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Viruses
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Flow Virometry: A New Tool for Studying Viruses
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Flow Virometry: A New Tool for Studying Viruses

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

Deadline for manuscript submissions: 15 July 2025 | Viewed by 7464

Special Issue Editor

Dr. John Yin

E-Mail Website
Guest Editor
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA
Interests: virus–host interactions; systems biology; quantitative virology; computational biology; origins of life; systems chemistry; prebiotic chemistry

Special Issue Information

Dear Colleagues,

We cordially invite you to contribute your groundbreaking research in the field of Flow Virometry to our upcoming issue of Viruses. As the Editor, I am excited to showcase your innovative work characterizing virus and virus-like particles at the frontiers of quantitative virology.

Flow Virometry promises to open new opportunities in the high-throughput physical, chemical, and biological characterization of viruses and virus-like particles, with impacts on diagnostics, viral pathogenesis, therapeutics, and vaccines. We welcome your manuscripts and offer you a platform to share your discoveries and insights with a broad audience. Your contributions will drive this emerging field forward and lead to a better understanding of viruses as readily quantifiable physical and chemical nanoparticles with encoded biology.

Submit your manuscripts by April 2024, and let us shape the future of virology together. Join us in this exciting journey of discovery.

Dr. John Yin
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

  • flow virometry
  • virus particle detection
  • virus counting
  • virus labeling/staining
  • virus-like particles
  • scanning ion occlusion sensing

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Published Papers (5 papers)

