Cryo-electron tomography of viral infection — from applications to biosafety


 Cellular cryo-electron tomography (cryo-ET) offers 3D snapshots at molecular resolution capturing pivotal steps during viral infection. However, tomogram quality depends on the vitrification level of the sample and its thickness. In addition, mandatory inactivation protocols to assure biosafety when handling highly pathogenic viruses during cryo-ET can compromise sample preservation. Here, we focus on different strategies applied in cryo-ET and discuss their advantages and limitations with reference to severe acute respiratory syndrome coronavirus 2 studies. We highlight the importance of virus-like particle (VLP) and replicon systems to study virus assembly and replication in a cellular context without inactivation protocols. We discuss the application of chemical fixation and different irradiation methods in cryo-ET sample preparation and acquisition workflows.



Introduction
Virus replication depends on host cells and although different viruses have developed distinct replication strategies, each replication cycle consists of the same stages: entry into the host cell, genome replication, assembly, and release.Traditionally, electron microscopy (EM) techniques have played a pivotal role in uncovering the structural details of virus-host interactions and in the mechanistic understanding of the virus replication cycle.From the first electron micrographs of isolated viruses taken by Helmut Ruska in 1938 [1] to today's state-of-the-art cryo-electron tomograms of infected cells, there has been a long journey of collective efforts to improve EM hardware, automatization of image acquisition, image processing, and particularly sample preparation (reviewed in [2][3][4][5]).
Cellular cryo-electron tomography (cryo-ET) is a powerful approach to not only obtain molecular details of virions inside the cellular environment that is remodeled to aid viral replication and assembly but also to study the mechanism of host defense machinery (reviewed in [6]).Cellular cryo-ET allows capturing 3D snapshots of the viral replication cycle directly inside host cells that have been physically immobilized by the rapid freezing preventing the formation of crystalline ice (vitrification), typically done by plungefreezing into liquid ethane.In cryo-ET, a series of lowdose projections is acquired by transmission electron microscopy (TEM) while tilting the sample stage typically from ± 60°.These images are computationally aligned to reconstruct a 3D tomogram (reviewed in [3]).However, TEM is limited to thin samples.Even at an accelerating voltage of 300 keV typically applied in cellular cryo-ET, electrons strongly interact with atoms of the sample.With increased sample thickness, the chance of inelastic and multiple electron scattering increases, which leads to a low signal-to-noise ratio in the final tomogram [7].Therefore, samples must be thin enough to have low probability for scattering events.Hence, an optimal sample thickness for cryo-ET is between 150 and 300 nm allowing 3D examination of a cellular volume without compromising on high-resolution information.Since cell lines permissive to viral infection can be up to 10-µm thick, early cellular cryo-ET studies were mainly restricted to a thin region of cellular periphery [8,9].To access the inner region of the cell body, the cell needs to be thinned.This can be done either by cryo-ultramicrotomy [10] or by a cryo-focused ion beam/scanning electron microscope (cryo-FIB/SEM), which is currently a method of choice to produce a thin and smooth slab (so-called lamella) inside the central part of an infected cell that can be studied by cryo-ET [11][12][13].
Cryo-EM/ET often consists of complex multistep workflows and choosing a suitable workflow depends not only on the biological questions of the virologist but is also dictated by sample thickness.Selection of a cryo-EM/ET technique or a workflow applied in virology can be divided into three

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common virology research applications: (i) structural analysis of released viruses, which utilizes either cryo-EM for structurally ordered virions and purified proteins or cryo-ET for pleomorphic isolated virions; (ii) virus entry, budding, and release at the plasma membrane, which relies on so-called whole-cell cryo-ET applied on the periphery (< 300-500 nm) of the infected cells; and (iii) virus replication and assembly in the nuclear or perinuclear region, which requires sample thinning by cryo-FIB milling to produce a lamella, here referred to as on-lamella cryo-ET.In this review, we mainly focus on cryo-ET applied to infected cells (cellular cryo-ET): whole-cell and on-lamella cryo-ET.In the advent of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we discuss different strategies for specific virological questions and associated challenges with cellular cryo-ET such as vitrification and biohazard considerations using mainly examples of studies on SARS-CoV-2 (Figure 1).

