key: cord-0882461-qwgde9uu authors: Hyatt, Alex D.; Eaton, Bryan T. title: Virological applications of the grid-cell-culture technique date: 1990-12-31 journal: Electron Microscopy Reviews DOI: 10.1016/0892-0354(90)90011-g sha: 032aa71cd58ababaf6e874f5bf58526541aa797f doc_id: 882461 cord_uid: qwgde9uu Abstract Whole mounts of intact virus-infected cells have been used for several decades to examine virus-cell relationships and virus structure. The general concept of studying virus structure in association with the host cell has recently been expanded to reveal interactions between viruses and the cytoskeleton. The procedure permits utilization of immuno-gold protocols using both the transmission and scanning electron microscopes. The grid-cell-culture technique is reviewed to explain how it can be exploited to provide valuable information about virus structure and replication in both diagnostic and research laboratories. The use of the technique at the research level is discussed using bluetongue virus as a model. The procedure can provide basic structural information about intact virions and additional data on the intracellular location of viruses and virus-specific structures and about the mode of virus release from infected cells. Application of immunoelectron microscopy reveals information on the protein composition of not only released virus particles but also cell surface and cytoskeletal-associated viruses and virus-specific structures. Collectively, this simple and physically gentle technique has provided information which would otherwise be difficult to obtain. and virus structure. The general concept of studying virus structure in association with the host cell has recently been expanded to reveal interactions between viruses and the cytoskeleton. The procedure permits utilization of immuno-gold protocols using both the transmission and scanning electron microscopes. The grid-cell-culture technique is reviewed to explain how it can be exploited to provide valuable information about virus structure and replication in both diagnostic and research laboratories. The use of the technique at the research level is discussed using bluetongue virus as a model. The procedure can provide basic structural information about intact virions and additional data on the intracellular location of viruses and virus-specific structures and about the mode of virus release from infected cells. Application of immunoelectron microscopy reveals information on the protein composition of not only released virus particles but also cell surface and cytoskeletal-associated viruses and virus-specific structures. Collectively, this simple and physically gentle technique has provided information which would otherwise be difficult to obtain. Eaton ('I rrl.. 1987. 1988 Such difficulties can be mainly attributed to the hydrophobic nature of the filmed substrate and can be rectified by glow discharging the filmed substrates (in air) or washing them with detergent such as 0.1% triton X-100 or 0.1% NP40 for several minutes prior to overnight incubation in tissue culture medium in the absence of ceils. The degree of cell confluency is also important, particularly for some subsequent preparative procedures. If, for example, the cell cultures are critical point dried, then high cell confluency has minimal effect except that little or no areas of substrate are available for released viruses to adsorb and this clearly limits the possibility of examining extracellular and surface cell-associated viruses. Alternatively, if the cells are to be negatively stained and air dried then the presence of too many cells will result in splitting and destruction of the supportive film due to severe shrinkage of large numbers of cells. This problem can be rectified by reducing the number of cells and/or the application of slightly thicker films. Cells are infected by adding a virus solution to the tissue culture dishes containing the filmed substrates. Alternatively if the virus stock solution is limited, each grid can be incubated in 20-50 ~1 droplets of virus suspension. The dilution of the virus suspension depends upon the desired multiplicity of infection and, when virus diagnosis is involved, the nature of the clinical sample (where cell toxicity may be a problem). Tissue culture dishes are placed at 37'C for 1 hr to permit virus adsorption following which the solution is replaced with fresh tissue culture growth or maintenance media. The major disadvantage of using whole mounts in TEM is the inability of the electron beam to penetrate the specimen. The use of critical point drying (De Harven et al., 1973; Buckley and Porter, 1975) and high voltage electron microscopy (HVEM) techniques (Kilarski et ul., 1976) have provided a partial solution to this problem. One of the earliest uses of critical point drying, in the study of animal viruses, involved purified oncornaviruses (De Harven et ~1.. 