per year. Low vaccine coverage and the occurrence of drug-resistant viral strains highlight the need for new and improved countermeasures. While influenza A virus (IAV) engineered to express a reporter gene may serve as a valuable tool for real-time tracking of viral infection, reporter gene insertion into IAV typically attenuates viral pathogenicity, hindering its ap ...[more]

PMID: 35210462

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Project description:Bioluminescence imaging (BLI) using engineered bioluminescent viruses has emerged as a powerful tool for real-time, noninvasive monitoring of viral replication in living animals. While traditional luciferase-based systems, such as firefly luciferase, have been widely used, the NanoLuc luciferase system offers distinct advantages, including its significantly smaller gene size, increased brightness, and independence from ATP as a cofactor, allowing for extracellular detection. However, the utility of NanoLuc has been limited by its traditional substrate, furimazine, which exhibits poor water solubility and potential cytotoxicity. In this study, we assessed fluorofurimazine (FFz), a novel substrate with improved water solubility and bioavailability, for tracking influenza A virus (IAV) replication in mice. Our findings demonstrate that FFz substantially enhances detection sensitivity in both respiratory organs and brain tissue without increasing toxicity, enabling more precise and sustained monitoring of IAV replication. In vitro, FFz generated higher photon flux at lower concentrations compared to furimazine, translating into superior in vivo sensitivity with reduced toxicity. Crucially, FFz did not alter the pathogenicity of IAV in mice, even at sublethal infectious doses, reinforcing its suitability for use in BLI-based viral pathogenicity studies. These results suggest that combining FFz with NanoLuc provides a more effective and less toxic approach for real-time tracking of viral infections in preclinical models.ImportanceMonitoring viral infections in living animals is a valuable approach for understanding how viruses replicate and cause disease. This study focuses on bioluminescent influenza A virus infection in a mouse model and evaluates fluorofurimazine, a new substrate that enhances bioluminescence imaging. Fluorofurimazine allows researchers to monitor viral spread more effectively than the traditional substrate, furimazine, which is often toxic and less reliable. It offers better sensitivity and lower toxicity, enabling longer and more accurate tracking of viral replication in the lungs and even the brain. Importantly, fluorofurimazine does not alter the pathogenicity of the virus, providing an unaltered representation of the infection process. This advancement has the potential to significantly improve how scientists study bioluminescent viral infections and evaluate antiviral drugs and vaccines, making it a valuable tool for research on influenza and other respiratory viruses.

Project description:RationaleExisting real-time surveillance of influenza morbidity, based primarily on time-trended U.S. hospitalization and death data, is inadequate. These surveillance methods do not accurately predict hospital resource requirements or sufficiently capture the public health impact of the current influenza season.ObjectivesTo determine the feasibility and potential usefulness of tracking surrogate markers of influenza morbidity among patients hospitalized with influenza.MethodsWe performed a pilot study at three tertiary care referral hospitals and retrospectively collected and analyzed data on patients admitted with influenza during the 2013-2014 influenza season. We analyzed traditional influenza surveillance metrics, including weekly statistics on admissions and deaths, as well as weekly rates and trends of intensive care unit (ICU), mechanical ventilation, and extracorporeal membrane oxygenation (ECMO) utilization.ResultsIn our three-hospital cohort, 431 patients were hospitalized with influenza and spent a total of 1,520 days in ICUs. Eighty-six (20%) of these patients required 1,080 days of mechanical ventilation, and 17 patients (4%) received 229 days of ECMO. Trends of ICU and mechanical ventilation use were similar but differed notably from trends of ECMO use, hospitalization, and death. In particular, at two hospitals, increases in utilization of ICU and mechanical ventilation among patients with influenza occurred several weeks after increases in hospitalization rates. Furthermore, ICU, mechanical ventilation, and ECMO utilization rates at the three-hospital network remained elevated for several weeks after the influenza-associated hospitalization rate declined.ConclusionsSurrogate markers of influenza severity were feasible to collect and revealed trends of ICU resource utilization that differed notably from trends of hospitalization and death given by traditional influenza surveillance metrics. A national network of sentinel hospitals that prospectively collects, time-trends, and reports additional influenza morbidity data would be useful to hospital administrators, hospital epidemiologists, infection preventionists, and public health officials.

Project description:BackgroundSevere fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that causes severe hemorrhagic fever in humans, but no FDA-approved specific antivirals or vaccines are available to treat or prevent SFTS.MethodsThe plasmids construction and transfection were performed to generate the recombinant SFTSV harboring the nanoluciferase gene (SFTSV-Nluc). Immunostaining plaque assay was performed to measure viral titers, and DNA electrophoresis and Sanger sequencing were performed to evaluate the genetic stability. Luciferase assay and quantitative RT-PCR were performed to evaluate the efficacy of antivirals in vitro. Bioluminescence imaging, titration of virus from excised organs, hematology, and histopathology and immunohistochemistry were performed to evaluate the efficacy of antivirals in vivo.FindingsSFTSV-Nluc exhibited high genetic stability and replication kinetics similar to those of wild-type virus (SFTSVwt), then a rapid high-throughput screening system for identifying inhibitors to treat SFTS was developed, and a nucleoside analog, 4-FlU, was identified to effectively inhibit SFTSV in vitro. SFTSV-Nluc mimicked the replication characteristics and localization of SFTSVwt in counterpart model mice. Bioluminescence imaging of SFTSV-Nluc allowed real-time visualization and quantification of SFTSV replication in the mice. 4-FlU was demonstrated to inhibit the replication of SFTSV with more efficiency than T-705 and without obvious adverse effect in vivo.InterpretationThe high-throughput screening system based on SFTSV-Nluc for use in vitro and in vivo revealed that a safe and effective antiviral nucleoside analog, 4-FlU, may be a basis for the strategic treatment of SFTSV and other bunyavirus infections, paving the way for the discovery of antivirals.FundingThis work was supported by grants from the National Key Research and Development Plan of China (2021YFC2300700 to L. Zhang, 2022YFC2303300 to L. Zhang), Strategic Priority Research Program of Chinese Academy of Sciences (XDB0490000 to L. Zhang), National Natural Science Foundation of China (31970165 to L. Zhang, U22A20379 to G. Xiao), the Science and Technology Commission of Shanghai Municipality (21S11903100 to Y. Xie), Hubei Natural Science Foundation for Distinguished Young Scholars (2022CFA099 to L. Zhang).

Dataset Information

Real-time tracking of bioluminescent influenza A virus infection in mice.

Publications

Real-time tracking of bioluminescent influenza A virus infection in mice.

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