Special Issue "Application of Advanced Imaging to the Study of Virus-Host Interactions"

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

Deadline for manuscript submissions: closed (30 November 2020) | Viewed by 21629

Special Issue Editor

Dr. Cristina Risco
E-Mail Website
Guest Editor
Cell Structure Laboratory, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, 28049 Madrid, Spain
Interests: virus-host interactions; microscopy; virus factories

Special Issue Information

Dear Colleagues,

All over the world, scientists are trying to understand how viruses infect hosts and cause illness. Viral infection can be studied at the level of single cells, organoids, tissues, and whole organisms. For all these studies, the combination of different imaging approaches is a very useful tool. Recent advances in light and electron microscopy are uncovering viral lifecycle events with a detail never before seen and providing knowledge to identify targets for new antiviral treatments. Viruses enter cells using a variety of pathways, and once inside, they assemble specialized organelles known as viral factories where they replicate their genome and build new infectious particles. The virus progeny is then often transferred to specific cell elements that mediate virus egress. Live cell imaging, fluorescence microscopy, electron microscopy, correlative microscopy, and in situ molecular mapping are powerful techniques to image virus entry, replication, morphogenesis, egress, and propagation. In addition, it is possible to study how cells and organisms defend themselves from viruses, to characterize how antiviral drugs work and understand why resistance to drugs is developed.

This Special Issue of Viruses will revise a variety of imaging techniques to study virus–host interactions. Scientists worldwide are welcome to contribute with articles, brief reports, technical notes, and reviews to show how they visualize virus infections. We aim to discuss how imaging helps to characterize and combat viruses that cause illness in plants, animals, and humans, but also how we can use viruses as tools in biotechnology and to cure disease.

Dr. Cristina Risco
Guest Editor

Manuscript Submission Information

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Keywords

  • virus-host
  • viral life cycle
  • viral replication
  • imaging
  • light microscopy
  • electron microscopy
  • correlative microscopy
  • molecular mapping in situ

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Published Papers (14 papers)

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Editorial

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Editorial
Application of Advanced Imaging to the Study of Virus–Host Interactions
Viruses 2021, 13(10), 1958; https://doi.org/10.3390/v13101958 - 29 Sep 2021
Viewed by 501
Abstract
Recent advances in light and electron microscopy are uncovering viral lifecycle events with a level of detail never before seen [...] Full article
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Research