1973) . The success in visualizing viruses adsorbed to the grid was attributed to a combination of positive staining with uranyl acetate and critical point drying. The viruses were easily identified and resembled those in thin sections. Variations of this approach followed, including those of Malech and Wivel (1976) who critical point dried purified murine intracisternal A particles which had been adsorbed to filmed grids and shadowed them with platinum-iridium. Replicas of critical point dried and freeze dried cells have also been used to analyze the interaction of vesicular stomatitis virus (Birdwell and Strauss, 1974) and murine mammary tumour virus (Sheffield. 1981) with cells. Overall, these procedures have revealed cell-associated and budding viruses which have provided valuable information on virus morphology and topography of cell surface-associated viruses during morphogenesis. Critical point drying and examination of whole cells at 100 kV can reveal the threedimensional structure of cultured cells (Kilarski and Koprowski, 1976) . The lack of embedding media facilitates these observations and enables many intracellular and extracellular virus-cell interactions to be studied. The advantanges of this technique in virological studies were noted by Iwaski (1978) and include (a) rapid screening of both intracellular and extracellular aspects of numerous virus-infected cells and (b) in situ fixation of both released virions and those in the process of being released from host cells. The noted disadvantages of this technically simple procedure included the uncertainty about the identity of some intracellular structures and poor visibility of structures within the vicinity of the nucleus due to the thick central nuclear region. HVEM has also been used to visualize viruses such as vaccinia, parainfluenza, herpes simplex type 1 and frog virus 3 within whole infected cells (Grimly, 1970; Kilarski ef al., 1976; Stokes, 1976a.b; Murti CJ~ NI., 1984; Yoshida rt (I/., 1986) . The USC of this technique however is limited by access to the specialized equipment. NCEM has also been used to examine whole cells infected with rabies, mouse mammary tumour, influenza and bluetongue viruses (Dales, 1962; Choppin, 1963 . Kramarsky et ul., 1970 Iwasnki, 197X; Hyatt CJ/ II/., 1978) . In the earl) studies virus-infected cells were either cultured in suspension or on glass substrates. Infected cells grown on substrates were trypsinizcd prior to further handling. The cells were, in the majority of studies. swollen using hypotonic saline, fixed. pelleted, washed. resuspended and then adsorbed to formvar-carbon coated grids prior to staining with 2% sodium phosphotungstatc. The information obtained from examination of these preparations must be considered with caution as deleterious ctfects on virus structure and virus cell interactions may bc consequential to either osmotic shock or cnLymatic treatment of the cells. Iwasaki (1978) cultured cells on formvar-carbon copper grids which had been placed on plastic cover slips. Although the copper grids were theoretically removed from direct contact with the cells and medium the procedure has the potential for GILISing a cell toxicity problem. Despite this. Iwasaki ( 197X) was successful in cultivating virus-infected cells and examining them by both critical point drying and NCEM. Hyatt (I/ rrl. (1987) seeded cells on to biologically inert carbon parlodion filmed gold grids. The cells were infected bq adding \iru\ suspension to lissuc culture filled petri dishes containing the grids. Upon first indications of ;I cytopathic elTect the grid-cell-cultures (OX) Lvere fixed in 2.5% glutaraldehyde. washed in isoosmotic butfer. post fixed in osmium tetroxidc. uushcd in distilled water and stained with 2" (1 potassium phosphotungstatc. These preparations were used. as described below. to examine various viruses and virus cell interactions. Nermut (19X2) cautions against stringent illterpretations of negatively stained preparations hccause. for example. enveloped viruses can colapse and distort due to the severe surface tensions generated by air drying. Such artifacts can bc manifested in the form of particle plcomorphism and "tails". However, if the preparations were fixed prior to air drying the images were more likely to represent in .situ structures and thuh "reasonable information can be obtained" (Ncrmast. 