Electron Microscopy in Discovery of Novel and Emerging Viruses from the Collection of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA)

Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been an important and widely used technique for discovery, identification and characterization of new viruses. Using TEM, viruses can be differentiated by their ultrastructure: shape, size, intracellular location and for some viruses, by the ultrastructural cytopathic effects and/or specific structures forming in the host cell during their replication. Ultrastructural characteristics are usually sufficient for the identification of a virus to the family level. In this review, we summarize 25 years of experience in identification of novel viruses from the collection of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA).

Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been one of the most important and widely used techniques for identification and characterization of new viruses. Two TEM techniques are generally used for this purpose: negative staining on an electron microscopic grid coated with a support film, or (ultra) thin-section TEM of infected cells, fixed, pelleted, dehydrated and embedded in epoxy plastic. Negative staining can be conducted on highly concentrated suspensions of purified virus or on cell culture supernatants. For some viruses, TEM can be conducted on contents of skin lesions (e.g., poxviruses and herpesviruses) or on concentrated stool material (rotaviruses and noroviruses). For successful detection of viruses in ultrathin sections of infected cells, at least 70% of cells must be infected, so either high multiplicity of infection (MOI) or rapid virus multiplication is required.
In many of these discoveries, transmission electron microscopy (TEM) was essential to determine the taxonomic position (family) of the virus, which will be demonstrated by the following examples.

Paramyxoviruses
Paramyxoviruses form at the cell surface using plasmalemma as their envelope. Usually they are pleomorphic or spherical in shape, varying in size from 100 nm to several hundred nanometers, but they can also be finger-like or filamentous ( Figure 1A). Filamentous virions are 55 nm to 85 nm in diameter and can reach up to 1 µm in length. Virions have spikes at the surface~7-10 nm long, which make their surface look "fuzzy" in ultrathin sections. They contain a helically packed nucleocapsid which in cross-sections looks like tubules~10 nm in diameter with~15 nm periodicity. At earlier stages of infection, cells can contain intracytosolic inclusions consisting of nucleocapsid strands 10 nm to 15 nm in diameter ( Figure 1B).
Viruses 2019, 11, x FOR PEER REVIEW 3 of 17 paramyxovirus [31], an insect-specific (capable of replication in insects but not in vertebrates) alphavirus [32], a new flavivirus genus [33] and other novel flaviviruses [34][35][36][37] and rhabdoviruses [38][39][40][41][42][43][44][45][46][47]. Lastly, WRCEVA scientists discovered the new family of mesoniviruses [37,48] and the new taxon of the negeviruses [49,50]. In the past decade, the WRCEVA has also been critical in contributing to the surveillance of dengue, chikungunya and Zika viruses during their spread through various parts of the world and identifying their sources and mechanisms of emergence. In many of these discoveries, transmission electron microscopy (TEM) was essential to determine the taxonomic position (family) of the virus, which will be demonstrated by the following examples.

Paramyxoviruses
Paramyxoviruses form at the cell surface using plasmalemma as their envelope. Usually they are pleomorphic or spherical in shape, varying in size from 100 nm to several hundred nanometers, but they can also be finger-like or filamentous ( Figure 1A). Filamentous virions are 55 nm to 85 nm in diameter and can reach up to 1 μm in length. Virions have spikes at the surface ~7-10 nm long, which make their surface look "fuzzy" in ultrathin sections. They contain a helically packed nucleocapsid which in cross-sections looks like tubules ~10 nm in diameter with ~15 nm periodicity. At earlier stages of infection, cells can contain intracytosolic inclusions consisting of nucleocapsid strands 10 nm to 15 nm in diameter ( Figure 1B).

