Paige Fletcher, Kyle L. O’Donnell, Joseph F. Rhoderick, Corey W. Henderson, Atsushi Okumura, Trenton Bushmaker, Ayato Takada, Chad S. Clancy, Gábor Kemenesi, and Andrea Marzi are all esteemed researchers contributing to the study.
Author affiliation: The authors are affiliated with the National Institute of Allergy and Infectious Diseases, located in Hamilton, Montana, USA (P. Fletcher, K.L. O’Donnell, J.F. Rhoderick, C.W. Henderson, A. Okumura, T. Bushmaker, C.S. Clancy, A. Marzi). Furthermore, Atsushi Okumura is associated with Hokkaido University in Sapporo, Japan (A. Takada), while Gábor Kemenesi represents the University of Pécs in Hungary.
Members of the Filoviridae family are known to be negative-sense, nonsegmented, single-stranded RNA viruses that exhibit a significant potential to induce severe hemorrhagic disease in both humans and nonhuman primates. Among this family, the Lloviu virus (LLOV) has emerged as a notable concern, being the sole member within the Cuevavirus genus (1). Initially, LLOV was discovered in 2002 in Schreiber’s bats (Miniopterus schreibersii) inhabiting Cueva del Lloviu in Spain (2); however, it wasn’t until 2016 that further reports of its presence emerged, this time identifying the virus again in Schreiber’s bats located in Hungary (3). It has since been detected in additional regions, specifically Bosnia-Herzegovina and Italy (4,5). Notably, the first infectious isolate of LLOV was successfully obtained in 2022 (6). While the pathogen’s potential to affect humans remains largely unexplored, it is important to note that LLOV is capable of infecting human cells, indicating a concerning possibility for zoonotic spillover (6). To date, no animal disease models have been established, which hampers our understanding of the pathogenicity of this relatively novel filovirus.
Given the recent utilization of the ferret model in evaluating the pathogenesis of wild-type filoviruses (7), our team selected domestic ferrets as subjects to delve into the pathogenic potential of LLOV. The focus on mucosal inoculation is particularly noteworthy, as LLOV RNA has been detected within lung samples of infected Schreiber’s bats, suggesting a respiratory route of infection (6). We assessed three different exposure routes: intranasal (IN), intramuscular (IM), and aerosol, to comprehensively compare their impact on LLOV infection.
All infectious work involving LLOV was conducted under strict adherence to standard operating procedures recognized by the Rocky Mountain Laboratories (RML) Institutional Biosafety Committee, specifically in the maximum containment laboratory operating within RML, part of the National Institute of Allergy and Infectious Diseases affiliated with the National Institutes of Health. The animal studies were performed in alignment with established guidelines detailed in the Guide for the Care and Use of Laboratory Animals, set forth by the National Institutes of Health, as well as the Office of Animal Welfare and the United States Department of Agriculture. The RML Animal Care and Use Committee granted approval for all animal work undertaken in this study. Procedures involving ferrets were conducted after the animals were anesthetized by skilled personnel under the careful supervision of veterinary staff. Throughout the study, ferrets were diligently monitored more than once a day for various clinical signs, encompassing their appearance, level of activity, body temperature, and bodyweight.
Figure 1
Figure 1. Clinical findings after experimental LLOV exposure in ferrets. Ferrets were inoculated through IM, IN, or aerosol (n = 6/group) routes with 1,000 focus-forming units of LLOV. A) Survival curve. B)…
Figure 2
Figure 2. Histopathologic findings after experimental LLOV exposure in ferrets. Ferret tissues demonstrated no abnormalities following exposure to LLOV at 21 days post-infection. A–C) Lung tissue; D–F) liver tissue; G–I) spleen tissue. Hematoxylin…
Dr. Paige Fletcher has conducted this vital research during her tenure as a postdoctoral fellow at the Rocky Mountain Laboratories, specifically within the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health. Her research endeavors are primarily focused on understanding the pathogenicity of lesser-known filoviruses and advancing the preclinical development of effective filovirus vaccines.
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The authors extend their gratitude to the members of the Rocky Mountain Veterinary Branch for their indispensable support throughout the ferret study. They also appreciate the assistance provided by the Rocky Mountain Research Technology Branch concerning virus stock sequencing and the histopathological analysis depicted in their findings.
This research received funding through the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health. Additionally, Gábor Kemenesi’s work is supported by the prestigious János Bolyai Research Scholarship awarded by the Hungarian Academy of Sciences.
