key: cord-0817977-s84b98j9
authors: Cortese, Mirko; Laketa, Vibor
title: Advanced microscopy technologies enable rapid response to SARS‐CoV‐2 pandemic
date: 2021-03-01
journal: Cell Microbiol
DOI: 10.1111/cmi.13319
sha: faab6b3b600df4630a157d9e97c24e1f2cb7c7f5
doc_id: 817977
cord_uid: s84b98j9

The ongoing SARS‐CoV‐2 pandemic with over 80 million infections and more than a million deaths worldwide represents the worst global health crisis of the 21th century. Beyond the health crisis, the disruptions caused by the COVID‐19 pandemic have serious global socio‐economic consequences. It has also placed a significant pressure on the scientific community to understand the virus and its pathophysiology and rapidly provide anti‐viral treatments and procedures in order to help the society and stop the virus spread. Here, we outline how advanced microscopy technologies such as high‐throughput microscopy and electron microscopy played a major role in rapid response against SARS‐CoV‐2. General applicability of developed microscopy technologies makes them uniquely positioned to act as the first line of defence against any emerging infection in the future.

In the last 20 years, three members of the betacoronaviruses have emerged from zoonotic reservoirs able to infect humans-severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 (Zhong et al., 2003) , Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 (De Wit, Van Doremalen, Falzarano, & Munster, 2016; Zaki, van Boheemen, Bestebroer, Osterhaus, & Fouchier, 2012) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019 . SARS-CoV-2 was first detected in Wuhan, Hubei province, China, from a cluster of atypical pneumonia diseases (later named coronavirus disease 2019, . While the spread of SARS-CoV and MERS-CoV has been successfully contained (De Wit et al., 2016) , SARS-CoV-2 caused a global pandemic with over a million deaths and devastating socio-economic consequences. SARS-CoV-2 also induced mobilisation of the global scientific community on an unprecedented scale in efforts to understand the virus, the underlying host-pathogen interactions, its pathophysiology and the immunological responses. Many of these efforts were aimed at developing anti-viral drugs, vaccines or therapeutic modalities in order to stop the virus spread and relieve the socioeconomic pressure. A PubMed query 'SARS-CoV-2' returned 52,926 scientific articles in the period from January 1, 2020 to December 31, 2020, which amounts to an incredible 145 articles per day.

Microscopy is a fundamental technology in modern bio-medical research because it is the only technology able to quantitatively address complex spatio-temporal dynamics of living systems at a sufficient resolution to provide the most realistic representations of the biological systems. Indeed, one can find light and electron microscopy imaging data in the majority of scientific articles published in biomedical fields (Jambor et al., 2020) . Additionally, microscopy has played a key role in infectious disease research since the discovery of the first microorganisms (Laketa, 2018) . Microscopy was essential for the discovery of infectious agents by direct observation as well as for testing the compliance with Koch postulates in order to identify new pathogens. It has also had a major role in infectious diseases diagnostics (Laketa, 2018) . The importance of microscopy in infectious disease research has continued in modern times, which is illustrated in the ongoing SARS-CoV-2 pandemic. The very first articles describing the isolation, identification and characterisation of SARS-CoV-2 strongly relied on light and electron microscopy data (in addition to next generation sequencing) to provide evidence of SARS-CoV-2 emergence (Zhou, Yang, et al., 2020) . This demonstrates how microscopy-derived evidence is still an essential component in identification of new pathogens.

Electron microscopy (EM) has long been the method of choice for direct visualisation of viruses. While recent advances in superresolution microscopy allowed to resolve the molecular distribution of viral proteins on the virion surface (Chojnacki et al., 2012; Muranyi, Malkusch, Müller, Heilemann, & Kräusslich, 2013) , EM still remains the only technique able to provide structural and morphological information. Due to its central role in virus research, EM has been extensively exploited during the 2020 COVID-19 pandemic to study different aspects of SARS-CoV-2 infection and pathogenesis. In addition to their scientific value, digitally enhanced EM images of SARS-CoV-2 isolated virions or virions bound to the host cell surface are used by the news media to accompany the daily news describing the evolution of the COVID-19 pandemic. Some of those images together with accurate illustrations generated from EM structural studies achieved widespread recognition among the general public and became an important resource to raise awareness on coronavirus diseases, fight disinformation and promote health recommendations to fight COVID-19 (Goodsell, Voigt, Zardecki, & Burley, 2020) . This review will focus on a discussion of the advanced microscopy technology modalities that have had the biggest impact and have the highest potential to limit the SARS-CoV-2 pandemic. A special emphasis is given to technologies whose employment directly enables transition from the basic research towards more translational aspects. With this in mind, we centre our discussion around two advanced microscopy technologies-(a) high-throughput microscopy and (b) electron microscopy with their roles in diagnostics, drug discovery and virus characterisation.