Rhabdoviruses
Rhabdoviruses have characteristic bullet or finger-like morphology with cross-striations 5-7 nm in periodicity, reflecting helical packaging of the nucleocapsid. In cross-sections, they appear ring-like in structure ( Figure 2C). The size of virions varies depending on virus, with diameters ranging from 45-80 nm and lengths of 120-200 nm; some virions may be much longer, reaching 900 nm ( Figure 2). Defective-interfering particles are shorter and wider, giving them a conical shape. Virions bud from host cell membranes either from the plasmalemma into extracellular space (Figure 2A,B) or into intracellular vacuoles of varying sizes ( Figure 2C,D). Inside vacuoles, virions can be packed in stacks of up to 1 µm ( Figure 2D). Some rhabdoviruses, especially in animal models, form intracytoplasmic inclusions consisting of nucleoprotein, sometimes with intravacuolar virions [16]. These inclusions correspond to Negri bodies seen in rabies virus infection.

Rhabdoviruses
Rhabdoviruses have characteristic bullet or finger-like morphology with cross-striations 5-7 nm in periodicity, reflecting helical packaging of the nucleocapsid. In cross-sections, they appear ringlike in structure ( Figure 2C). The size of virions varies depending on virus, with diameters ranging from 45-80 nm and lengths of 120-200 nm; some virions may be much longer, reaching 900 nm ( Figure 2). Defective-interfering particles are shorter and wider, giving them a conical shape. Virions bud from host cell membranes either from the plasmalemma into extracellular space (Figure 2A,B) or into intracellular vacuoles of varying sizes ( Figure 2C,D). Inside vacuoles, virions can be packed in stacks of up to 1 μm ( Figure 2D). Some rhabdoviruses, especially in animal models, form intracytoplasmic inclusions consisting of nucleoprotein, sometimes with intravacuolar virions [16]. These inclusions correspond to Negri bodies seen in rabies virus infection.
As for other orthomyxoviruses, virions form at the cell surface. THOV and DHOV exhibit significant variations in morphology: they can be pleomorphic, spherical or filamentous, 65 nm in diameter and up to 300 nm long. Other viruses that may be assigned to this genus are mostly spherical but display some polymorphism in sizes: virions of ABV can be 75-200 nm in diameter ( Figure 3A), UPOV -75-100 nm ( Figure 3B), JOSV -100-140 nm, ARAV -110-40 nm, having spikes at their surface ~7 nm long. Virions of Bourbon virus are mostly pleomorphic, but can be spherical or filamentous [54].
The genus Quaranjavirus currently includes two named viruses: Quaranfil virus (QRFV) and Johnston Atoll virus (JAV). Lake Chad virus (LKCV) [51], Wellfleet Bay virus (WFBV), Cygnet River virus [55] and Tjulok (Tyulek or Тюлёк) virus (TLKV) [56] are also probable members of the genus. These viruses are mostly spherical but also display size polymorphism: JAV virions are 140-160 nm in diameter, LKCV -95-105 nm, WFBV -85-100 nm ( Figure 3C). Most virions of TLKV are ~140 nm in diameter but their size can vary from 105-165 nm ( Figure 3D). In ultrathin sections of some virions, up to seven dense granules can be observed representing ribonucleoprotein complexes [55]. The morphology and size of the virions initially led to their misidentification as bunyaviruses or arenaviruses [22,57,58]. Virions mostly form at the cell surface, but with LKCV and TLKV, virions have been observed inside intracytosolic vacuoles with single or two to four virions in a tight vacuole.  The genus Quaranjavirus currently includes two named viruses: Quaranfil virus (QRFV) and Johnston Atoll virus (JAV). Lake Chad virus (LKCV) [51], Wellfleet Bay virus (WFBV), Cygnet River virus [55] and Tjulok (Tyulek or Тюлёк) virus (TLKV) [56] are also probable members of the genus. These viruses are mostly spherical but also display size polymorphism: JAV virions are 140-160 nm in diameter, LKCV -95-105 nm, WFBV -85-100 nm ( Figure 3C). Most virions of TLKV are~140 nm in Viruses 2019, 11, 477 6 of 17 diameter but their size can vary from 105-165 nm ( Figure 3D). In ultrathin sections of some virions, up to seven dense granules can be observed representing ribonucleoprotein complexes [55]. The morphology and size of the virions initially led to their misidentification as bunyaviruses or arenaviruses [22,57,58]. Virions mostly form at the cell surface, but with LKCV and TLKV, virions have been observed inside intracytosolic vacuoles with single or two to four virions in a tight vacuole.