1983 ). Air drying of negatively stained GC'(' has been used in our laboratory to identify progeny viruses amplified in GC'C, the parental \irus ot which were submitted to the laboratory in the form of diagnostic clinical samples. (Nagai et al., 1983) . From these thickened areas "rigid", irregular protusions are frequently observed (Fig. 1B) . The presence of these structures can be confirmed by examination of ultra-thin sections of similarly infected cells. Surface projections in many of these negatively stained viruses can be difficult to observe. It is believed that prefixation with glutaraldehyde or osmium tetroxide can produce a disorganizing effect on similar projections in other viruses (Nermut, 1972; von Bonsdorff and Harrison, 1975; Nermut, 1982) . We have found that this problem is more pronounced when viruses (fixed in situ) were in the process of budding. Examination of cells infected with avian influenza virus (AIV, Orthomyxoviridae) do not exhibit peripheral areas of membrane thickening and the budding virions generally approximate a filamentous shape (of uniform width) from which virions appear to "pinch" (Fig. 2) . These (NDV and AIV) are examples of viruses which derive their envelope from the plasma membrane. The in situ fixation minimizes the pleomorphic appearance of viruses and thus further enables virus identification based on virus morphology. Alphaviruses and flaviviruses are reported to be morphologically similar (Palmer and Martin, 1982) . They are spherical (40-70 nm) with alphaviruses being the larger. Flaviviruses although accepted to be smaller have been reported in the size range of 40--60 nm (Murphy, 1980) . They are also reported to have 7 nm rings on the virion surface and possess an intracellular-derived envelope, whereas purified alphaviruses have a conspicuous T = 4 icosahedral structure and derive their envelope from the plasma membrane. If problems arise in the identification of viruses within this size group (40-70 nm), then examination of virus particles by GCC can reveal details of the virion surface structure and the derivation of the associated envelope (intracellular or plasma membrane viruses against a clean background (Fig. 3C) . The viruses can also be observed in association with the plasma membrane (Fig. 311 ). The structure of these did not differ significantly from that of released or adsorbed particles. Extensive examination of the infected cells did not reveal budding viruses (that is, viruses acquiring a membrane from the plasma membrane) however. viruses with associated envelopes could be observed crossing the cell membrane. 0th~ membrane associated viruses. but with well defined shape, which have been detected with thi\ technique are shown in Figs 4 and S. Non-enveloped viruses can also be visualized with CCC. Representatives of Reoviridae, Adenoviridae. Parvoviridae and Birnaviridae have been identified in this laboratory using this technique. Representative viruses from some of these families are discussed below. Bluetongue virus (BTV), which is a member of the orbivirus genus in the family Reoviridae, is a 60-70 nm icosahedral virus with a fibrillar outer coat (Hyatt et ul., 1987; Hyatt and Eaton, 1988) . When crude preparations are used for NCEM the intact virion may be difficult to identify amongst the background material. Alternatively when the viruses are subjected to laboratory manipulation (for example, ultracentrifugation through density gradients) the outer coat often is partially or totally removed to reveal a capsid with large ring-shaped capsomeres (Fig. 6A) . Therefore the morphology of orbiviruses may be altered by the preparative techniques used. Diagnosticians should be aware of these variations or alternatively use a less disruptive technique such as the grid-cell-culture technique. Analysis of BTV-infected CCC revealed numerous virions adsorbed to the grid substrate, underlying the cell surface and being released from the cell. The virus surface was not disrupted and exhibited a fine fibrillar outer coat (Fig. 7A) . Similar virions were observed intracellularly (Fig. 7B) and were occasionally associated with cellular filaments. Virions were also observed exiting the cell by extrusion and budding from the surface membrane (Figs 7C, D) . Collectively the data provided valuable information for virus diagnosis (virus size. surface topography, association with cellular components and the modes of virus release). The sensitivity of the grid-cell-culture technique for other non-enveloped viruses such as birnaviruses, adenoviruses and adeno-associated viruses (AAV) is shown in Fig. 8 . The small I8 28 nm icosahedral AAV were obvious against a clean background which indicated the suitability of the technique for the detection of parvoviruses. Adenoviruses could also be observed on the grid substrate and entering host cells; Fig. 8B shows a particle entering a cell presumably by endocytosis. were also examples of lytic viruses, that is viruses which gain release from host cells following cell lysis. It is possible to locate these particles and associated structures on the grid substrate (Fig. 8) . When lytic viruses are being used it may be necessary to leave the harvesting of GCC until advanced CPE has occurred as this would optimize the chance of virus detection but have the disadvantage of producing a comparatively high background (Fig. 8A ). This disadvantage was not severe if the viruses involved are icosahedral non-enveloped particles. 