2 | HIGH-THROUGHPUT MICROSCOPY IN SARS-COV-2 RESEARCH 2.1 | High-throughput microscopy in anti-viral drug development

High-throughput microscopy is a method that typically involves automated microscopy, robotics and quantification by image analysis in order to test a large number of reagents for their activity in biological assays. It can incorporate various imaging modalities and generally employs cellbased assays combined with fluorescently labelled structures or molecular components. The method has been extensively used in academia to perform genome-wide genetic screens using RNA interference or overexpression (Boutros, Heigwer, & Laufer, 2015) , as well as in industry at all stages of the drug discovery and development processes (Bickle, 2010) . In the context of infectious disease research, it plays an important role in studying host-pathogen interactions and in the discovery of anti-microbial compounds (Brodin & Christophe, 2011) . The main advantage of high-throughput microscopy over traditional biochemical methods in drug discovery is the fact that employment of cellular assays is more predictive of the in vivo situation compared to biochemical assays, especially concerning cell penetration and susceptibility to intracellular metabolism. In addition, the ability to multiplex many readouts in one assay allows assessment of toxicity, modes of action and multiple drug profiling, essentially replacing the need for secondary and tertiary assays (Simm et al., 2018) .

With the emergence of SARS-CoV-2, it is more obvious than ever why rapid drug development is necessary. The key to utilising high- replication kinetics to the wild-type virus .

Another milestone in application of high-throughput microscopy to fight the SARS-CoV-2 pandemic is the development of an appropriate cellular assay to monitor virus infectivity ( Figure 1 ). As it is common for all coronaviruses, viral protease 3CL pro is cleaving the viral polyproteins into individual non-structural proteins, and it is essential for the viral replication (Harcourt et al., 2004; Thiel et al., 2003) . (Froggatt, Heaton, & Heaton, 2020) . Changes in fluorescence reflect 3CL pro activity and, in turn, can be used to evaluate SARS-CoV-2 infection. Also relying on the 3CL pro activity, a parallel assay was developed that employed an endoplasmic reticulum (ER)anchored GFP molecule tagged with a 3CL pro cleavage site and nuclear localization signal sequence, which promotes GFP translocation from the ER to the nucleus upon SARS-CoV-2 infection (Pahmeier et al., 2020) . Importantly, both assays allow for anti-viral drug screening in fixed or living cells (Froggatt et al., 2020; Pahmeier et al., 2020) . In addition, these assays enable discovery of direct inhibitors of 3CL pro , one of the main targets for pharmacological intervention against SARS-CoV-2, to be conducted under biosafety level 1 or 2 (BSL1 or BSL2) containment, which is critical for rapid drug discovery. Repurposing of known and clinically evaluated drugs CoV-2 serology studies mainly as a confirmatory assay to provide increased specificity of detection in the setting of low antibody prevalence (Stringhini et al., 2020; Tönshoff et al., 2021) . Increased specificity potential of the microscopy-based assays compared to ELISA and others is probably due to expression of viral antigens in the cellular context ensuring correct protein folding and appropriate posttranslational modifications. A study using 293 T-cells expressing the spike protein of SARS-CoV-2 and using FACS for a readout has shown increased specificity of that assay compared to other approaches (Grzelak et al., 2020) .

The presence of specific anti-viral antibodies in patient sera does not always correlate with neutralising capacity. Therefore, additional assays are used to assess the antibody neutralisation capacity, often by using time-consuming plaque reduction neutralisation tests (Perera et al., 2020) . Muruato et al. have developed a microscopy-based virus neutralisation assay using SARS-CoV-2 construct harbouring mNeonGreen fluorescent protein .

This approach has shortened the assay turnaround time by several days and increased sensitivity compared to a standard plaque assay.

This is an important step towards the successful vaccine development, and the assay has been used to support clinical trials for COVID-19

vaccine candidates (Mulligan et al., 2020) . Furthermore, neutralising antibodies as well as COVID-19 convalescent patient plasma have shown clinical benefits underscoring the importance of fast neutralisation capacity assessment via microscopy-based assays. (Krammer, 2020) . SARS-CoV-2 S protein is also the primary antigenic component responsible for inducing host immune response, including neutralising antibody production and protective immunity against viral infection (Krammer, 2020) . Cryo-EM studies have been instrumental in defining the structure of the S protein under different conformational states (pre and post fusion), under different pH conditions, in complex with its cellular receptor (angiotensin convertase enzyme 2 [ACE2]), and with neutralising antibodies or antibody fragments (Benton et al., 2020; Cai et al., 2020; Custódio et al., 2020; Lv et al., 2020; . Moreover, several studies used cryoelectron tomography to define the structure of the S protein and other SARS-CoV-2 structural proteins in situ, using purified virus preparations or cells infected directly on EM grids (Ke et al., 2020; Turoňová et al., 2020; Yao et al., 2020) (Figure 2) . These in situ studies revealed the presence of flexible hinges in the stalk region of the S protein homotrimer that allowed the protein to be tilted up to 90