Phleboviruses
From the largest family of RNA viruses (Bunyaviridae), we illustrate the morphology of new and emerging tick-borne viruses that are likely to be assigned to the genus Phlebovirus. Phleboviruses are spherical, enveloped viruses 75-95 nm in diameter ( Figure 4) sometimes with a dense nucleocapsid core ( Figure 4D). Virion envelopes are formed either from the host cell plasmalemma ( Figure 4A,D) or from the membrane of small intracytosolic vesicles ( Figure 4B,C), usually originating in the Golgi. Envelope glycoproteins are organized into small hollow cylindrical units~10 nm in diameter arranged in an icosahedral surface lattice revealed in negatively stained virions [59].

Phleboviruses
From the largest family of RNA viruses (Bunyaviridae), we illustrate the morphology of new and emerging tick-borne viruses that are likely to be assigned to the genus Phlebovirus. Phleboviruses are spherical, enveloped viruses 75-95 nm in diameter ( Figure 4) sometimes with a dense nucleocapsid core ( Figure 4D). Virion envelopes are formed either from the host cell plasmalemma ( Figure 4A,D) or from the membrane of small intracytosolic vesicles ( Figure 4B,C), usually originating in the Golgi. Envelope glycoproteins are organized into small hollow cylindrical units ~10 nm in diameter arranged in an icosahedral surface lattice revealed in negatively stained virions [59].

Flaviviruses
Flaviviruses are spherical enveloped viruses ~40 nm in diameter in ultrathin sections, usually with a dense nucleocapsid core. Immature virions are larger in size (~60 nm) and are located inside the cisterns of granular endoplasmic reticulum within infected cells ( Figure 5). Replicating flaviviruses induce formation of characteristic intracytoplasmic membraneous structures: convoluted membranes ( Figure 5C), paracrystalline arrays and smooth membrane structures (SMS) within usually expanded cisterns of granular endoplasmic reticulum ( Figure 5A,B), visible in conventional (epoxy plastic-embedded) ultrathin sections, or vesicle packets in ultrathin cryo-sections [60]. These structures apparently are connected with each other and the membrane system of the cell

Flaviviruses
Flaviviruses are spherical enveloped viruses~40 nm in diameter in ultrathin sections, usually with a dense nucleocapsid core. Immature virions are larger in size (~60 nm) and are located inside the cisterns of granular endoplasmic reticulum within infected cells ( Figure 5). Replicating flaviviruses induce formation of characteristic intracytoplasmic membraneous structures: convoluted membranes ( Figure 5C), paracrystalline arrays and smooth membrane structures (SMS) within usually  Figure 5A,B), visible in conventional (epoxy plastic-embedded) ultrathin sections, or vesicle packets in ultrathin cryo-sections [60]. These structures apparently are connected with each other and the membrane system of the cell (endoplasmic reticulum and Golgi) and serve as sites of virus RNA replication and processing thus representing flavivirus replication complexes [61,62]. The presence of even one of these structures can serve as an identifier of an infection of the cell with a flavivirus. SMS can be spherical, 50-65 nm in diameter, or slightly elongated ( Figure 5A,B) but in some viruses they can be extremely long, up to 2 µm ( Figure 5D). representing flavivirus replication complexes [61,62]. The presence of even one of these structures can serve as an identifier of an infection of the cell with a flavivirus. SMS can be spherical, 50-65 nm in diameter, or slightly elongated ( Figure 5A,B) but in some viruses they can be extremely long, up to 2 μm ( Figure 5D).

