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Viruses
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Microscopy Methods for Virus Research
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Microscopy Methods for Virus Research

A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "General Virology".

Deadline for manuscript submissions: 31 May 2025 | Viewed by 14131

Special Issue Editor

Dr. Krishanu Ray

E-Mail Website
Guest Editor
Institute of Human Virology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
Interests: single-molecule fluorescence spectroscopy; super-resolution imaging; fluorescence lifetime imaging microscopy; HIV envelope; SARS-CoV-2; Fc-receptor; DNA packaging motor; plasmonics

Special Issue Information

Dear Colleagues,

Significant advances in microscopy methods for biomedical applications have been made in the past two decades, and these methods are now being actively pursued with regard to virus research. Implementing quantitative advanced microscopic methods, including optical and EM imaging, can provide vital information regarding the mechanism and dynamics of viral infection and aid in the identification of targets for antiviral strategies. These methods are opening novel avenues for research in microbiology, particularly with respect to viruses. Improvements in microscopy methods have allowed researchers to directly examine single viruses in their natural forms, generating new insights into their structure, function, and mechanism of action over time and/or in three dimensions. Specifically, recent progress in several super-resolved fluorescence microscopy, single-molecule, and in vivo optical imaging methods, as well as novel fluorescence tags and electron microscopy methods including Cryo-EM and Cryo-ET, has revealed the possibility of tracking viruses in host–pathogen interactions and evaluating dynamics at the molecular level. This promises to provide unprecedented information for developing intervention and therapeutic strategies. In this Special Issue, we welcome the submission of reviews and original research articles that focus on the progress and application of microscopy methods and imaging in the broad area of virus research.

Dr. Krishanu Ray
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Viruses is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • viruses
  • fluorescence imaging
  • single-molecule imaging
  • super-resolution imaging
  • intra-vital imaging
  • electron microscopy
  • Cryo-EM
  • Cryo-ET

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Published Papers (5 papers)

