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Updated: May 28, 2026
Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry
Published on: November 4, 2015
Foot-and-mouth disease virus particles inactivated with binary ethylenimine are efficiently internalized into
Miguel A Martín-Acebes1, Angela Vázquez-Calvo, Mónica González-Magaldi
1Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Cantoblanco 28049, Madrid, Spain.
Abstract:
Conventional foot-and-mouth disease (FMD) vaccines are produced from virus grown in cell culture that is chemically inactivated by using binary ethylenimide (BEI). Here, we show that BEI treatment preserves both the architecture of FMDV particles, as inactivated viral particles showed by electron microscopy characteristics similar to those of infectious virions, as well as the general features of infectious virus internalization. Binding of inactivated particles to BHK-21 cells was blocked by preincubation with either a FMDV-specific monoclonal antibody or a synthetic peptide spanning the integrin-binding viral motif Arg-Gly-Asp (RGD). In addition, these particles were internalized into cultured cells through endocytosis, being directed to early endosomes, as indicated by their colocalization with the marker protein Rab5. When purified BEI-inactivated virions were labelled and their interaction with live cultured cells analyzed by time-lapse fluorescence microscopy, a major subpopulation of virus particles, about 80%, was shown to undergo internalization into a static endosome population, insensitive to the microtubule depolymerization exerted by nocodazole, while the remaining subpopulation (about 20%) was dynamic and sensitive to this drug. Thus, BEI-inactivated particles provide an interesting tool to study early steps in FMDV-cell interactions enabling a distinction between FMDV internalization and productive infection. Possible implications for FMDV immune response elicited following vaccine administration are discussed.
Insights
Binary ethylenimide (BEI) inactivated foot-and-mouth disease virus (FMDV) particles retain their structure and cellular interaction features. This inactivation method allows for studying FMDV cell entry mechanisms and immune responses.
Area of Science:
- Virology
- Immunology
- Cell Biology
Background:
- Conventional foot-and-mouth disease (FMD) vaccines utilize virus inactivated by binary ethylenimide (BEI).
- Understanding the early steps of virus-cell interaction is crucial for vaccine development and studying viral pathogenesis.
Purpose of the Study:
- To investigate the structural integrity and cellular interaction properties of BEI-inactivated FMDV particles.
- To explore the potential of BEI-inactivated FMDV as a tool for studying virus internalization and immune responses.
Main Methods:
- Electron microscopy to assess viral particle architecture.
- Cell binding assays using FMDV-specific antibodies and RGD peptide.
- Confocal microscopy and time-lapse fluorescence microscopy to track virus internalization and endosome trafficking (Rab5, nocodazole treatment).
Main Results:
- BEI treatment preserved FMDV particle architecture and general internalization features.
- Inactivated particles bound to cells via integrins (RGD motif) and were internalized via endocytosis into Rab5-positive early endosomes.
- Two subpopulations of internalized particles were observed: 80% in static endosomes and 20% in dynamic, microtubule-dependent endosomes.
Conclusions:
- BEI-inactivated FMDV particles serve as a valuable tool for dissecting FMDV-cell interactions, distinguishing internalization from productive infection.
- The distinct endosomal trafficking pathways suggest complex mechanisms of FMDV entry and potential implications for vaccine-induced immunity.