1985; Bell et al., 1988) . The cytoskeleton is generally defined as a three-dimensional network of microtubules, intermediate filaments and microfilaments consisting of specific core proteins and filament-specific associ-ated proteins (Bell et al., 1988 Many methods have been used to observe the cytoskeleton. These include TEM of ultra-thin sections, whole and detergent-extracted cells and platinum-carbon replicas of frozen and fractured cells, NCEM and scanning electron microscopy (SEM) of detergent extracted cells and HVEM of whole and extracted cells (Bell et al., 1988) . With the grid-cell-culture technique satisfactory cytoskeletons can be prepared by washing the GCC with a mixture of 1% triton X-100 or 1% NP40 in 0. I '%I glutaraldehyde (in buffer of choice) for 2 min and critical point drying from carbon dioxide (Hyatt et trl., 1987) . The preparations remain attached to the filmed-grid substrate and can be coated or viewed directly at 50 kV in a transmission electron microscope (Fig. 9) . The preparation of cytoskeletons including conditions required for minimizing depolymerization and extraction of cytoskeletal proteins are described by Small ( 1988) . A common problem with whole mount cytoskeletons is that interpretation of conventional two-dimensional photographic images can often be difficult as they represent a three-dimensional network of interconnecting filaments. The use of htereopairs can help avoid the problem by revealing the spatial organization of and viral association with the cytoskclton. Cytoskeletons prepared and recorded in the above manner can prove beneficial in diagnostic virology and virus morphogenesis studies. The main advantages are (a) conventional thin sections, which provide comparatively poor detail on cytoskeletal organization and take the major part of a working day to prepare. are not required for the identification of intracellular viruses and related structures; (b) preparation time is less than 90 min and (c) the inter-relationship between virus and cell can be studied in ;I threc-dmensional matrix which facilitates in identification and localization of structural components of the cytoskeleton and any associated viral components. Figures are involved, IEM (Section V) may be used for their identification. It should be noted that cytoskeletons can also provide valuable information on the location of viruses, that is whether they are found predominantly in the soluble or insoluble phase of the cell. Orbiviruses, for example, are associated with the cytoskeleton (insoluble phase) of infected cells and thus any procedures involving purification of these viruses may incorporate a step which shears the particles from the cytoskeletal filaments. Both unextracted and extracted GCC can be used for IEM. Currently, colloidal gold probes are being successfully used for microbiological im-munocytochemistry (Beesley, 1988; Carrascosa, 1988) . These probes are generally complexed to protein A, protein G, monospecific or polyspecific antibodies and streptavidin. In practice they are used directly or indirectly for the detection of specific antigens by a range of methodologies including post-embedding, pre-embedding, immuno-negative stain and immuno-replica techniques. GCC can be used as alternatives to the classical immuno-negative stain and plastic embedding techniques. Figure 12 illustrates the use of the technique in the gold labelling of unextracted BTV-infected cells. Gold-labelled viruses can be observed on the filmed grid substrate and on the surface of the infected host cell. Labelling was achieved by the use of gold-complexed monoclonal antibody (Mab) to a virus outer coat protein, VP2. Although the preparation had been pretreated with 0.1% glutaraldehyde (to maintain cell structure and prevent removal of cells from the grid during washing procedures) no detrimental effects due to (a) free IEM can also be performed on extracted cells. aldehyde groups (manifested as non-specific la-The technique is rapid (2 4 hr) but has the belling) or (b) poor labelling due to possible adlimitation that only the insoluble fraction of a cell verse effect on the reactivity of the Mab to specific has the potential to be labelled. Despite this aldehyde-fixed antigenic sites, were apparent. The limitation the concept has been used by Bohn micrograph indicates that the labelling is specific P [ ~1. (1986) to study the involvement of actin and intense. Similar results were routinely obtained filaments in the budding of measles virus and in this laboratory for a range of viruses includ-Eaton et cri. (1987, 1988) to study the association ing herpesviruses, alphaviruses, coronaviruses, and protein composition