Application of electron microscopy in virus diagnosis and advanced therapeutic approaches

Electron microscopy in virus discovery and identification

In 1886, Adolph Meyer made a groundbreaking discovery concerning tobacco mosaic disease, revealing its transmission from an infected plant to a healthy one through liquid plant extracts. Subsequently, in 1892, Dmitri Ivanowski demonstrated the persistence of this disease even after the removal of all viable bacteria using the Chamberland-Pasteur filter. However, it took several years to establish that these infectious agents were a novel class of disease-causing entities known as viruses. Typically ranging in size from 20 to 250 nanometers (nm), viral particles remained elusive until the advent of the electron microscope in the 1940s. This technological breakthrough allowed scientists to obtain a comprehensive view of the structure of the tobacco mosaic virus and other viruses for the first time [1,2].

The first transmission electron microscope (TEM) was invented by Max Knoll and Ernst Ruska in the early 1930s. The invention of the electron microscope, with magnification and resolution powers several orders of magnitude better than those of optical instruments, opened up possibilities for biological applications. Helmut Ruska, the younger brother of Ernst Ruska, characterized the morphology of several viruses and determined the size and shape of various viral particles using TEM. Ruska’s discovery of the utility of TEM in clinical virology quickly led to the application of this technique for diagnosing smallpox and chickenpox, caused by the variola and varicella-zoster viruses, respectively [3,4]. These early studies found the varicella-zoster virus, which is within the herpes family of viruses, to have a spherical shape and a diameter within the range of 140 nm to 150 nm. However, TEM is a highly time-consuming technique. Biological samples, such as cells and tissues, need to be embedded in a hard resin, sectioned into 50–70 nm slices with an ultramicrotome before imaging. Furthermore, the analysis of TEM images can be particularly laborious and requires a significant amount of knowledge about viral morphology by the individual. Despite these disadvantages, its utility in providing an accurate diagnosis maintained its clinical use until the 1990s. As technology continued to advance, several novel molecular techniques, including enzyme-linked immunosorbent assays (ELISAs) and polymerase chain reactions (PCRs), have also emerged as alternatives to TEM [5–7].

Although a few other methods are available for the detection of viruses, TEM remains very useful in medical diagnosis. TEM can be employed to confirm a diagnosis that was previously established with molecular techniques. During the 2003 severe acute respiratory syndrome (SARS) pandemic in China, TEM was used to characterize the specific etiology of the coronavirus responsible for the outbreak. At almost the same time, TEM was also utilized to identify a poxvirus known as the monkeypox virus, which was causing widespread illness among prairie dogs in the United States. More recently, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was imaged by electron microscopy. The data produced by electron microscopy techniques confirmed that SARS-CoV-2 closely resembled both the SARS virus and the Middle East respiratory syndrome coronavirus (MERS-CoV). Electron microscopy also offers the unique ability to simultaneously detect multiple infections caused by one or more viruses, which could otherwise be missed in conventional molecular or antigen tests.

Nearly all lethal viral outbreaks in the past two decades were caused by newly emerging viruses. Viral contamination of biotechnology products may arise from the original source of the cell lines or from adventitious introduction of virus during production processes. TEM is routinely used for fast virus detection and identification in diagnostic settings. Federal agencies including the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) continue to recommend the use of TEM as a complementary tool for various in vitro assays and molecular tests. These agencies specify the use of TEM to assess the viral safety of biopharmaceutical products including cell lines, culture supernatants, and fermenter bulk harvests.

According to recommendations as in the WHO TRS 878 Annex 1 published in 2010 (‘Recommendations for the evaluation of animal cell substrates for the manufacture of biological medical products and for the characterization of cell banks’). At least 200 cells from the MCB or WCB, and ECB are examined by TEM for evidence of contamination with microbial agents. Methods include negative staining and thin section. TEM results for cell substrates include the number of cells examined and evaluation of morphology of the particles to further follow-up for evaluating potential risk (for retroviruses, it would also include particle type (e.g. intracisternal A type, type C, etc.). If there are particles detected, then different magnification of photographs is included to indicate the subcellular location of the particles and detailed morphology of particles provided (e.g. in case of retroviruses to distinguish nonenveloped, intracisternal A type and enveloped type C, budding from the membrane). For cell substrates, since the number of particles is difficult to quantify, it can be provided as a range (e.g. 1–5, etc.) or none detected. In case of determination of particles from the supernatant, quantification can be helpful in demonstrating viral clearance. In case of quantification, the particle counts and the calculations for particles per ml can be tabulated. Additional follow-up studies may be required if particles are identified using TEM.