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Research

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19 pages, 3858 KiB  
Article
Flow Virometry in Wastewater Monitoring: Comparison of Virus-like Particles to Coliphage, Pepper Mild Mottle Virus, CrAssphage, and Tomato Brown Rugose Fruit Virus
by Melis M. Johnson, C. Winston Bess, Rachel Olson and Heather N. Bischel
Viruses 2025, 17(4), 575; https://doi.org/10.3390/v17040575 - 16 Apr 2025
Viewed by 352
Abstract
Flow virometry (FVM) offers a promising approach for monitoring viruses and virus-like particles (VLPs) in environmental samples. This study compares levels of non-specific VLPs across a wastewater treatment plant (WWTP) with levels of somatic coliphage, (F+) specific coliphage, Pepper Mild Mottle Virus (PMMoV), [...] Read more.
Flow virometry (FVM) offers a promising approach for monitoring viruses and virus-like particles (VLPs) in environmental samples. This study compares levels of non-specific VLPs across a wastewater treatment plant (WWTP) with levels of somatic coliphage, (F+) specific coliphage, Pepper Mild Mottle Virus (PMMoV), CrAssphage (CrAss), and Tomato Brown Rugose Fruit Virus (ToBRFV). All targets were quantified in influent, secondary-treated effluent, and tertiary-treated effluent at the University of California, Davis Wastewater Treatment Plant (UCDWWTP) over 11 weeks. We established an FVM-gating boundary for VLPs using bacteriophages T4 and ϕ6 as well as four phages isolated from wastewater. We then utilize T4 alongside three submicron beads as quality controls in the FVM assay. Coliphage was measured by standard plaque assays, and genome copies of PMMoV, CrAss, and ToBRFV were measured by digital droplet (dd)PCR. FVM results for wastewater revealed distinct microbial profiles at each treatment stage. However, correlations between VLPs and targeted viruses were poor. Trends for virus inactivation and removal, observed for targeted viruses during wastewater treatment, were consistent with expectations. Conversely, VLP counts were elevated in the WWTP effluent relative to the influent. Additional sampling revealed a decrease in VLP counts during the filtration treatment step following secondary treatment but a substantial increase in VLPs following ultraviolet disinfection. Defining application boundaries remain crucial to ensuring meaningful data interpretation as flow cytometry and virometry take on greater significance in water quality monitoring. Full article
(This article belongs to the Special Issue Flow Virometry: A New Tool for Studying Viruses)
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17 pages, 3611 KiB  
Article
Characterization of Nanobody Binding to Distinct Regions of the SARS-CoV-2 Spike Protein by Flow Virometry
by Mariam Maltseva, Martin A. Rossotti, Jamshid Tanha and Marc-André Langlois
Viruses 2025, 17(4), 571; https://doi.org/10.3390/v17040571 - 15 Apr 2025
Viewed by 330
Abstract
Nanobodies, or single-domain antibodies (VHHs) from camelid heavy-chain-only antibodies, offer significant advantages in therapeutic and diagnostic applications due to their small size and ability to bind cryptic protein epitopes inaccessible to conventional antibodies. In this study, we examined nanobodies specific to [...] Read more.
Nanobodies, or single-domain antibodies (VHHs) from camelid heavy-chain-only antibodies, offer significant advantages in therapeutic and diagnostic applications due to their small size and ability to bind cryptic protein epitopes inaccessible to conventional antibodies. In this study, we examined nanobodies specific to regions of the SARS-CoV-2 spike glycoprotein, including the receptor-binding domain (RBD), N-terminal domain (NTD), and subunit 2 (S2). Using flow virometry, a high-throughput technique for viral quantification, we achieved the efficient detection of pseudotyped viruses expressing the spike glycoprotein. RBD-targeting nanobodies showed the most effective staining, followed by NTD-targeting ones, while S2-specific nanobodies exhibited limited resolution. The simple genetic structure of nanobodies enables the creation of multimeric formats, improving binding specificity and avidity. Bivalent VHH-Fc constructs (VHHs fused to the Fc region of human IgG) outperformed monovalent formats in resolving viral particles from background noise. However, S2-specific monovalent VHHs demonstrated improved staining efficiency, suggesting their smaller size better accesses restricted antigenic sites. Furthermore, direct staining of cell supernatants was possible without virus purification. This versatile nanobody platform, initially developed for antiviral therapy against SARS-CoV-2, can be readily adapted for flow virometry applications and other diagnostic assays. Full article
(This article belongs to the Special Issue Flow Virometry: A New Tool for Studying Viruses)
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21 pages, 5836 KiB  
Article
Applying Flow Virometry to Study the HIV Envelope Glycoprotein and Differences Across HIV Model Systems
by Jonathan Burnie, Claire Fernandes, Ayushi Patel, Arvin Tejnarine Persaud, Deepa Chaphekar, Danlan Wei, Timothy Kit Hin Lee, Vera A. Tang, Claudia Cicala, James Arthos and Christina Guzzo
Viruses 2024, 16(6), 935; https://doi.org/10.3390/v16060935 - 9 Jun 2024
Cited by 3 | Viewed by 1877
Abstract
The HIV envelope glycoprotein (Env) is a trimeric protein that facilitates viral binding and fusion with target cells. As the sole viral protein on the HIV surface, Env is important both for immune responses to HIV and in vaccine designs. Targeting Env in [...] Read more.
The HIV envelope glycoprotein (Env) is a trimeric protein that facilitates viral binding and fusion with target cells. As the sole viral protein on the HIV surface, Env is important both for immune responses to HIV and in vaccine designs. Targeting Env in clinical applications is challenging due to its heavy glycosylation, high genetic variability, conformational camouflage, and its low abundance on virions. Thus, there is a critical need to better understand this protein. Flow virometry (FV) is a useful methodology for phenotyping the virion surface in a high-throughput, single virion manner. To demonstrate the utility of FV to characterize Env, we stained HIV virions with a panel of 85 monoclonal antibodies targeting different regions of Env. A broad range of antibodies yielded robust staining of Env, with V3 antibodies showing the highest quantitative staining. A subset of antibodies tested in parallel on viruses produced in CD4+ T cell lines, HEK293T cells, and primary cells showed that the cellular model of virus production can impact Env detection. Finally, in addition to being able to highlight Env heterogeneity on virions, we show FV can sensitively detect differences in Env conformation when soluble CD4 is added to virions before staining. Full article
(This article belongs to the Special Issue Flow Virometry: A New Tool for Studying Viruses)
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20 pages, 11056 KiB  
Article
Premature Activation of the HIV-1 Protease Is Influenced by Polymorphisms in the Hinge Region
by Caroline O. Tabler, Sarah J. Wegman, Najwa Alhusaini, Nicole F. Lee and John C. Tilton
Viruses 2024, 16(6), 849; https://doi.org/10.3390/v16060849 - 26 May 2024
Cited by 1 | Viewed by 1453
Abstract
HIV-1 protease inhibitors are an essential component of antiretroviral therapy. However, drug resistance is a pervasive issue motivating a persistent search for novel therapies. Recent reports found that when protease activates within the host cell’s cytosol, it facilitates the pyroptotic killing of infected [...] Read more.
HIV-1 protease inhibitors are an essential component of antiretroviral therapy. However, drug resistance is a pervasive issue motivating a persistent search for novel therapies. Recent reports found that when protease activates within the host cell’s cytosol, it facilitates the pyroptotic killing of infected cells. This has led to speculation that promoting protease activation, rather than inhibiting it, could help to eradicate infected cells and potentially cure HIV-1 infection. Here, we used a nanoscale flow cytometry-based assay to characterize protease resistance mutations and polymorphisms. We quantified protease activity, viral concentration, and premature protease activation and confirmed previous findings that major resistance mutations generally destabilize the protease structure. Intriguingly, we found evidence that common polymorphisms in the hinge domain of protease can influence its susceptibility to premature activation. This suggests that viral heterogeneity could pose a considerable challenge for therapeutic strategies aimed at inducing premature protease activation in the future. Full article
(This article belongs to the Special Issue Flow Virometry: A New Tool for Studying Viruses)
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Review

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11 pages, 967 KiB  
Review
Analysis of Individual Viral Particles by Flow Virometry
by Caroline O. Tabler and John C. Tilton
Viruses 2024, 16(5), 802; https://doi.org/10.3390/v16050802 - 18 May 2024
Cited by 3 | Viewed by 2350
Abstract
This review focuses on the emerging field of flow virometry, the study and characterization of individual viral particles using flow cytometry instruments and protocols optimized for the detection of nanoscale events. Flow virometry faces considerable technical challenges including minimal light scattering by small [...] Read more.
This review focuses on the emerging field of flow virometry, the study and characterization of individual viral particles using flow cytometry instruments and protocols optimized for the detection of nanoscale events. Flow virometry faces considerable technical challenges including minimal light scattering by small viruses that complicates detection, coincidental detection of multiple small particles due to their high concentrations, and challenges with sample preparation including the inability to easily “wash” samples to remove unbound fluorescent antibodies. We will discuss how the field has overcome these challenges to reveal novel insights into viral biology. Full article
(This article belongs to the Special Issue Flow Virometry: A New Tool for Studying Viruses)
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