Reoviruses
In ultrathin sections of infected cells virions of arthropod-borne reoviruses appear as spherical particles 45-70 nm in diameter with central dense cores (Figures 6C,D and 7A). Family Reoviridae includes two subfamilies, Spinareovirinae and Sedoreovirinae, according to morphology of virion surface which can be visualized in negatively stained preparations of purified viruses. Virions of viruses assigned to the Spinareovirinae have short flat spikes or turrets. In Fako virus (unassigned; probable genus Dinovernavirus), recently isolated from mosquitoes in Cameroon [29], these turrets are ~7 nm tall and ~15 nm wide ( Figure 6A). In the Sedoreovirinae, virions are devoid of spikes but can display round surface capsomere subunits ~7 nm in diameter ( Figure 6B). Reoviruses reproduce in cytoplasmic inclusions -virus factories, or viroplasms which appear in ultrathin sections as medium density masses composed of fine fibrils and granules and containing virus particles of different stages of maturity and empty shells ( Figures 6C,D and 7A). In coltiviruses (genus Coltivirus) and orbiviruses (genus Orbivirus), virus factories are often associated with randomly distributed fibrils and/or microtubules as demonstrated by F.A. Murphy [58,63].

Reoviruses
In ultrathin sections of infected cells virions of arthropod-borne reoviruses appear as spherical particles 45-70 nm in diameter with central dense cores ( Figure 6C,D and Figure 7A). Family Reoviridae includes two subfamilies, Spinareovirinae and Sedoreovirinae, according to morphology of virion surface which can be visualized in negatively stained preparations of purified viruses. Virions of viruses assigned to the Spinareovirinae have short flat spikes or turrets. In Fako virus (unassigned; probable genus Dinovernavirus), recently isolated from mosquitoes in Cameroon [29], these turrets are~7 nm tall and~15 nm wide ( Figure 6A). In the Sedoreovirinae, virions are devoid of spikes but can display round surface capsomere subunits~7 nm in diameter ( Figure 6B). Reoviruses reproduce in cytoplasmic inclusions -virus factories, or viroplasms which appear in ultrathin sections as medium density masses composed of fine fibrils and granules and containing virus particles of different stages of maturity and  Figure 7A). In coltiviruses (genus Coltivirus) and orbiviruses (genus Orbivirus), virus factories are often associated with randomly distributed fibrils and/or microtubules as demonstrated by F.A. Murphy [58,63].

Arenaviruses
Arenavirus virions are spherical and pleomorphic with variable sizes, from 80 nm to 200 nm in diameter ( Figure 7B). They are surrounded by an envelope ~10 nm thick covered with spikes and typically contain several ribosomes 15 nm to 20 nm in diameter. Virions bud from the cell surface ( Figure 7B).

Alphaviruses
Alphavirus virions are icosahedral particles which appear spherical in ultrathin sections, 50-70 nm in diameter with dense nucleocapsid core ( Figure 7C). Alphavirus replication complexes are associated with specific vesicles -spherules formed as invaginations of either plasma membrane (in vertebrate cells), [64] or limiting membranes of endosomes (occasionally in vertebrate but mostly in mosquito cells) [65,66]. These spherule-loaded endosomes (and/or lysosomes) are termed "type 1 cytopathic vacuoles" [67] and are very characteristic for alphavirus infection ( Figure 7D) although recently they have been encountered in negevirus-infected C6/36 mosquito cells ( Figure 8D). Alphavirus dense nucleocapsid cores ~40 nm in diameter can accumulate around cytopathic vacuoles [64] and can be found in the cytoplasm close to plasma membrane ( Figure 7C, arrows). Alphavirus virions form by budding from the plasma membrane and can form paracrystalline arrays at the cell surface ( Figure 7C). Electron microscopy was instrumental in the recent discovery of the insectspecific alphavirus, Eilat virus ( Figure 10B,C), which cannot replicate in mammalian cells [68], allowing it to serve as a safe platform for vaccine development [69,70].

Mesoniviruses
Mesoniviruses were recently classified as a family [71,72] in the order Nidovirales, being distantly related to coronaviruses (family Coronaviridae, order Nidovirales). They appear to be very common and widespread in mosquito populations from different geographical and ecological locations [48]. Mature virions are spherical ~50 nm in diameter with a dense nucleocapsid core ~40 nm in diameter

Arenaviruses
Arenavirus virions are spherical and pleomorphic with variable sizes, from 80 nm to 200 nm in diameter ( Figure 7B). They are surrounded by an envelope~10 nm thick covered with spikes and typically contain several ribosomes 15 nm to 20 nm in diameter. Virions bud from the cell surface ( Figure 7B).