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Research

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26 pages, 11150 KiB  
Article
Expanding Insights: Harnessing Expansion Microscopy for Super-Resolution Analysis of HIV-1–Cell Interactions
by Annett Petrich, Gyu Min Hwang, Laetitia La Rocca, Mariam Hassan, Maria Anders-Össwein, Vera Sonntag-Buck, Anke-Mareil Heuser, Vibor Laketa, Barbara Müller, Hans-Georg Kräusslich and Severina Klaus
Viruses 2024, 16(10), 1610; https://doi.org/10.3390/v16101610 - 15 Oct 2024
Viewed by 5225
Abstract
Expansion microscopy has recently emerged as an alternative technique for achieving high-resolution imaging of biological structures. Improvements in resolution are achieved by physically expanding samples through embedding in a swellable hydrogel before microscopy. However, expansion microscopy has been rarely used in the field [...] Read more.
Expansion microscopy has recently emerged as an alternative technique for achieving high-resolution imaging of biological structures. Improvements in resolution are achieved by physically expanding samples through embedding in a swellable hydrogel before microscopy. However, expansion microscopy has been rarely used in the field of virology. Here, we evaluate and characterize the ultrastructure expansion microscopy (U-ExM) protocol, which facilitates approximately four-fold sample expansion, enabling the visualization of different post-entry stages of the HIV-1 life cycle, focusing on nuclear events. Our findings demonstrate that U-ExM provides robust sample expansion and preservation across different cell types, including cell-culture-adapted and primary CD4+ T-cells as well as monocyte-derived macrophages, which are known HIV-1 reservoirs. Notably, cellular targets such as nuclear bodies and the chromatin landscape remain well preserved after expansion, allowing for detailed investigation of HIV-1–cell interactions at high resolution. Our data indicate that morphologically distinct HIV-1 capsid assemblies can be differentiated within the nuclei of infected cells and that U-ExM enables detection of targets that are masked in commonly used immunofluorescence protocols. In conclusion, we advocate for U-ExM as a valuable new tool for studying virus–host interactions with enhanced spatial resolution. Full article
(This article belongs to the Special Issue Microscopy Methods for Virus Research)
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21 pages, 6333 KiB  
Article
Multiplex Microscopy Assay for Assessment of Therapeutic and Serum Antibodies against Emerging Pathogens
by Nuno Sartingen, Vanessa Stürmer, Matthias Kaltenböck, Thorsten G. Müller, Paul Schnitzler, Anna Kreshuk, Hans-Georg Kräusslich, Uta Merle, Frauke Mücksch, Barbara Müller, Constantin Pape and Vibor Laketa
Viruses 2024, 16(9), 1473; https://doi.org/10.3390/v16091473 - 17 Sep 2024
Viewed by 2916
Abstract
The emergence of novel pathogens, exemplified recently by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the need for rapidly deployable and adaptable diagnostic assays to assess their impact on human health and guide public health responses in future pandemics. In this [...] Read more.
The emergence of novel pathogens, exemplified recently by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the need for rapidly deployable and adaptable diagnostic assays to assess their impact on human health and guide public health responses in future pandemics. In this study, we developed an automated multiplex microscopy assay coupled with machine learning-based analysis for antibody detection. To achieve multiplexing and simultaneous detection of multiple viral antigens, we devised a barcoding strategy utilizing a panel of HeLa-based cell lines. Each cell line expressed a distinct viral antigen, along with a fluorescent protein exhibiting a unique subcellular localization pattern for cell classification. Our robust, cell segmentation and classification algorithm, combined with automated image acquisition, ensured compatibility with a high-throughput approach. As a proof of concept, we successfully applied this approach for quantitation of immunoreactivity against different variants of SARS-CoV-2 spike and nucleocapsid proteins in sera of patients or vaccinees, as well as for the study of selective reactivity of monoclonal antibodies. Importantly, our system can be rapidly adapted to accommodate other SARS-CoV-2 variants as well as any antigen of a newly emerging pathogen, thereby representing an important resource in the context of pandemic preparedness. Full article
(This article belongs to the Special Issue Microscopy Methods for Virus Research)
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20 pages, 3592 KiB  
Article
Discordant Antigenic Properties of Soluble and Virion SARS-CoV-2 Spike Proteins
by Sameer Kumar, Souradip Dasgupta, Mohammad M. Sajadi, Greg A. Snyder, Anthony L. DeVico and Krishanu Ray
Viruses 2024, 16(3), 407; https://doi.org/10.3390/v16030407 - 6 Mar 2024
Cited by 1 | Viewed by 2232
Abstract
Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as [...] Read more.
Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2. Full article
(This article belongs to the Special Issue Microscopy Methods for Virus Research)
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12 pages, 11849 KiB  
Article
An Improved Workflow for the Quantification of Orthohantavirus Infection Using Automated Imaging and Flow Cytometry
by Laura Menke and Christian Sieben
Viruses 2024, 16(2), 269; https://doi.org/10.3390/v16020269 - 8 Feb 2024
Cited by 3 | Viewed by 1487
Abstract
Determination of the infectious titer is a central requirement when working with pathogenic viruses. The plaque or focus assay is a commonly used but labor- and time-consuming approach for determining the infectious titer of orthohantavirus samples. We have developed an optimized virus quantification [...] Read more.
Determination of the infectious titer is a central requirement when working with pathogenic viruses. The plaque or focus assay is a commonly used but labor- and time-consuming approach for determining the infectious titer of orthohantavirus samples. We have developed an optimized virus quantification approach that relies on the fluorescence-based detection of the orthohantavirus nucleocapsid protein (N) in infected cells with high sensitivity. We present the use of flow cytometry but highlight fluorescence microscopy in combination with automated data analysis as an attractive alternative to increase the information retrieved from an infection experiment. Additionally, we offer open-source software equipped with a user-friendly graphical interface, eliminating the necessity for advanced programming skills. Full article
(This article belongs to the Special Issue Microscopy Methods for Virus Research)
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Review

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13 pages, 3324 KiB  
Review
Zooming in and out: Exploring RNA Viral Infections with Multiscale Microscopic Methods
by Cheng-An Lyu, Yao Shen and Peijun Zhang
Viruses 2024, 16(9), 1504; https://doi.org/10.3390/v16091504 - 23 Sep 2024
Viewed by 1498
Abstract
RNA viruses, being submicroscopic organisms, have intriguing biological makeups and substantially impact human health. Microscopic methods have been utilized for studying RNA viruses at a variety of scales. In order of observation scale from large to small, fluorescence microscopy, cryo-soft X-ray tomography (cryo-SXT), [...] Read more.
RNA viruses, being submicroscopic organisms, have intriguing biological makeups and substantially impact human health. Microscopic methods have been utilized for studying RNA viruses at a variety of scales. In order of observation scale from large to small, fluorescence microscopy, cryo-soft X-ray tomography (cryo-SXT), serial cryo-focused ion beam/scanning electron microscopy (cryo-FIB/SEM) volume imaging, cryo-electron tomography (cryo-ET), and cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) have been employed, enabling researchers to explore the intricate world of RNA viruses, their ultrastructure, dynamics, and interactions with host cells. These methods evolve to be combined to achieve a wide resolution range from atomic to sub-nano resolutions, making correlative microscopy an emerging trend. The developments in microscopic methods provide multi-fold and spatial information, advancing our understanding of viral infections and providing critical tools for developing novel antiviral strategies and rapid responses to emerging viral threats. Full article
(This article belongs to the Special Issue Microscopy Methods for Virus Research)
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