Microscopy in Virology

Greb, Christoph

July 13, 2020

Microscopy in Virology

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The coronavirus SARS-CoV-2, causing the Covid-19 disease effects our world in all aspects. Research to find immunization and treatment methods, in other words to fight this virus, gained highest priority in 2020. Microscopes play a significant role in this kind of research. To understand the underlaying principles of receptor binding, genome release, replication, assembly, and virus budding, as well as the response of our immune system, different methodologies and microscopes can be used. This article summarizes why microscopy is an important tool in virology and infection biology and gives examples for different microscopy technologies and their applications in these research fields.

Broad range of microscopy applications for virology

The application range of light microscopy for virologists is broad. Most of them are based on fluorescence. Fluorescence microscopy can be roughly subdivided into immunofluorescence and the utilization of fluorescent proteins. Immunofluorescence utilizes fixed cells or tissues to stain the protein of interest with fluorescently marked antibodies, while fluorescent proteins can also be utilized for live-cell imaging.

Microscopy methods used in virology range from widefield microscopy, through confocal microscopy, to super-resolution microscopy. Moreover, there is also the applicability of Laser Microdissection.

The following references will give you an overview of prominent microscopy methods and their application in virology:

Influence of the cellular lipid metabolism on coronavirus replication

Müller et al. studied the influence of the cellular lipid metabolism on coronavirus replication. They infected Huh-7 cells with the coronavirus HCov-229E and stained these cells with antibodies against dsRNA, the coronavirus N protein, and the coronavirus NSP8 protein. Moreover, they marked lysophospholipids with the help of the BODIPY phospholipid PC-A2. Imaging was then executed with a TCS SP5 confocal microscope equipped with a 63x Plan-Apochromat objective. The researchers were interested in colocalization events of dsRNA and PC-A2.

The cytosolic phospholipase A2α (cPLA2α) produces phospholipids. When they treated the cells with an inhibitor of cPLA2α, they could detect a significant effect on viral RNA and protein accumulation in human coronavirus cells. In more detail, they observed that RTC (viral replication/transcription complex) co-localized with lysophospholipid containing membrane structures. For this reason, they conclude that cPLA2α could be a target for an antiviral drug.

Viruses have their own RNA or DNA. Due to this fact, they can be detected by fluorescence in situ hybridization (FISH) where a fluorescent probe is used to target viral nucleotides.

Fig. 1: Typical components of a virion: proteins, e.g. Polymerase, nucleic acid (RNA or DNA), lipid bilayer (enveloped), envelope proteins, ribonucleoprotein, and a capsid.

Bacterial infection in the mouse mucosal barrier

Jarret et al. did not investigate viral but rather bacterial infection in the mouse mucosal barrier. By using single-molecule fluorescence in situ mRNA hybridization (smFISH; THUNDER Imager 3D Live Cell) they discovered that intestinal neurons produce the cytokine IL-18. With it, they could show that neuron derived IL-18 signaling plays a major role in intestinal immunity which was not yet discovered.

Monitor markers for immunosuppressive mechanisms

Immunosuppression can be studied with the help of viral infected mouse models. Andrew Beppu used large multi-channel tile scans of mouse lungs to monitor markers for immunosuppressive mechanisms. The mice were infected with the influenza virus and then investigated for regression of lung basal-like structures (THUNDER Imager 3D Cell Culture).

Fig. 4: Immunofluorescence, Keratin-5 (green), PDL-1 (red): Thin section of mouse lung where the mouse was inoculated with the Puerto Rico 8 strain of influenza virus to induce injury to the lung epithelium. Image courtesy of Andrew Beppu, Cedars Sinai Medical Center. — Fig. 2: Immunofluorescence, Keratin-5 (green), PDL-1 (red): Thin section of mouse lung where the mouse was inoculated with the Puerto Rico 8 strain of influenza virus to induce injury to the lung epithelium. Image courtesy of Andrew Beppu, Cedars Sinai Medical Center.

DENV induced gene expression effects

Sim et al. studied Dengue virus (DENV) induced gene expression effects in the salivary gland of the yellow fever mosquito (Aedes aegypti). The authors found out that upon DENV infection, the transcriptome of the mosquito salivary gland was changed. By using microarrays for gene expression analysis, they could show for the first time that some genes were expressed which have influence on the mosquito’s host-seeking and probing behavior. They used immunofluorescence microscopy to identify regions in the mosquito’s chemosensory organs where the virus was abundant (see Fig. 3).
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1002631#s4

Fig. 3: Sim et al. examined the salivary gland of Aedes aegypti after infection with the Dengue virus.