towards the viral membrane. The large conformational space might influence the interaction between the S protein and the ACE2 receptor or hinder the access of antibodies to epitopes located in the stalk region. In addition, they allowed for a precise definition of the glycan shell that coats the S protein surface and might therefore shield important epitopes from the binding of neutralising antibodies (Ke et al., 2020; Turoňová et al., 2020) . Together, this structural information has provided one of the major focal points for the rapid devel- Finally, a major breakthrough in in situ structural microscopy was provided by combining cryo-ET with FIB milling. Conventional cryo-EM is limited to specimens thinner than approximately 300 nm thin but cannot be used to visualise entire cells whose thickness far exceeds this limit. In cryo-FIB approaches, thin lamellae are milled at cryogenic temperatures within specific regions of the vitrified specimen, allowing for subsequent analysis through transmission electron microscopy (TEM) and tomography. This allows to image the cell interior and obtain structural information of macromolecular complexes in their cellular context (Villa, Schaffer, Plitzko, & Baumeister, 2013) . In situ cryo-ET performed on cryo-FIB-milled lamellae, allowed for the visualisation of ROs, from chemically inactivated SARS-CoV-2, in close-to-native conditions (Klein et al., 2020) Thao et al., 2020; Xie et al., 2020 ) together with cellular model system (Froggatt et al., 2020; Pahmeier et al., 2020) and highthroughput microscopy have enabled the testing of reagents for their inhibitory effect against SARS-CoV-2. Cryo-electron tomography has been used to determine virion and viral protein structures within months of the first observed SARS-CoV-2 infections, enabling structure-guided drug design and giving rise to therapeutic antibodies with affinities in femtomolar range and neutralisation capacity in picomolar range (Schoof et al., 2020) . Studies on SARS-CoV-2-induced cellular ultrastructural changes have been vital in understanding viral impact on infected host cells and have provided clues for potential therapeutic procedures Ogando et al., 2020; Wolff et al., 2020) . Microscopy-based serological assays enabled specific, sensitive and quantitative assessment of SARS-CoV-2-specific antibodies and proved to be especially useful in situations where commercial assays are either not yet developed or in short supply due to a high demand . Moreover, the employment of microscopy-based virus neutralisation assays paved the way to fast vaccine development (Mulligan et al., 2020; Muruato et al., 2020) . However, high expertise is needed for proper identification of the viral ultrastructural morphologies, and great care has to be taken to avoid misidentifying common cellular structures as virions. The COVID-19 pandemic has clearly demonstrated the importance of rapid communication of scientific data to provide early visibility and broad dissemination among the scientific community and the general public. Nevertheless, one has to be careful to prevent ambiguous information finding their way into the scientific literature. Given the technical requirement for both proper image acquisition and the complexity of image analysis with advanced microscopy techniques, it is vital to take special care when interpreting these studies. The publication of potentially ambiguous EM micrographs derived from COVID-19 patient samples has prompted researchers to request that authors and editors conduct more rigorous assessment of EM data in order to prevent the spread of misleading or false information Miller & Goldsmith, 2020) . In this direction, dedicated web resources have been developed to sort, organise and curate COVID-19-related information deposited in the public databases (Brzezinski et al., 2021) , such as the structural data of SARS-CoV-2 proteins deposited in the PDB server. Overall, the pressure that the current pandemic has exerted on the scientific community favoured the long-sought evolution towards a more open, transparent and direct way of communicating the research results. For the microscopy community, this should translate to the practise of making raw datasets available for published work. This will allow not only for a more transparent evaluation during the peer-review process, but also permits other groups to re-analyse those datasets, thus fostering novel and potentially unanticipated discoveries. Cortese et al. have taken a step in this direction by sharing all the raw data associated with our EM analysis of SARS-CoV-2-infected cells. Data have been uploaded on EMPIAR, a public repository for EM datasets where direct access and visualisation through the MoBIE plugin of the FIJI software, a framework for sharing and browsing of multimodal big image data (Vergara et al., 2020) was provided. Big-data viewers such as MoBIE do not require the download of the data to visualise the dataset content, a great advantage considering that EM data can easily reach terabytes size, and thus combine date share with a user-friendly experience.

In summary, during SARS-CoV-2 pandemic, advanced microscopy technologies have emerged to be especially relevant for rapid response by facilitating drug and vaccine discovery, serological testing and the accumulation of knowledge surrounding virus pathophysiology. Furthermore, many of the developed microscopy-based procedures are not necessarily SARS-CoV-2 specific but can be made generally applicable. This makes microscopy technologies uniquely positioned to act as the first line of defence against any emerging infection in the future.

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We are grateful to Dr. Christopher J. Neufeldt for the useful suggestions and the critical reading of our manuscript. We would like to thank Deutsches Zentrum fuer Infektionsforschung (DZIF) (VL: project TTU 04.705) for funding. Individual images used in the Figure 1 courtesy of medical illustrations database-https://smart.servier.com/. The authors declare they have no conflicts of interest.

Data sharing not applicable to this article as no datasets were generated or analysed during the current study ORCID Vibor Laketa https://orcid.org/0000-0002-9472-2738