Negative staining TEM (nsTEM)

Sydney Brenner and Robert Horne developed the negative staining method for TEM in 1959. In negative staining, aqueous-based suspensions of biological particle samples are deposited onto coated grids. Heavy metal salts such as uranyl acetate or phosphotungstic acid are then used to stain the grids to enhance the visibility of the biological particles. Negative staining not only allowed researchers to distinguish viral particles from the background, but also their unique structural characteristics. The using of nsTEM was successful for isolation and identification of numerous viruses including adenoviruses, enteroviruses, paramyxoviruses, rotaviruses, and reoviruses in the 1970s and 1980s. This method continues to have significant clinical impacts on patients for viral detections (Figure 1).

Figure 1. A representative nsTEM image of lentivirus vectors at 10kx. Scale bar = 200 nm.

For more than two decades now, nsTEM has been used for direct observation of the particles and a clear discrimination between different particle populations as they appear in the image. In a general preparation, 400 mesh copper grids coated with a carbon film, overlayed with a Formvar film were hydrophilized using a glow discharger. A glow discharged grid was mounted on tweezers and 3 μl of sample were placed onto the grid. After an adsorption time of about 10 seconds, the excess of liquid present on grid was blotted-off using a piece of filter paper. Grids were then stained with 2% aqueous uranyl acetate or an alternative reagent.

The sensitivity and specificity of nsTEM for the detection of viruses was significantly improved after the application of direct ultracentrifugation to deposit samples onto a specimen grid. Ultracentrifugation was also used to quantitatively evaluate a test article for the presence of viral particles. A selected volume (typically 100 μl) of test article is diluted with a selected volume (typically 100 μl) of water. The mixture is then ultracentrifuged at 10–20 PSI for 5 minutes in a Beckman airfuge. This centrifugation deposits the test article directly onto coated grids. The grids were recovered, dried with bibulous paper, stained with uranyl acetate, and dried again with bibulous paper. A total of 500–1000 particles from two different grids were counted, with at least five different areas per grid. The level of sensitivity of this procedure is approximately 10⁶ particles/ml.

Thin sections TEM (tsTEM)

Ultramicrotomy is a method for cutting specimens into extremely thin slices, called thin sections or ultrathin sections, that can be studied and documented at different magnifications by TEM. Tissues or cultured cells are fixed with aldehydes and osmium tetroxide, dehydrated through a graded series of alcohol to remove water, and finally embedded in a plastic resin. The resin-embedded material is sectioned using either glass or diamond knives. Sections can be either cut thick (0.5–2.0 µm) for light microscopy or ultrathin (50 to 100 nm) for TEM. It is a very common practice to view thick sections in a light microscope first, before proceeding with ultramicrotomy or thin sectioning. These thick sections are also known as survey sections and are viewed in a light microscope to determine whether the right area of the specimen is in a position for thin sectioning. For best resolutions, thin sections should be from 30 to 60 nm. This is roughly the equivalent to splitting a 0.1 mm-thick human hair into 2,000 slices along its diameter or cutting a single red blood cell into 100 slices.

The use of tsTEM allows for the visualization of cellular components, which help in the identification of cell type and may aid in describing any cellular changes that could occur during biopharmaceutical production. Preparation of thin sections of virus-infected cells and tissues is an indispensable technique for the study of those aspects of virus–cell interaction that are accessible to direct examination by electron microscopy. Thin sectioning is also of value in elucidating the structure of viruses; the information obtained often complements that provided by a nsTEM. This method can be utilized to visualize a variety of viral types including retroviruses, herpesviruses, adenoviruses, picornaviruses, parvoviruses, orthomyxo- and paramyxoviruses, reoviruses, and many other common viral agents. Contamination by other microbial agents such as yeast, fungi, and bacteria may also be detected (Figure 2).

Figure 2. Retrovirus-like viral particles in the cytoplasm of murine cells. The cells were prepared and imaged with tsTEM. Scale bar = 200 nm.