Structural analysis of isolated virions
While cryo-EM combined with single-particle analysis (SPA) is a method of choice to study the structure of ordered virions, cryo-ET of released or purified virions is instrumental to obtain the 3D structure of membraneenveloped virions [15] that do not follow high-symmetry rules and show internal structural variability (pleomorphy, also see [16]).It is important to note that cryo-ET by itself provides only limited 3D information and resolution due to the restricted tilting range due to slablike sample geometry and limited electron dose per tomogram, respectively (reviewed in [17]).To increase the resolution of structural components within the virions, subtomogram averaging (STA) can be applied.In this computational approach, individual viral components are computationally extracted, iteratively aligned, and averaged (reviewed in [18,19]).The high-resolution structure of the purified SARS-CoV-2 spike (S) protein ectodomain was determined by cryo-EM and SPA [20,21].However, cryo-ET combined with STA provided the structure of the full-length SARS-CoV-2 protein directly on the surface of the virions [22][23][24][25].This allowed estimating the number of S protein per virion and revealed the striking flexibility of the S protein due to the presence of three hinges in the S-protein stem region [22][23][24].In addition, this approach provided the first low-resolution structure and organization of viral ribonucleoproteins inside SARS-CoV-2 virions [24,25].

Virus entry, budding, and release at the plasma membrane
Whole-cell cryo-ET is a straightforward approach to structurally investigate the budding of virions.It can also be applied to visualize virus membrane fusion at the plasma membrane or to study virus uptake and uncoating in the cell periphery that is thinner than 300-500 nm (Figure 2a-c).The infected cells cultured on EM grids [26] are vitrified by plunge-freezing into liquid ethane at a temperature of − 183 °C [27,28].Since whole-cell tomography is applicable only to thin regions of cells, fibroblasts and cells that tend to spread and have large lamellipodia and filopodia are particularly useful for this approach.To capture early replication steps such as viral entry or membrane fusion, synchronized infection at a high multiplicity of infection is typically used [29].In such an approach, viruses are adsorbed onto surface cells at 4 °C to prevent entry.After a rapid temperature shift to 37 °C, cells are plunge-frozen in a time course from a few minutes to an hour post infection, which allows capturing snapshots of dynamic processes such as membrane fusion pathway.Whole-cell cryo-ET was instrumental to study SARS-CoV-2 at the later stages of infection.It allowed determining the structure of SARS-CoV-2 virions that remain attached to the plasma membrane of infected cells after their release by exocytosis [25,30] (Figure 2a-c).SARS-CoV-2 plasma membrane virion retention is not yet fully understood and does not seem to depend only on the ACE2 receptor.Since cryo-ET also revealed accumulations of SARS-CoV-2 virions on filopodia and tunneling nanotubes [31], the virion retention at the plasma membrane at late stages of infection likely plays an important role in the cell-to-cell spread and syncytium formation.Interestingly, the level of SARS-CoV-2 virion retention at the plasma membrane varies in different cell lines [25], possibly due to different expression levels of cellular receptors at the plasma membrane.

Virus replication and assembly in the nuclear or perinuclear region
To investigate nuclear or perinuclear stages of viral infection, cryo-FIB milling is applied to generate thin lamellae.In this approach, infected cells grown on EM grids are typically vitrified by plunge-freezing and subsequently transferred into a cryo-FIB/SEM equipped with gallium ions and electron probe beams with scanning capabilities.Accelerated and focused gallium ions are used to sputter away cellular material and thereby mill a thin lamella within the cell body [12].A grid with multiple lamellae is transferred into a cryo-TEM where the lamellae are first imaged at an intermediate magnification to localize sites of viral assembly or replication for subsequent cryo-ET.Cryo-FIB followed by on-lamella cryo-ET (Figure 2d-f) allowed the discovery of the molecular pore spanning the double-membrane vesicles (DMVs) that serve as replication sites for coronaviruses [32,33].While DMVs have been described previously using thin-section EM of resin-embedded samples, the level of structural preservation was not sufficient to detect the pore [34][35][36].In addition, on-lamella cryo-ET was used to capture the assembly of SARS-CoV-2 inside the endoplasmic reticulum-Golgi apparatus intermediate compartment (ERGIC) [25,30].This revealed that the S proteins do not play a major role in inducing membrane curvature during virus budding and it indicated that the S proteins undergo lateral redistribution after the completion of viral membrane scission [25].However, how SARS-CoV-2 structural proteins orchestrate viral budding and membrane scission remains to be investigated.