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Article
Bromodeoxyuridine Labelling to Determine Viral DNA Localization in Fluorescence and Electron Microscopy: The Case of Adenovirus
Viruses 2021, 13(9), 1863; https://doi.org/10.3390/v13091863 - 18 Sep 2021
Cited by 1 | Viewed by 681
Abstract
The localization of viral nucleic acids in the cell is essential for understanding the infectious cycle. One of the strategies developed for this purpose is the use of nucleotide analogs such as bromodeoxyuridine (BrdU, analog to thymine) or bromouridine (BrU, analog of uridine), [...] Read more.
The localization of viral nucleic acids in the cell is essential for understanding the infectious cycle. One of the strategies developed for this purpose is the use of nucleotide analogs such as bromodeoxyuridine (BrdU, analog to thymine) or bromouridine (BrU, analog of uridine), which are incorporated into the nucleic acids during replication or transcription. In adenovirus infections, BrdU has been used to localize newly synthesized viral genomes in the nucleus, where it is key to distinguish between host and viral DNA. Here, we describe our experience with methodological variations of BrdU labeling to localize adenovirus genomes in fluorescence and electron microscopy. We illustrate the need to define conditions in which most of the newly synthesized DNA corresponds to the virus and not the host, and the amount of BrdU provided is enough to incorporate to the new DNA molecules without hampering the cell metabolism. We hope that our discussion of problems encountered and solutions implemented will help other researches interested in viral genome localization in infected cells. Full article
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Article
Single-Molecule Super-Resolution Imaging of T-Cell Plasma Membrane CD4 Redistribution upon HIV-1 Binding
Viruses 2021, 13(1), 142; https://doi.org/10.3390/v13010142 - 19 Jan 2021
Cited by 3 | Viewed by 2332
Abstract
The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. [...] Read more.
The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For one of the first times, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host CD4+ T cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function. Full article
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Article
Unpicking the Secrets of African Swine Fever Viral Replication Sites
Viruses 2021, 13(1), 77; https://doi.org/10.3390/v13010077 - 08 Jan 2021
Cited by 2 | Viewed by 2565
Abstract
African swine fever virus (ASFV) is a highly contagious pathogen which causes a lethal haemorrhagic fever in domestic pigs and wild boar. The large, double-stranded DNA virus replicates in perinuclear cytoplasmic replication sites known as viral factories. These factories are complex, multi-dimensional structures. [...] Read more.
African swine fever virus (ASFV) is a highly contagious pathogen which causes a lethal haemorrhagic fever in domestic pigs and wild boar. The large, double-stranded DNA virus replicates in perinuclear cytoplasmic replication sites known as viral factories. These factories are complex, multi-dimensional structures. Here we investigated the protein and membrane compartments of the factory using super-resolution and electron tomography. Click IT chemistry in combination with stimulated emission depletion (STED) microscopy revealed a reticular network of newly synthesized viral proteins, including the structural proteins p54 and p34, previously seen as a pleomorphic ribbon by confocal microscopy. Electron microscopy and tomography confirmed that this network is an accumulation of membrane assembly intermediates which take several forms. At early time points in the factory formation, these intermediates present as small, individual membrane fragments which appear to grow and link together, in a continuous progression towards new, icosahedral virions. It remains unknown how these membranes form and how they traffic to the factory during virus morphogenesis. Full article
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Article
Live Cell Microscopy of Murine Polyomavirus Subnuclear Replication Centers
Viruses 2020, 12(10), 1123; https://doi.org/10.3390/v12101123 - 02 Oct 2020
Cited by 1 | Viewed by 1087
Abstract
During polyomavirus (PyV) infection, host proteins localize to subnuclear domains, termed viral replication centers (VRCs), to mediate viral genome replication. Although the protein composition and spatial organization of VRCs have been described using high-resolution immunofluorescence microscopy, little is known about the temporal dynamics [...] Read more.
During polyomavirus (PyV) infection, host proteins localize to subnuclear domains, termed viral replication centers (VRCs), to mediate viral genome replication. Although the protein composition and spatial organization of VRCs have been described using high-resolution immunofluorescence microscopy, little is known about the temporal dynamics of VRC formation over the course of infection. We used live cell fluorescence microscopy to analyze VRC formation during murine PyV (MuPyV) infection of a mouse fibroblast cell line that constitutively expresses a GFP-tagged replication protein A complex subunit (GFP-RPA32). The RPA complex forms a heterotrimer (RPA70/32/14) that regulates cellular DNA replication and repair and is a known VRC component. We validated previous observations that GFP-RPA32 relocalized to sites of cellular DNA damage in uninfected cells and to VRCs in MuPyV-infected cells. We then used GFP-RPA32 as a marker of VRC formation and expansion during live cell microscopy of infected cells. VRC formation occurred at variable times post-infection, but the rate of VRC expansion was similar between cells. Additionally, we found that the early viral protein, small TAg (ST), was required for VRC expansion but not VRC formation, consistent with the role of ST in promoting efficient vDNA replication. These results demonstrate the dynamic nature of VRCs over the course of infection and establish an approach for analyzing viral replication in live cells. Full article
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Review