Alphaviruses
Alphavirus virions are icosahedral particles which appear spherical in ultrathin sections, 50-70 nm in diameter with dense nucleocapsid core ( Figure 7C). Alphavirus replication complexes are associated with specific vesicles -spherules formed as invaginations of either plasma membrane (in vertebrate cells), [64] or limiting membranes of endosomes (occasionally in vertebrate but mostly in mosquito cells) [65,66]. These spherule-loaded endosomes (and/or lysosomes) are termed "type 1 cytopathic vacuoles" [67] and are very characteristic for alphavirus infection ( Figure 7D) although recently they have been encountered in negevirus-infected C6/36 mosquito cells ( Figure 8D). Alphavirus dense nucleocapsid cores~40 nm in diameter can accumulate around cytopathic vacuoles [64] and can be found in the cytoplasm close to plasma membrane ( Figure 7C, arrows). Alphavirus virions form by budding from the plasma membrane and can form paracrystalline arrays at the cell surface ( Figure 7C). Electron microscopy was instrumental in the recent discovery of the insect-specific alphavirus, Eilat virus ( Figure 10B,C), which cannot replicate in mammalian cells [68], allowing it to serve as a safe platform for vaccine development [69,70].

Mesoniviruses
Mesoniviruses were recently classified as a family [71,72] in the order Nidovirales, being distantly related to coronaviruses (family Coronaviridae, order Nidovirales). They appear to be very common and widespread in mosquito populations from different geographical and ecological locations [48]. Mature virions are spherical~50 nm in diameter with a dense nucleocapsid core~40 nm in diameter and surrounding envelope ( Figure 8A). Usually they are found within large vacuoles often filling their entire space ( Figure 8A) but can be localized in individual small vacuoles and at the cell surface ( Figure 8B). Some infected cells had intravacuolar paracrystalline arrays consisting of empty and full virus particles but with less electron density than mature virions. At the periphery of these arrays, mature virions could be observed either free in cytosol or inside vacuoles [48].
Viruses 2019, 11, x FOR PEER REVIEW 10 of 17 and surrounding envelope ( Figure 8A). Usually they are found within large vacuoles often filling their entire space ( Figure 8A) but can be localized in individual small vacuoles and at the cell surface ( Figure 8B). Some infected cells had intravacuolar paracrystalline arrays consisting of empty and full virus particles but with less electron density than mature virions. At the periphery of these arrays, mature virions could be observed either free in cytosol or inside vacuoles [48].

Negeviruses
Insect-specific viruses isolated recently from mosquitoes and phlebotomine sandflies have been characterized and proposed to represent a new genus (Negevirus) related to genera of mite-infecting plant viruses (Blunervirus, Cilevirus, and Higrevirus) in the new family Kitaviridae [49,73], or novel members of Entomobirnavirus, family Birnaviridae ( Figure 10D). They appear to be very common and widespread in insect populations on different continents as isolates have been obtained from pools of mosquitoes and sandflies collected in Israel, North and South America, Africa, and Indonesia. In negatively stained preparations of Piura virus, isolated from Culex sp. mosquitoes in Peru, spherical particles with diameters of ~45 nm and ~55 nm were found. The striking feature of negevirus infection

Negeviruses
Insect-specific viruses isolated recently from mosquitoes and phlebotomine sandflies have been characterized and proposed to represent a new genus (Negevirus) related to genera of mite-infecting plant viruses (Blunervirus, Cilevirus, and Higrevirus) in the new family Kitaviridae [49,73], or novel members of Entomobirnavirus, family Birnaviridae ( Figure 10D). They appear to be very common and widespread in insect populations on different continents as isolates have been obtained from pools of mosquitoes and sandflies collected in Israel, North and South America, Africa, and Indonesia. In negatively stained preparations of Piura virus, isolated from Culex sp. mosquitoes in Peru, spherical particles with diameters of~45 nm and~55 nm were found. The striking feature of negevirus infection of C6/36 mosquito cells are the cytopathic effects -enormous expansions of perinuclear space -a granular endoplasmic reticulum system which becomes filled with vesicles and/or microtubules ( Figure 8C, Figure 9A,B andFigure 10A). They have a diameter of 20-25 nm but can be of different lengths. The shortest can resemble a rice grain in morphology and can be arranged in rosettes in cross-sections of ER. Others can be very long, reaching up to several micrometers ( Figures 8C and 9A,B). Expanded perinuclear space can occupy almost the whole cytoplasm of the cell. Some negeviruses cause long protrusions of the nucleus and deep invaginations of perinuclear membranes leading to the fragmentation of the nucleus ( Figure 9B). Another peculiarity of negevirus infection of C6/36 cells is the formation of cytopathic vacuoles with spherule-like structures 45-55 nm in diameter at the inner periphery of their limiting membrane ( Figure 8C,D). These vacuoles can reach 1.4 µm in diameter and are morphologically similar to type 1 cytopathic vacuoles in alphaviruses. They have been found in all 10 described negeviruses [49] and later in many others that have not yet been fully characterized. Their role in negevirus replication is not known.

Conclusion
The WRCEVA collection at the University of Texas Medical Branch in Galveston, Texas, plays an important role in virology research as a depository of natural viral biodiversity, and as a valuable resource for knowledge of viruses that are not only pathogenic for animals and humans but also are naturally occurring in arthropods. Propagation in cell cultures and subsequent examination of virus morphology by electron microscopy have been useful initial steps in the identification and characterization of novel viruses, giving indications for their further genetic characterization.
Author Contributions: All authors contributed sections for the completion of this manuscript.

Mixed Infections
Sometimes, after inoculation of C6/36 mosquito cells with homogenates of mosquito pools, several viruses can be observed in the culture, or even in the same cell. Figure 9C illustrates mixed infection with Karang Sari virus (genus Alphamesonivirus) (virions 55 nm in diameter inside ER cisterns) and an unknown flavivirus (smaller virions, 40 nm in diameter in different ER cisterns). Figure 9D demonstrates a co-infection with a Kamphaeng Phet virus (unclassified; probable genus Alphamesonivirus; virions 55 nm in diameter) and an unknown reovirus (virions 45 nm in diameter with a characteristic dark nucleocapsid core). We have also observed co-infection with Bontag Baru virus (unclassified; probable genus Alphamesonivirus) and an unknown flavivirus, Ngewontan (unclassified; proposed genus Negevirus) and Nam Dinh virus (genus Alphamesonivirus); Eilat virus (genus Alphavirus) ( Figure 10B,C) and Negev virus (unclassified; proposed genus Negevirus) ( Figure 10A). Three viruses have also been observed to infect the same culture: Bontag Baru virus (unclassified; probable genus Alphamesonivirus), an unknown flavivirus and an unknown rhabdovirus, the latter budding into an ER cistern occupied by a flavivirus virion. It is most likely that these viruses originate from different individual mosquitoes in the pool, but the possibility of a co-infection of one mosquito with several different viruses cannot be excluded.

Conclusions
The WRCEVA collection at the University of Texas Medical Branch in Galveston, Texas, plays an important role in virology research as a depository of natural viral biodiversity, and as a valuable resource for knowledge of viruses that are not only pathogenic for animals and humans but also are naturally occurring in arthropods. Propagation in cell cultures and subsequent examination of virus morphology by electron microscopy have been useful initial steps in the identification and characterization of novel viruses, giving indications for their further genetic characterization.
Author Contributions: All authors contributed sections for the completion of this manuscript.
Funding: This work was supported in part by NIH grant R24 AI120942 and contract HHSN272004/D04. The funding agencies had no involvement in the writing of the report or in the decision to submit this article for publication.