Challenges in sample vitrification and targeting viral infection sites
One of the main limitations of the on-lamella cryo-ET approach is that plunge-freezing into liquid ethane provides inefficient vitrification of the perinuclear and nuclear region of the cell due to its greater thickness (∼10 µm).Vitrification becomes even more challenging when large cells, organoids, or tissues are used as a model system to study viral infection (Figure 3).Bäuerlein et al. showed that the addition of 10% glycerol as a cryo-protectant to the sample shortly before plunge-freezing improves vitrification and does not seem to alter cell morphology [37].Although further studies are needed to evaluate the impact of cryo-protectants on the morphology of membrane organelles, this is a promising approach.Alternatively, propane-jet freezing allows improved vitrification of cells [38][39][40] but needs to be adapted for cells grown on EM grids.When vitrification of large specimens is needed, high-pressure freezing (HPF), which allows vitrifying samples up to 200 µm in depth, is the only method of choice.While cryo-FIB milling of large specimens vitrified by HPF is still timeconsuming and laborious, recently developed approaches termed cryo-FIB lift-out and Waffle Method [41,42] will facilitate the investigation of viral infection in more relevant model systems such as organoids and tissue biopsies.Besides vitrification, automation and an increase in throughput are needed to streamline lamellae production.Although automated milling routines have been established [43,44], with the automation of cryo-ET data collection [45] and development of parallel cryo-electron tomography [46], the preparation of lamellae remains a time-consuming procedure, especially for bulky specimens.However, recent data on cryoplasma FIB show successful lamella production at an increased milling rate when compared with gallium FIB, which will enhance milling throughput [47,48].
Finally, capturing an infection site within the volume of milled lamellae can be difficult.This is particularly challenging when 3D analysis of a rare or transient infection event (e.g.virus entry) is of interest.Considering that the final lamella has approximate dimensions of 15 µm × 10 µm × 200 nm, the volume of lamella represents only about 3% of the total cell volume.To increase the chances of capturing the region of interest, correlative light and electron microscopy at cryogenic temperature (cryo-CLEM) [14,[49][50][51][52] can be applied when fluorescently labeled reporter viruses are available.CLEM relies on a precise correlation of LM and TEM data.Since the development of cryo-immersion lenses has proven to be technically difficult, most cryo-LM rely on long-distance air objectives that have a low numerical aperture.This results in low axial resolution of cryo-LM, which limits 3D correlation or targeting precision.Recent studies showed that cryo-confocal LM equipped with modern detectors is sensitive enough to detect a fluorescent signal directly on milled lamella, which circumvents the need for 3D correlation and allows for precise 2D correlation [53].In addition, ongoing developments in cryo-structured illumination microscopy [54], cryo-super-resolution microscopy (reviewed in [55]), and finally integration of cryo-LM directly into cryo-FIB/ SEM microscope [56,57] will enable more precise targeting.

Biosafety considerations in cryo-electron microscopy of viral infection
The major advantage of cryo-EM methods is the high preservation of structural details within the cellular environment in the absence of chemical fixatives.However, this poses a problem in the structural investigation of biohazardous agents, as vitrification of pathogens or pathogen-infected cells does not lead to their inactivation.Hence, stringent biosafety regulations must be considered when cryo-EM is applied in studies involving pathogenic viruses during both sample preparation and data acquisition in the microscope.Based on the level of risk to human health and the environment, pathogens are classified into four biosafety levels (BSL-1-4).Although in most cases, the classification is universal per the recommendation of different institutions such as the Centers for Disease Control and Prevention, European Centre for Disease Prevention and Control, and World Health Organisation (WHO), there can be country-specific variations in the biosafetylevel classification of certain pathogens.While multiple cryo-EM facilities are certified for work with BSL-2 pathogens, there are only a handful of sites where BSL-3 pathogens can be handled and to our knowledge, there is not an institution housing a cryo-EM in BSL-4 containment likely since both are high-maintenance facilities.A list of current and planned BSL-3-certified facilities and available cryo-EM instruments is provided in Table 1.Owing to a limited number of BSL-3 cryo-EM facilities, the application of aldehydes as inactivation agents before plunge-freezing and cryo-EM of BSL-3 [58] and BSL-4 viruses [59][60][61] is mandatory.To facilitate safe and effective cryo-EM research on pathogenic viruses, several avenues and their combinations are possible: (i) improvement and certification of inactivation protocols tailored for cryo-EM; (ii) application of noninfectious virus-like particles (VLPs), attenuated viruses, and replicon platforms as a model system; (iii) establishment of national cryo-EM facilities that can operate at BSL-3 with implemented microscope decontamination protocols.Overview of cryo-EM approaches to study viruses and viral infection based on sample thickness.The selection of the appropriate approach depends on the sample thickness and the site of the replication event of interest.Structural analysis of isolated viruses or viral proteins is studied by cryo-EM or cryo-ET.Virus entry, budding, and release at the plasma membrane can be studied using whole-cell cryo-ET applied on the cell periphery with thicknesses up to 300-500 nm.Virus replication and assembly in the nuclear and perinuclear regions require sample thinning using cryo-FIB/SEM followed by on-lamella cryo-ET [12].Samples thicker than 10 µm must be vitrified by HPF to achieve vitrification, which is followed by thinning of the sample using cryo-FIB/SEM with the lift-out [42] or Waffle Method [41].Addition of 10% glycerol to cells before plunge-freezing to increase vitrification depth [37].Depending on the sample, different EM grid types can be used.For purified viruses or protein, copper grids are used, whereas gold or titanium grids are needed to seed and grow cells directly on the grid.