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Review
FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist
Viruses 2021, 13(4), 611; https://doi.org/10.3390/v13040611 - 02 Apr 2021
Cited by 5 | Viewed by 1997
Abstract
The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric “volume electron microscopy” or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a “Goldilocks zone” in vEM: iterative [...] Read more.
The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric “volume electron microscopy” or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a “Goldilocks zone” in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures. Full article
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Review
Imaging Techniques to Study Plant Virus Replication and Vertical Transmission
Viruses 2021, 13(3), 358; https://doi.org/10.3390/v13030358 - 25 Feb 2021
Cited by 1 | Viewed by 1025
Abstract
Plant viruses are obligate parasites that need to usurp plant cell metabolism in order to infect their hosts. Imaging techniques have been used for quite a long time to study plant virus–host interactions, making it possible to have major advances in the knowledge [...] Read more.
Plant viruses are obligate parasites that need to usurp plant cell metabolism in order to infect their hosts. Imaging techniques have been used for quite a long time to study plant virus–host interactions, making it possible to have major advances in the knowledge of plant virus infection cycles. The imaging techniques used to study plant–virus interactions have included light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopies. Here, we review the use of these techniques in plant virology, illustrating recent advances in the area with examples from plant virus replication and virus plant-to-plant vertical transmission processes. Full article
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Review
Single-Molecule FRET Imaging of Virus Spike–Host Interactions
Viruses 2021, 13(2), 332; https://doi.org/10.3390/v13020332 - 21 Feb 2021
Cited by 6 | Viewed by 1922
Abstract
As a major surface glycoprotein of enveloped viruses, the virus spike protein is a primary target for vaccines and anti-viral treatments. Current vaccines aiming at controlling the COVID-19 pandemic are mostly directed against the SARS-CoV-2 spike protein. To promote virus entry and facilitate [...] Read more.
As a major surface glycoprotein of enveloped viruses, the virus spike protein is a primary target for vaccines and anti-viral treatments. Current vaccines aiming at controlling the COVID-19 pandemic are mostly directed against the SARS-CoV-2 spike protein. To promote virus entry and facilitate immune evasion, spikes must be dynamic. Interactions with host receptors and coreceptors trigger a cascade of conformational changes/structural rearrangements in spikes, which bring virus and host membranes in proximity for membrane fusion required for virus entry. Spike-mediated viral membrane fusion is a dynamic, multi-step process, and understanding the structure–function-dynamics paradigm of virus spikes is essential to elucidate viral membrane fusion, with the ultimate goal of interventions. However, our understanding of this process primarily relies on individual structural snapshots of endpoints. How these endpoints are connected in a time-resolved manner, and the order and frequency of conformational events underlying virus entry, remain largely elusive. Single-molecule Förster resonance energy transfer (smFRET) has provided a powerful platform to connect structure–function in motion, revealing dynamic aspects of spikes for several viruses: SARS-CoV-2, HIV-1, influenza, and Ebola. This review focuses on how smFRET imaging has advanced our understanding of virus spikes’ dynamic nature, receptor-binding events, and mechanism of antibody neutralization, thereby informing therapeutic interventions. Full article
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Review
Application of Super-Resolution and Advanced Quantitative Microscopy to the Spatio-Temporal Analysis of Influenza Virus Replication
Viruses 2021, 13(2), 233; https://doi.org/10.3390/v13020233 - 02 Feb 2021
Cited by 3 | Viewed by 1780
Abstract
With an estimated three to five million human cases annually and the potential to infect domestic and wild animal populations, influenza viruses are one of the greatest health and economic burdens to our society, and pose an ongoing threat of large-scale pandemics. Despite [...] Read more.
With an estimated three to five million human cases annually and the potential to infect domestic and wild animal populations, influenza viruses are one of the greatest health and economic burdens to our society, and pose an ongoing threat of large-scale pandemics. Despite our knowledge of many important aspects of influenza virus biology, there is still much to learn about how influenza viruses replicate in infected cells, for instance, how they use entry receptors or exploit host cell trafficking pathways. These gaps in our knowledge are due, in part, to the difficulty of directly observing viruses in living cells. In recent years, advances in light microscopy, including super-resolution microscopy and single-molecule imaging, have enabled many viral replication steps to be visualised dynamically in living cells. In particular, the ability to track single virions and their components, in real time, now allows specific pathways to be interrogated, providing new insights to various aspects of the virus-host cell interaction. In this review, we discuss how state-of-the-art imaging technologies, notably quantitative live-cell and super-resolution microscopy, are providing new nanoscale and molecular insights into influenza virus replication and revealing new opportunities for developing antiviral strategies. Full article
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Review
Application of Advanced Light Microscopy to the Study of HIV and Its Interactions with the Host
Viruses 2021, 13(2), 223; https://doi.org/10.3390/v13020223 - 01 Feb 2021
Cited by 1 | Viewed by 1105
Abstract
This review highlights the significant observations of human immunodeficiency virus (HIV) assembly, release and maturation made possible with advanced light microscopy techniques. The advances in technology which now enables these light microscopy measurements are discussed with special emphasis on live imaging approaches including [...] Read more.
This review highlights the significant observations of human immunodeficiency virus (HIV) assembly, release and maturation made possible with advanced light microscopy techniques. The advances in technology which now enables these light microscopy measurements are discussed with special emphasis on live imaging approaches including Total Internal Reflection Fluorescence (TIRF), high-resolution light microscopy techniques including PALM and STORM and single molecule measurements, including Fluorescence Resonance Energy Transfer (FRET). The review concludes with a discussion on what new insights and understanding can be expected from these measurements. Full article
Review
Imaging Viral Infection by Fluorescence Microscopy: Focus on HIV-1 Early Stage
Viruses 2021, 13(2), 213; https://doi.org/10.3390/v13020213 - 30 Jan 2021
Cited by 5 | Viewed by 1491
Abstract
During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. [...] Read more.
During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step. Full article
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Review
Multiscale Electron Microscopy for the Study of Viral Replication Organelles
Viruses 2021, 13(2), 197; https://doi.org/10.3390/v13020197 - 28 Jan 2021
Cited by 3 | Viewed by 1623
Abstract
During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human [...] Read more.
During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs. Full article
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Review
Nuclear Import of Adeno-Associated Viruses Imaged by High-Speed Single-Molecule Microscopy
Viruses 2021, 13(2), 167; https://doi.org/10.3390/v13020167 - 22 Jan 2021
Cited by 3 | Viewed by 1278
Abstract
Understanding the detailed nuclear import kinetics of adeno-associated virus (AAV) through the nuclear pore complex (NPC) is essential for the application of AAV capsids as a nuclear delivery instrument as well as a target for drug development. However, a comprehensive understanding of AAV [...] Read more.
Understanding the detailed nuclear import kinetics of adeno-associated virus (AAV) through the nuclear pore complex (NPC) is essential for the application of AAV capsids as a nuclear delivery instrument as well as a target for drug development. However, a comprehensive understanding of AAV transport through the sub-micrometer NPCs in live cells calls for new techniques that can conquer the limitations of conventional fluorescence microscopy and electron microscopy. With recent technical advances in single-molecule fluorescence microscopy, we are now able to image the entire nuclear import process of AAV particles and also quantify the transport dynamics of viral particles through the NPCs in live human cells. In this review, we initially evaluate the necessity of single-molecule live-cell microscopy in the study of nuclear import for AAV particles. Then, we detail the application of high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy in tracking the entire process of nuclear import for AAV particles. Finally, we summarize the major findings for AAV nuclear import by using SPEED microscopy. Full article
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Other

