Imaging flow cytometry reveals the mechanism of equine arteritis virus entry and internalization.
Abstract: The process of viral entry into host cells is crucial for the establishment of infection and the determination of viral pathogenicity. A comprehensive understanding of entry pathways is fundamental for the development of novel therapeutic strategies. Standard techniques for investigating viral entry include confocal microscopy and flow cytometry, both of which provide complementary qualitative and quantitative data. Imaging flow cytometry, which integrates the advantages of both methodologies, offers significant potential in virological studies. In this investigation, we employed imaging flow cytometry coupled with immunostaining to monitor the entry of equine arteritis virus EAV into Vero cells via the endosomal trafficking route. Analysis provided an insight into the early infection dynamics across thousands of cells, revealing statistically significant alterations in internalization and uncoating process. Moreover, we evaluated the effectiveness of two inhibitors targeting cellular factors involved in facilitating viral entry: ammonium chloride, which disrupts endocytosis, and camostat mesylate, which inhibits the activity of serine proteases. The results demonstrated a clear distinction between effective and ineffective inhibitors. This study highlighted the potential of imaging flow cytometry to advance the study of viral entry and the evaluation of antiviral agents.
© 2025. The Author(s).
Publication Date: 2025-01-25 PubMed ID: 39863680PubMed Central: PMC11762754DOI: 10.1038/s41598-025-87080-xGoogle Scholar: Lookup
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- Journal Article
Summary
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The research explores the process of how the equine arteritis virus (EAV) enters and becomes active in host cells, utilizing imaging flow cytometry and immunostaining. The findings provide comprehensive insight into its entry, infection cycle, and effectiveness of potential inhibitors.
Methodology
- The researchers used imaging flow cytometry, a method that combines microscopy and flow cytometry, to analyze the entry of EAV into Vero cells. This technology allows simultaneous capture of high-resolution images and measurement of light scatter and fluorescence of single particles in a flow system.
- Immunostaining techniques were also employed to visualize and trace the virus’ entry process.
Findings
- The analysis offered a deep understanding of the early stages of infection in a large number of cells. It showed significant changes in the process of virus internalization and disassembly (uncoating).
- The research revealed that the virus enters host cells via the endosomal trafficking route, a pathway inside cells used for transport of substances.
Evaluation of Inhibitors
- The effectiveness of ammonium chloride and camostat mesylate were evaluated. The former disrupts the process of endocytosis (the process by which cells intake large particles), and the latter inhibits the activity of serine proteases (enzymes that cut protein).
- The results demonstrated a clear difference in the performance of the two inhibitors, suggesting that one was more effective than the other in preventing viral entry or internalization.
Implications
- This study underscored the potential of using imaging flow cytometry for advancing the study of viral entry.
- The methodology could also provide valuable data for the development and evaluation of new antiviral drugs or therapies.
Cite This Article
APA
Kublicka A, Lorek D, Mikołajczyk-Martinez A, Chodaczek G, Chwirot A, Bażanów B, Matczuk AK.
(2025).
Imaging flow cytometry reveals the mechanism of equine arteritis virus entry and internalization.
Sci Rep, 15(1), 3246.
https://doi.org/10.1038/s41598-025-87080-x Publication
Researcher Affiliations
- Department of Pathology, Division of Microbiology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-375, Wroclaw, Poland.
- Department of Immunology, Pathophysiology and Veterinary Preventive Medicine, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-375, Wroclaw, Poland.
- Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-375, Wroclaw, Poland.
- Laboratory of Confocal Microscopy, Łukasiewicz Research Network - PORT Polish Center For Technology Development, Wroclaw, Poland.
- Department of Pathology, Division of Microbiology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-375, Wroclaw, Poland.
- Department of Pathology, Division of Microbiology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-375, Wroclaw, Poland.
- Department of Pathology, Division of Microbiology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-375, Wroclaw, Poland. anna.matczuk@upwr.edu.pl.
MeSH Terms
- Animals
- Virus Internalization / drug effects
- Equartevirus / physiology
- Equartevirus / drug effects
- Chlorocebus aethiops
- Flow Cytometry / methods
- Vero Cells
- Endocytosis / drug effects
- Endosomes / metabolism
- Endosomes / virology
- Ammonium Chloride / pharmacology
- Arterivirus Infections / virology
Conflict of Interest Statement
Declarations. Competing interests: The authors declare no competing interests.
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