Retroviruses can be classified based on their morphological types by tsTEM as A-type, B-type, C-type, and D-type. The A-type viruses bud intracellularly, either into the cytoplasm or within endoplasmic reticulum, are not considered to be infectious, and have an electron lucent core. These are endogenous viruses, and some animal species have thousands of copies of these A-type viruses in their chromosomal DNA. Their function remains unknown. B-type viruses have an eccentric core and the mammary tumor viruses exclusively have this structure. These viruses exist as endogenous and exogenous viruses in some animals and when expressed can cause mammary tumors. C-type viruses have a central electron-dense core, and most of the oncoviruses and endogenous viruses are of this type. The D-type viruses have a rod-shaped core and Lentiviruses are of this type.

Cellular structures versus viral particles?

Viral DNA or RNA genomes are contained within a protein coat (capsid). The nucleic acid together with the protein coat forms the nucleocapsid, which can be membrane bound (enveloped viruses) or without a membrane (naked viruses). The coronavirus is an enveloped RNA virus that infects cells after it binds to the cell surface and is internalized in endocytic vesicles. Cells have many organelles comparable in size and structure to virus particles. Interpretation of electron micrographs requires integration of morphology and biology. This is especially important for sample examinations that may be compromised by low resolution and poor tissue preservation in autopsy samples. Even in those samples with proper fixation and preservation of ultrastructure, consideration should be given to the mechanism of virus production, including the location inside of cells, as well as the appearance (size, shape, internal pattern of the nucleocapsid, and surface spikes). Care should be taken to prevent mistaking cell organelles for viral particles.

Bullock HA et al. (Emerg Infect Dis. 2021) summarized structures commonly misidentified as coronaviruses in 2021 [8]. They performed a literature search for reports published during March 1–30 November 2020, that used EM to identify coronavirus directly in patient specimens. In 27 reports with EM findings, 23 articles revealed a pattern of subcellular structures misidentified as virus, including clathrin-coated vesicles (CCVs), multivesicular bodies (MVBs), circular cross-sections through vesiculated RER, spherical invaginations of RER, and other nonviral structures.

Cellular vesicles can be difficult to classify based on morphology alone but can be deduced from their relationship with other membranes in the cell. CCVs are major carriers for cargo transport in cells. They carry cargo in three pathways: plasma membrane to endosome, from endosome to endosome, and between endosomes and trans-Golgi network (TGN). CCVs seen budding from the plasma membrane are about 100 to 150 nm in diameter, surrounded by an electron-dense coat. Vesicles that measure approximately 60 to 100 nm in diameter, have similar spiculated electron-dense coats, are found in the vicinity of ER and Golgi, and bud from these organelles are likely coatamer-coated (COPI and COPII). Other vesicles or granules identified in the cell cytoplasm can be difficult to classify based on ultrastructural morphology alone.

Evaluations of viral vectors by nsTEM in gene therapies

Gene therapy is the treatment of a genetic disease by the introduction of specific cell function-altering genetic material into a patient. The key step in gene therapy is efficient gene delivery to the target tissue/cells, which is carried out by gene delivery vehicles called vectors. There are two types of vectors: viral and non-viral. Viral vectors are nature’s gene delivery machines that can be optimized to allow for tissue-specific targeting, site-specific chromosomal integration, and efficient long-term infection of dividing and non-dividing cells. Contemporary viral vector-based gene therapy is achieved by in vivo delivery of the therapeutic gene into the patient by vectors based on retroviruses, adenoviruses (Ads) or adeno-associated viruses (AAVs). AAVs are commonly used as vectors for gene therapy given their ability to deliver genes to non-dividing cells where the gene has a long-term therapeutic impact. Alternatively, a therapeutic transgene can be delivered ex vivo, whereby cells of a patient are extracted and cultured outside of the body. Cells are then genetically modified by introduction of a therapeutic transgene and are then re-introduced back into the patient. This approach is also called cell therapy.

The development and production of very high-quality vectors are one of the most important steps leading to delivery of the desired genes. Transmission electron microscopy, combined with optimized procedure of negative staining for virus, offers a unique means to generate visual data of the vector preparation; and it has gained increasing application to help assess the quality of vectors. The nsTEM technique is particularly suited for investigating particle overall morphology, size distribution, and purity. Additionally, it can be used for assessing particle integrity. Due to penetration of the staining solution into a broken particle, its interior has a dark and inhomogeneous appearance surrounded by a well-defined exterior, while an intact particle’s interior appears bright and homogenous.