Brief Report
New Look at RSV Infection: Tissue Clearing and 3D Imaging of the Entire Mouse Lung at Cellular Resolution
Viruses 2021, 13(2), 201; https://doi.org/10.3390/v13020201 - 28 Jan 2021
Cited by 2 | Viewed by 1159
Abstract
Background: Respiratory Syncytial Virus (RSV) is the major cause of severe acute respiratory tract illness in young children worldwide and a main pathogen for the elderly and immune-compromised people. In the absence of vaccines or effective treatments, a better characterization of the pathogenesis [...] Read more.
Background: Respiratory Syncytial Virus (RSV) is the major cause of severe acute respiratory tract illness in young children worldwide and a main pathogen for the elderly and immune-compromised people. In the absence of vaccines or effective treatments, a better characterization of the pathogenesis of RSV infection is required. To date, the pathophysiology of the disease and its diagnosis has mostly relied on chest X-ray and genome detection in nasopharyngeal swabs. The development of new imaging approaches is instrumental to further the description of RSV spread, virus–host interactions and related acute respiratory disease, at the level of the entire lung. Methods: By combining tissue clearing, 3D microscopy and image processing, we developed a novel visualization tool of RSV infection in undissected mouse lungs. Results: Whole tissue analysis allowed the identification of infected cell subtypes, based on both morphological traits and position within the cellular network. Furthermore, 3D imaging was also valuable to detect the cytoplasmic viral factories, also called inclusion bodies, a hallmark of RSV infection. Conclusions: Whole lung clearing and 3D deep imaging represents an unprecedented visualization method of infected lungs to allow insight into RSV pathophysiology and improve the 2D histology analyses. Full article
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