Since SARS-CoV-2 is worldwide classified as a BSL-3 pathogen, cryo-EM studies on this pathogen have been performed after virus inactivation mainly utilizing aldehydes (formaldehyde, glutaraldehyde) or more rarely beta-propiolactone.Aldehydes react with N-terminal amino acid residue and the side-chain amino groups of arginine, lysine, and histidine, as well as with nucleic acids and with some phospholipids, for example, phosphatidylethanolamine [62].While some studies report that aldehyde inactivation is compatible with high-resolution cryo-EM studies on, for example, Venezuelan equine encephalitis virus [63] and SARS-CoV-2 S protein [22,23], aldehyde fixation of infected cells and virions can lead to morphological changes, which should be taken into consideration during data interpretation of membrane-enveloped virions in particular.A typical example of aldehyde-fixation artifact is poor preservation of membrane envelope ('moth-eaten membrane') in Ebola and Marburg viruses after chemical fixation [64].As another example, the SARS-CoV-2 DMV pore is not well resolved by cryo-ET in the sample after chemical fixation that included 0.1% glutaraldehyde in addition to 4% formaldehyde [25].In contrast, SARS-CoV-2 DMV pore was better preserved in SARS-CoV-2-infected cells chemically fixed with 3% formaldehyde [32].Compared with unfixed cells infected with murine hepatitis virus, which is a BSL-2 pathogen, SARS-CoV-2-infected cells chemically fixed with formaldehyde showed reduced contrast along with morphological alterations in organelle morphology [32].Importantly, aldehyde fixation leads to alterations of pH, and when not performed in the presence of isotonic buffers, it can lead to osmotic changes in the cell that alter organelle morphology.However, we need additional comparative studies to evaluate the effect of aldehyde fixation on organelle structures using cryo-ET on FIB-milled cells.In addition, combination of cryo-protectants with chemical fixation in an isotonic buffer will provide improved vitrification of cells infected with highly pathogenic viruses.Some variants of SARS-CoV-2 might be more sensitive to chemical fixation presumably because of amino acid changes in membrane (M), envelope (E), or nucleocapsid (N) proteins that are the main structural components of the virion.Recent cryo-ET work showed that virions of the SARS-CoV-2 Delta variant contain envelope invaginations when inactivated with 3% formaldehyde [65].However, another work using formaldehyde fixation has not detected this phenomenon but instead reported that SARS-CoV-2 Delta virions have a discoidal shape [66].Both studies used formaldehyde to inactivate the virus but applied it either before [65] or after virion purification [66] by ultracentrifugation using either culturing medium (final concentration of 3% formaldehyde) or a buffer (final concentration of 4% formaldehyde) as a solvent for the aldehyde.Interestingly, the number of envelope invaginations dramatically decreased when inactivation using electron beam irradiation (E-beam) at 2 kGy was used instead of fixation by aldehydes, indicating that invaginations were caused by formaldehyde fixation.The E-beam inactivation additionally increased the number of S protein in post-fusion conformation compared with the number of S protein in post-fusion conformation of formaldehyde-inactivated virions [65].This suggests that E-beam inactivates virions by changing the conformation of S. In addition, chemical fixation and ultracentrifugation to concentrate the virus as part of cryo-EM sample preparation workflow had an impact on the S conformations as well as on the ratio of open and closed forms of the SARS-CoV-2 S protein [22,67].Furthermore, the inactivation of SARS-CoV-2 by betapropiolactone leads to the conversion of all S proteins to post-fusion conformation [67].Alternatively, using sizeexclusion chromatography may result in more native distribution of prefusion S protein as well as infectivity preservation compared with sucrose pelleting experiments, which leads to loss of infection yield [68]. Overall, this shows that virus purification combined with chemical fixation may result in different morphological changes of virions depending on the applied protocol.When in doubt whether purification protocols cause alteration of virus structure, cellular cryo-ET can be applied to investigate virion structure immediately after release from host cells.Overall, comparative studies are needed to optimize inactivation protocols compatible with cell infection and virus purification methods to deliver best preservation.
SARS-CoV-2 cryo-ET studies showing that chemical fixation can alter virion structure necessitate the development of alternative inactivation protocols that do not interfere with the structure of the proteins and virus morphology.Ideally, such protocols should allow the inactivation of viruses independently of their structure and concentration before vitrification and data acquisition at cryo-EM facilities.This is particularly important for highly pathogenic viruses since the installation of high-end cryo-EM instruments into BSL-4 containment would come at a very high cost.Moreover, the operation and maintenance of such a facility would likely be inefficient due to stringent regulations.As a promising alternative to chemical fixation, irradiation using electromagnetic rays of different energies has proven to be an efficient way to inactivate pathogens as part of sterilization protocols in research [69] and industry and thus could be implemented in cryo-EM workflows either before or after vitrification (Figure 4a, c).A recent study by Depelteau et al. provided an elegant solution by using ultraviolet-C (UV-C) radiation to inactivate vitrified samples directly in liquid nitrogen after plungefreezing [70].Direct absorption of the UV-C photons by nucleic acid basis and/or capsid proteins leads to the generation of photoproducts that inactivate the virus [71].Hence, UV-C would offer an alternative inactivation protocol for cryo-ET sample preparation of other BSL-3 or BSL-4 viruses, but the compatibility with high-resolution cryo-EM needs to be validated.While this method showed that it is possible to obtain the highresolution structure of an inactivated bacteriophage, it would require performing plunge-freezing in a biosafety containment when applied to highly pathogenic viruses.However, most of the BSL-3 or BSL-4 laboratories do not operate a plunge-freezing device and current devices operate with tweezers that are not permitted in some biosafety labs.Hence, recently developed fully automated plunge-freezing devices [40,72,73] could be combined with integrated UV-C inactivation.Alternatively, UV-C radiation could be applied already before vitrification.Such protocols are, for example, used for the inactivation of blood transfusion-relevant viruses [74].UV-C irradiation is effective in inactivating SARS-CoV-2 replication, even at very high-input concentrations (up to a multiplicity of infection 1000) [75].Further studies need to be performed to evaluate the structure of isolated virions and infected cells that were inactivated by UV-C.In addition to UV-C and E-beam, other types of radiation could be explored such as γ-rays, which can be used to inactivate SARS-CoV-2 [76].Whether this approach is suitable for structural analysis using cryo-ET remains to be seen as the highly energetic γ-rays will likely cause damage to the protein structure.
Alternatively, virus inactivation can be completely circumvented by using VLPs, attenuated viruses [77], or single-cycle infectious virions that are permitted in lower biosafety conditions.Currently, several SARS-CoV-2 replicon platforms exist and trans-complementation of the deleted structural gene S [78], N [79] or E, and ORF3a genes [80] enables the production of single-cycle infectious virions handled at BSL-2 conditions.Using transfection of a single plasmid encoding SARS-CoV-2 nsp3 and nsp4 genes, we were able to determine the minimum components of the pore-spanning DMVs [33].Similar systems are also available for BSL-4 pathogens such as the Ebola virus [81] and collectively will play an important role to study highly pathogenic viruses.
