Browsing by Author "Duprex, W. Paul"

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    A reporter Oropouche virus expressing ZsGreen from the M segment enables pathogenesis studies in mice
    (American Society for Microbiology, 2024) Gunter, Krista; Omoga, Dorcus; Bowen, James M.; Gonzalez, Lorimar Robledo; Severt, Sydney; Davis, Mackenzie; Szymanski, Megan; Sandusky, George; Duprex, W. Paul; Tilston-Lunel, Natasha L.; Microbiology and Immunology, School of Medicine
    Oropouche fever caused by Oropouche virus (OROV) is a significant zoonosis in Central and South America. Despite its public health significance, we lack high-throughput diagnostics, therapeutics, and a comprehensive knowledge of OROV biology. Reporter viruses are valuable tools to rapidly study virus dynamics and develop neutralization and antiviral screening assays. OROV is a tri-segmented bunyavirus, which makes generating a reporter virus challenging, as introducing foreign elements into the viral genome typically affects fitness. We previously demonstrated that the non-structural gene NSm on the OROV medium (M) segment is non-essential for replication in vitro. Taking advantage of this, we have now generated a recombinant OROV expressing fluorescent protein ZsGreen in place of NSm. This reporter OROV is both stable and pathogenic in IFNAR-/- mice and provides a powerful tool for OROV pathogenesis studies and assay development.IMPORTANCEEmerging and reemerging infectious agents such as zoonotic bunyaviruses are of global health concern. Oropouche virus (OROV) causes recurring outbreaks of acute febrile illness in the Central and South American human populations. Biting midges are the primary transmission vectors, whereas sloths and non-human primates are their reservoir hosts. As global temperatures increase, we will likely see an expansion in arthropod-borne pathogens such as OROV. Therefore, developing reagents to study pathogen biology to aid in identifying druggable targets is essential. Here, we demonstrate the feasibility and use of a fluorescent OROV reporter in mice to study viral dynamics and pathogenesis. We show that this reporter OROV maintains characteristics such as growth and pathogenicity similar to the wild-type virus. Using this reporter virus, we can now develop methods to assist OROV studies and establish various high-throughput assays.
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    FeMV is a cathepsin-dependent unique morbillivirus infecting the kidneys of domestic cats
    (National Academy of Sciences, 2022) Nambulli, Sham; Rennick, Linda J.; Acciardo, Andrew S.; Tilston-Lunel, Natasha L.; Ho, Gregory; Crossland, Nicholas A.; Hardcastle, Kathy; Nieto, Betsy; Bainbridge, Graeme; Williams, Tracey; Sharp, Claire R.; Duprex, W. Paul; Microbiology and Immunology, School of Medicine
    Feline morbillivirus (FeMV) has been classified as a morbillivirus despite lacking several biological features shared by all other known viruses in the genus. We confirm that FeMV uses CD150 as a cellular receptor and employs a different protease to furin to process the fusion glycoprotein. As such, FeMV may represent an important evolutionary intermediate between morbilliviruses and the zoonotic henipaviruses. Feline chronic kidney disease is the leading cause of morbidity and mortality in cats and has no clear etiology. FeMV has been postulated to be a causative agent. We dissected FeMV pathogenesis using recombinant, fluorescent protein expressing viruses based on an unpassaged clinical isolate. This sheds light on the primary target cells infected and possible mechanisms of host-to-host transmission.
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    Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses
    (American Association for the Advancement of Science, 2021-05-26) Nambulli, Sham; Xiang, Yufei; Tilston-Lunel, Natasha L.; Rennick, Linda J.; Sang, Zhe; Klimstra, William B.; Reed, Douglas S.; Crossland, Nicholas A.; Shi, Yi; Duprex, W. Paul; Microbiology and Immunology, School of Medicine
    Globally, there is an urgency to develop effective, low-cost therapeutic interventions for coronavirus disease 2019 (COVID-19). We previously generated the stable and ultrapotent homotrimeric Pittsburgh inhalable Nanobody 21 (PiN-21). Using Syrian hamsters that model moderate to severe COVID-19 disease, we demonstrate the high efficacy of PiN-21 to prevent and treat SARS-CoV-2 infection. Intranasal delivery of PiN-21 at 0.6 mg/kg protects infected animals from weight loss and substantially reduces viral burdens in both lower and upper airways compared to control. Aerosol delivery of PiN-21 facilitates deposition throughout the respiratory tract and dose minimization to 0.2 mg/kg. Inhalation treatment quickly reverses animals' weight loss after infection, decreases lung viral titers by 6 logs leading to drastically mitigated lung pathology, and prevents viral pneumonia. Combined with the marked stability and low production cost, this innovative therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.
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    Sustained Replication of Synthetic Canine Distemper Virus Defective Genomes In Vitro and In Vivo
    (American Society for Microbiology, 2021) Tilston-Lunel, Natasha L.; Welch, Stephen R.; Nambulli, Sham; de Vries, Rory D.; Ho, Gregory W.; Wentworth, David E.; Shabman, Reed; Nichol, Stuart T.; Spiropoulou, Christina F.; de Swart, Rik L.; Rennick, Linda J.; Duprex, W. Paul; Microbiology and Immunology, School of Medicine
    Defective interfering (DI) genomes restrict viral replication and induce type I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity, and determine their long-term effects. To address this, we generated a recombinant canine distemper virus (rCDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques, and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate that these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues, which are a major site of CDV replication. Our established pipeline for detection, generation, and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome. IMPORTANCE: Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes might be harnessed therapeutically. Negative-strand RNA virus research has provided a rich pool of natural DI genomes over many years, and they are probably the best understood in vitro. Here, we demonstrate the identification, synthesis, production, and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection.