key: cord-302716-wfla3l20
authors: Popov, Vsevolod L.; Tesh, Robert B.; Weaver, Scott C.; Vasilakis, Nikos
title: Electron Microscopy in Discovery of Novel and Emerging Viruses from the Collection of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA)
date: 2019-05-25
journal: Viruses
DOI: 10.3390/v11050477
sha: 
doc_id: 302716
cord_uid: wfla3l20

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.

Viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location, or from the ultrastructural cytopathic effects and specific structures forming in the host cell Upolu, Aransas Bay [22] , Sinu [23] , and Trinity [24] orthobunyaviruses [25] [26] [27] , nyamiviruses [28] , a new reovirus from Cameroon (Fako virus) [29] and Colombia [30] , a new 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 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 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 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 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. 

The family Nyamiviridae includes six genera. Nyamanini virus (NYMV), Midway virus, and Sierra Nevada virus (SNVV) were recently assigned to the genus Nyavirus [17] [18] [19] . They are mostly spherical in shape and bud from the cell surface. NYMV virions are 100-160 nm in diameter ( Figure  1C ), SNVV is larger and more variable in size, its virions measuring 300-750 nm in cross-sections ( Figure 1D ). 

The family Nyamiviridae includes six genera. Nyamanini virus (NYMV), Midway virus, and Sierra Nevada virus (SNVV) were recently assigned to the genus Nyavirus [17] [18] [19] . They are mostly spherical in shape and bud from the cell surface. NYMV virions are 100-160 nm in diameter ( Figure 1C ), SNVV is larger and more variable in size, its virions measuring 300-750 nm in cross-sections ( Figure 1D ).

Arboviruses assigned to the family Orthomyxoviridae are predominantly tick-borne and recently classified into the genera Thogotovirus and Quaranjavirus [51] . The genus Thogotovirus currently comprises 2 viruses: Thogoto virus (THOV) and Dhori virus (DHOV). Probable members also include Batken virus and its variants, Araguari virus (ARAV) [52] , Jos virus (JOSV) [53] , Upolu virus (UPOV), Aransas Bay virus (ABV) [22] and the recently isolated Bourbon virus [54] .

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] . comprises 2 viruses: Thogoto virus (THOV) and Dhori virus (DHOV). Probable members also include Batken virus and its variants, Araguari virus (ARAV) [52] , Jos virus (JOSV) [53] , Upolu virus (UPOV), Aransas Bay virus (ABV) [22] and the recently isolated Bourbon virus [54] .

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.

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] . 

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 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 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 ). 

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] . 

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] . 

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 ). 

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 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 

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 ).

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 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] . 

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 

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. 

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. 

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.

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.

The foundations of virology -discoverers and discoveries, inventors and inventions, developers and technologies

The Foundations of Virology -Discoverers and Discoveries, Inventors and Inventions, Developers and Technologies

Electron microscopy of viral infections

Diagnosis of viral infections by electron microscopy

Hsiung's Diagnostic Virology as Illustrated by Light and Electron Microscopy

Atlas of Invertebrate Viruses

Electron Microscopy in Viral Diagnosis

An Atlas of Mammalian Viruses

Electron Microscopy in Diagnostic Virology: A Practical Guide and Atlas

The Atlas of Insect and Plant Viruses: Including Mycoplasmaviruses and Viroids

Ultrastructure of Animal Viruses and Bacteriophages: An Atlas

Atlas of Anatomy and Ontogenesis of Human and Animal Viruses

Virus Morphology

Modern uses of electron microscopy for detection of viruses

Cell culture and electron microscopy for identifying viruses in diseases of unknown cause

Morphologic differentiation of viruses beyond the family level

Electron microscopy methods for virus diagnosis and high resolution analysis of viruses

Continued evolution of west nile virus

Associations between two mosquito populations and west nile virus in harris county, texas

West nile virus infection among the homeless

Comprehensive molecular detection of tick-borne phleboviruses leads to the retrospective identification of taxonomically unassigned bunyaviruses and the discovery of a novel member of the genus phlebovirus

Characterization of the bhanja serogroup viruses (bunyaviridae): A novel species of the genus phlebovirus and its relationship with other emerging tick-borne phleboviruses

Upolu virus and aransas bay virus, two presumptive bunyaviruses, are novel members of the family orthomyxoviridae

Sinu virus, a novel and divergent orthomyxovirus related to members of the genus thogotovirus isolated from mosquitoes in colombia

Characterization of triniti virus supports its reclassification in the family peribunyaviridae

Itaya virus, a novel orthobunyavirus associated with human febrile illness

Characterization of five unclassified orthobunyaviruses (bunyaviridae) from africa and the americas

Whole genome analysis of sierra nevada virus, a novel mononegavirus in the family nyamiviridae

A newly isolated reovirus has the simplest genomic and structural organization of any reovirus

Genome sequence of chiqui virus, a novel reovirus isolated from mosquitoes collected in colombia

Identification of a new newcastle disease virus isolate from indonesia represents an ancestral lineage of class ii genotype xiii

Eilat virus, a unique alphavirus with host range restricted to insects by rna replication

Divergent viruses discovered in arthropods and vertebrates revise the evolutionary history of the flaviviridae and related viruses

Insect-specific viruses detected in laboratory mosquito colonies and their potential implications for experiments evaluating arbovirus vector competence

Mercadeo virus: A novel mosquito-specific flavivirus from panama

Characterization of three new insect-specific flaviviruses: Their relationship to the mosquito-borne flavivirus pathogens

Genomes of viral isolates derived from different mosquitos species

A proposed new genus in the rhabdoviridae has a strong ecological association with bats

Koolpinyah and yata viruses: Two newly recognised ephemeroviruses from tropical regions of australia and africa

Almendravirus: A proposed new genus of rhabdoviruses isolated from mosquitoes in tropical regions of the americas

Kolente virus, a rhabdovirus species isolated from ticks and bats in the republic of guinea

Characterization of farmington virus, a novel virus from birds that is distantly related to members of the family rhabdoviridae

Arboretum and puerto almendras viruses: Two novel rhabdoviruses isolated from mosquitoes in peru

Niakha virus: A novel member of the family rhabdoviridae isolated from phlebotomine sandflies in senegal

Malpais spring virus is a new species in the genus vesiculovirus

Genomic characterisation of cuiaba and charleville viruses: Arboviruses (family rhabdoviridae, genus sripuvirus) infecting reptiles and amphibians

Evolution of genome size and complexity in the rhabdoviridae

Mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range

Negevirus: A proposed new taxon of insect-specific viruses with wide geographic distribution

Genetic characterization, molecular epidemiology, and phylogenetic relationships of insect-specific viruses in the taxon negevirus

Quaranfil, johnston atoll, and lake chad viruses are novel members of the family orthomyxoviridae

Araguari virus, a new member of the family orthomyxoviridae: Serologic, ultrastructural, and molecular characterization

Genomic and antigenic characterization of jos virus

Novel thogotovirus associated with febrile illness and death, united states

Cyclic avian mass mortality in the northeastern united states is associated with a novel orthomyxovirus

Taxonomic status of the tyulek virus (tlkv) (orthomyxoviridae, quaranjavirus, quaranfil group) isolated from the ticks argas vulgaris filippova, 1961 (argasidae) from the birds burrow nest biotopes in the kyrgyzstan

Tick-borne viruses structurally similar to orthomyxoviruses

Electron microscopy and antigenic studies of uncharacterized viruses. I. Evidence suggesting the placement of viruses in families arenaviridae, paramyxoviridae, or poxviridae

Generation and analysis of infectious virus-like particles of uukuniemi virus (bunyaviridae): A useful system for studying bunyaviral packaging and budding

Ultrastructure of kunjin virus-infected cells: Colocalization of ns1 and ns3 with double-stranded rna, and of ns2b with ns3, in virus-induced membrane structures

Wrapping things up about virus RNA replication

Modification of intracellular membrane structures for virus replication

Genus coltivirus (family reoviridae): Genomic and morphologic characterization of old world and new world viruses

Functional sindbis virus replicative complexes are formed at the plasma membrane

Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes

The host range phenotype displayed by a sindbis virus glycoprotein variant results from virion aggregation and retention on the surface of mosquito cells

Cytoplasmic structures associated with an arbovirus infection: Loci of viral ribonucleic acid synthesis

Eilat virus host range restriction is present at multiple levels of the virus life cycle

Novel insect-specific eilat virus-based chimeric vaccine candidates provide durable, mono-and multivalent, single-dose protection against lethal alphavirus challenge

A chikungunya fever vaccine utilizing an insect-specific virus platform

An insect nidovirus emerging from a primary tropical rainforest

Identification and characterization of genetically divergent members of the newly established family mesoniviridae

The authors thank Julie Wen and Zhixia Ding for expert assistance in electron microscopy and Dora Salinas for excellent help in preparation of the 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.

The authors declare no conflicts of interest.

The authors thank Julie Wen and Zhixia Ding for expert assistance in electron microscopy and Dora Salinas for excellent help in preparation of the manuscript.

The authors declare no conflict of interest.