Since the majority of human viruses can be studied at BSL-3 or lower, the establishment of additional national cryo-EM facilities certified for work with pathogens up to BSL-3 is needed.Such facilities would offer their services for infectious disease research and diagnostic purposes.An important consideration for running such facilities is the decontamination of cryo-EM instruments, which is required before service or maintenance.Several strategies using either chemical-or heat-based decontamination protocols have been tested.TEM column fumigation with strong oxidizers such as vaporized hydrogen peroxide or chlorine dioxide is effective but may cause condensation or corrosion and its implementation comes at an excessive cost [82].Based on personal communication with Dr. Michael Scherman (University of Texas Medical Branch), no issues were observed with the microscope performance after using chlorine dioxide decontamination.As an alternative decontamination approach, heat inactivation may provide both an effective and compatible avenue for the decontamination of electron microscopes.For example, CM300 cryo-TEM (FEI, now Thermo Fisher Scientific, Hillsboro, OR, USA) was decontaminated at 100 °C for 2 h and in a high vacuum after acquiring cryo-EM data on Hantaan virus classified as BSL-3 pathogen [83].Thermo Fisher Scientific company, which manufactures and services high-end cryo-EM instruments, now offers an installation of heating to 60 °C into the parts that come in contact with the samples.However, it is important to note that not all viruses have the same thermal stability and depending on their structure, some viruses are much more heat-resistant.While heating to 60 °C is sufficient to inactivate most of the membrane-enveloped viruses, it is not sufficient to inactivate nonenveloped viruses, for example, murine norovirus (MNV-1) (Figure 4b).Further studies are needed to evaluate the contribution of microscope vacuum, which could presumably decrease the effective inactivation temperature and time.Some viruses are inactivated by dehydration on various material surfaces, including metals even at room temperature.For example, influenza-A virus (IAV) infectivity is not recovered from nonporous materials after 24 h at room temperature [84].Hence, for work with less thermostable viruses, a prolonged cryo-cycle (warm-up cycle of the autoloader and column from cryo to room temperature), which is typically implemented in cryo-TEMs, can be applied to decontaminate the microscope if heating is not available.In summary, a microscope equipped with a stage and autoloader decontamination cycle with adjustable temperatures ranging from 60° to 90 °C would be needed to deactivate a broad range of viruses.Overall, inactivation strategies depend on the level of biosafety and different inactivation steps could be implemented at various steps of cryo-EM biosafety workflows (Figure 4c).

Conclusions and perspectives
Cryo-ET applied to study the viral replication directly in a cellular environment is an essential method that with a continuing development in improved data quality, throughput, and STA approaches will provide new opportunities for the development of antiviral interventions.In particular, on-lamella cryo-ET provides access to scrutinize the viral replication cycle on the molecular level as the technique provides a link between virus-host interactions and high-resolution structure determination.
Current main limitations pertain to inactivation protocols, improper vitrification, a limited model system such as a cell culture system, and finally infection targeting.Hence, improvement of inactivation protocols, implementation of HPF, and improved model systems such as mini-organoids are needed.Overall, suitable inactivation protocols tailored to cryo-EM would also allow using cryo-EM methods in diagnostics.Current EM-based diagnostic relies mainly on negative staining EM of chemically inactivated pathogens, which offers only limited interpretability and can lead to wrong virus classification.Hence, cryo-EM could replace negative staining EM-based diagnostics in reference laboratories and become a part of modern virus diagnostic workflows.Since cryo-EM allows for better structural preservation than negative staining, it could provide more accurate diagnostics based on morphology and would aid diagnostics done by PCR.In addition, automated medium magnification screening procedures provided by SerialEM [45] combined with the development of deep learning particle recognition algorithms would allow automated screening of viral isolates containing a small number of virions.Chemical fixation bears the risk of altering the structure of proteins and membranes, especially membrane proteins.Suitable inactivation protocols using UV-C such as those shown in Depelteau et al. need to be established and validated for future cryo-ET studies of infectious samples.Currently, the majority of cryo-EM facilities are located in BSL-2 containments and the establishment of BSL-3 cryo-EM facilities will facilitate research and vaccine development against highly pathogenic viruses.This is particularly important since the WHO's Blueprint of prioritized infectious diseases for R&D in public health emergency contexts lists mainly BSL-3 and BSL-4 viruses.Finally, virus attenuation, VLP, and replicon systems provide invaluable tools to study the structure and assembly of highly pathogenic viruses in the absence of inactivation.

Figure 1 Current
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Figure 2 Current
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Figure 3 Current
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Figure 4 Current
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Table 1 Current and planned cryo-EM facilities operating at BSL-3.
EM sites operating at increased or high BSL of cryo-EM instruments around the world can be found here: http://tiny.cc/cryoem_map.