The morphology of Hendra virus infected cells

Monaghan et al. characterized the morphology of Hendra virus infected cells with the help of microscopy. Confocal images (SP5) revealed the intracellular viral protein distribution, whereas super-resolution microscopy (SR-GSD) even gave a closer look into the protein distribution inside virions.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254186/

Cellular RNA helicase binding to La Crosse virus (LACV) nucleocapsids

Super-resolution microscopy was utilized by Weber et al. to study cellular RNA helicase binding to La Crosse virus (LACV) nucleocapsids (SR-GSD). For this purpose, immunofluorescence was applied to stain the viral LACV N protein and cellular RNA helicase RIG-I in infected A549 cells. Super-resolution microscopy supported biochemistry data that RIG-I binds the nucleocapsids via the 5’ppp dsRNA panhandle.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515363/

Natural killer (NK) cells

Maze and Orange investigated natural killer (NK) cells which are important counterplayers of viruses. They belong to the innate immune system and surveil virally infected and tumorigenic cells. Directed secretion of specialized secretory lysosomes via the immunological synapse kills the virally infected cells. STED nanoscopy (TCS SP8 STED) was used to decipher the interaction of lytic granules with the cytoskeleton of the NK cells.

Fig. 4: The viral replication cycle starts with receptor binding at the surface of the host cell. After endocytosis, the viral content is released into the host cell. RNA (RNA viruses) can be translated directly into proteins, whereas DNA (DNA viruses) must be transcribed first. Moreover, the viral genome is replicated in the nucleus or so-called virus factories. Afterwards, the viral components are assembled to build an intact virion again, which buds through the plasma membrane of the host cell to infect other cells. Lytic viruses do not bud, but they destroy their host cell for release.

Image the immunological synapse with STED nanoscopy

Another publication by Zheng et al. describes the technical procedure which can be used to image the immunological synapse with STED nanoscopy (TCS SP8 STED).
https://www.jove.com/video/52502/super-resolution-imaging-natural-killer-cell-immunological-synapse-on

Shed light on parasitology problems with STED nanoscopy

In addition to virology and immunology, STED nanoscopy can also shed light on parasitology problems, such as merozoite invasion in erythrocytes. 3D STED (TCS SP8 STED) reveals spatial information of the protein components which are involved in Plasmodium infection.

Influence of coronaviruses on cellular NF-κB signaling and chromatin landscape

Poppe et al. investigated the influence of coronaviruses on cellular NF-κB signaling and chromatin landscape. With the help of Laser Microdissection (LMD6000), they isolated cells expressing the coronavirus N protein and extracted their entire RNA. By utilizing RT-qPCR and microarray analysis, they found out which mRNAs were over- or under-represented in the cells. Furthermore, they used immunofluorescence of the coronavirus N protein to monitor viral infection and its spread in A546 cells (DMIRE2, DMi8).
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006286#sec002)

Brightfield microscopy plays a direct, but only minor role in virology, because fluorescence microscopy fulfills better the demands of researchers. Nevertheless, there are opportunities for brightfield microscopy when animal tissues are investigated. For example, researchers can check tissues for morphology changes after viral infection, etc.

In addition, brightfield microscopy is used in cell culture labs to check the health and growth status of cells (see Fig. 5) which are already infected or will be at a later time (DM IL, DMi1).

Fig. 5: Brightfield image of MDCK cells, a polarized epithelial cell line, which are utilized in virology amongst other cell lines.

This list of microscopy methods is not meant to be comprehensive. There are other techniques which are also used to visualize viruses, but are not in the scope of this article. For example, electron microscopy (EM) can resolve virus particles. Single molecule detection and Fluorescence Lifetime Imaging (FLIM) (STELLARIS 8 FALCON), as well as multiphoton microscopy (SP8 DIVE) are additional methods, which are suitable for virology (see references).

Fig. 6: Sample preparation for electron microscopy. Image taken from https://www.youtube.com/watch?v=dtKT0FUKKxA at EMBL, Heidelberg.

SARS-CoV and SARS-CoV-2 were able to infect enterocytes

Lamers et al. investigated, if the coronavirus SARS-CoV-2 infects not only the respiratory system, but also the human gut. By using confocal microscopy (SP8) and electron microscopy (EM sample preparation: EM UC7) with human small intestine organoids, they could show that SARS-CoV and SARS-CoV-2 were able to infect enterocytes.

To find out more about electron microscopy, please refer to the examples given in the references below.