of BTV-specific structures paramyxoviruses and bunyviruses; some of these attached to the cytoskelton. A major benefit ot are illustrated in Fig. 13 . The labelling depicted in using extracted GCC for IEM is the ability the electron micrographs cover direct and indirect of antibodies and gold probes to freely diffuse procedures utilizing gold-labelled monoclonal anti-through the cytoskeletal matrix and interact with bodies and gold-complexed protein A. The success the intact virus structures. Thus antibody antigen of this technique can be attributed to its simplicity interactions will be optimized in a biological and its overall non-destructive nature which main-system where normally, with post-embedding tains surface-associated antigens. Details of the techniques, the antigenic mass of the viruses methodology are given in Hyatt et ni. (1987) . or associated structures is so low that feu antibody molecules will interact with their specific targets. Representative results obtained with IEM of extracted GCC are illustrated in Fig. 14. The labelling protocol is detailed by Hyatt et ul. (1987) . Problems with immuno-gold labelling of cytoskeletal preparations can occur. The most common problem involves "sticky" gold probes, especially the smaller (6 nm) probes which may interact with the cytoskeletal matrix'. This problem of non-specific binding is discussed in detail by Birrell et al. (I 987) and Hyatt (I 989) and is largely solved by substituting fish gelatin for the conventional bovine serum albumin stabilizer or by pre-adsorbing the antibody with non-infected cells. The potential of the grid-cell-culture technique to preserve virus ultrastructure and to produce large numbers of adsorbed progeny viruses presents the virologist with an unique opportunity to undertake quantitative studies on comparative epitope density, efficacy of antibody binding and antibody competition (Eaton et al.. 1988; Gould et N/., 1988; Hyatt et ul., 1988) . The studies can involve either single or double immuno-gold labelling. Figure 15 illustrates double-labelling of surface epitopes on a bunyavirus (Akabane) and an orbivirus (BTV). The results in Fig. 1% demonstrate that two neutralizing Mabs were directed to either the same or separate but neighbouring epitopes where the binding of one antibody inhibited the binding of the second (Hyatt et ul., 1988) . Figure 15B demonstrates the "co-expression" of two BTV proteins namely VP2 and VP7 on the surface of the virus in an unstained critical point dried GCC preparation. The second example demonstrates the importance of virus structural preservation during preparative procedures as VP2 is a surface-associated protein easily removed during ultracentrifugation and VP7 is the major if not sole constituent of the underlying capsomers of the virus core particle. It should be noted that spurious results can be generated from double-labelling experiments unless various sources of error such as cross-contamination and degree of saturation of antigenic sites are determined. These sources of error are discussed by Hyatt et al. (I 988) and Hyatt (1989) . GCC can also be used for cell-surface studies involving topographic distribution of viruses during release and distribution of specific receptor sites. The examination of such preparations by standard SEM may be pertinent when the cells are too thick for the detection of gold-labelled surfaceassociated antigens by conventional TEM. SEM can be used to detect colloidal gold probes by either secondary (SEI) or back-scattered electron imaging (BEI). The detection of smaller (5515 nm) gold particles by SE1 and standard instrumentation is difficult especially when the complex topography of the cell surface and the frequent presence of contaminating protein aggregates are taken into consideration. Detection of these smaller gold probes can be made with BEI in conjunction with higher resolution instruments (Hodges rr ul., 1987). Alternatively larger probes (2040 nm) can be used in conventional SEM and visualized independently or with a combination of BEI and SE1 signals (Fig. 16A) . Unfortunately the use of larger probes for the detection of virus specific antigens will sterically preclude a one-toone correspondence of target to marker molecule and thus effectively decrease the overall labelling intensity. To avoid this problem 12 nm gold probes can be used and physically enhanced in size using the immuno-gold-silver staining (IGSS) technique (Hyatt rt ul., 1989) . The IGSS protocol is similar to that described for GCC (Hyatt et ul. 1987) except that a physical developing solution is included in the final steps (Hyatt et al., 1989) . The colloidal gold catalyzes the reduction of silver ions in the developing solution to metallic silver resulting in the physical growth of the electron-dense gold markers. The major advantages of this adaptation are that the silver enhanced probes are easily observed at low magnifications by a conventional scanning electron microscope utilizing SE1 and/or BE1 (Fig. 16B ) and. as stated above, the avoidance of steric interference. Comparison between Figs 16A and 16B illustrate the increase in labelling obtained with the smaller probes. Details on post-fixation and coating procedures for these preparations are discussed by Hodges rt cd. When GCC are used in the above manner. analogous preparations can be viewed by TEM (Fig. 17) . For viral applications this will indicate the specificity of the "background" gold ~silver labelling. For example, the particles present on the filmed substrate in Fig. 16B are gold&silver-labelled BTV and not the end product of indiscriminate background labelling. The techniques described above can also be used in basic virological studies. In this section we shall describe how GCC have been used to investigate the surface-associated protein composition of BTV and related structures in addition to specific events in BTV morphogenesis. A detailed discussion of BTV morphogenesis is beyond the scope of this review but can be found in reviews by Eaton and Hyatt (1989) and Eaton et ul. ( 1989) . BTV consists of an icosahedra core containing two major proteins, VP3 and VP7 and three minor proteins VPI. VP4 and VP6. Th core is surrounded by an outer fibrillar coat con sisting of two proteins. VP2 and VPS. The virus contains 10 segments of double-strandard RNA which code for the seven structural proteins and three non-structural proteins NSI, NS2 and NS3 (Verwoerd et d.. 1972; Huismans c'/ (I/., 19X7: Eaton and Hyatt, 1989) . Until recently studies on the structure of BTV have used purified virus. As mentioned above (Section V. A) the biophysical and biochemical procedures employed during purification of \,iru\ particles such as BTV can lead to distortion OI disruption and the removal of some proteins. It i< therefore important, in any experiment involving the identification of virus substructure. to maintain the original structural composition and for this the grid-cell-culture technique is ideal. We have used the technique to examine the surface "expression" of intrinsic cirion proteins (Eaton et (I/., 1988; Hyatt and Eaton. 19xX) . Viruses adsorbed to the grid substrate Lvere probed with available Mabs to BTV proteins VP?. VP7. NSI and VP3. Single and double-labelling cxperlments were attempted. The results showed the co-localization of VP2 with VP7 and VP? with NSI (Fig. 15) . VP3 was not detected on the \,irion surface, These results in conjuction with con~lltional experiments utilizing purified BTV core\ inferred that VP7 [known, from biochemical data. to be a major constituent of core capsomcrcb. Huismans rt ~1. (1987)j and NSI (previousI> thought to be solely an intracellular non-structural protein) extended beyond the core into the outer tibrillar coat where they were detected by spccilic Mabs. CCC were used to invcstigatc the association of BTV with the cytoskclctnn. Released viruses were probed with :I 6 nm goldlabelled anti-VP? Mab. following \s hich the cells WC'I'C extracted and viruses probed with I2 nm gold-labelled antibody, Thus only viruses adsorbed to the grid substrate outside the cell and labelled solely Mith I2 nm gold probes have been released from the qtosol follov8ng cell Iysis. The binding profiles (Fig. 20) indicate that there is a difYerence bct\+een the three populations in their ability to bind the antibody. Cytoplasmic \,irus bound most and released \.irus the leas; amount of gold-labelled antibody. These results infer that either VP2 is rcmod from intracellular \,irions during morphogenesis or alternatiwl~ the difrerences represent conf~~rmational changes in VP2 which obscure the reacti\,e epitopcs in some VP2 molecules. Electron microscopic examination of thin sections show that the cytoplasm of BTV-infected cells contains three major viral specific structures; \ iruses (discussed abo\,e). virus inclusion bodies (the site of virus core particle synthesis) and virusspecific tubules. the function of v, hich is unknown. Cytoskeletons of BTV-infected GCC were characterized by the presence of dense areas in juxtanuclear positions. The majority of \ irus inclusion bodies and virus tubules were obsewed in these locations. Virus tubules were observed in bundles of upto 20 tubules and in advanced stages of infection were also localized in the peripheral areas of the cq'toskcletal matrix. The \.irus inclusion bodies appeared to be penetrated bq the complex anastomoslng network of qtoskcletal filaments (Hyatt. unpublished obser\,ations) lvhereas the association of virus tubules kvith the cytoskelctal filaments remains unclear. Although the results indicated that these virus specific structures were associated with the qtoskeleton it is important to Lerif!, that the results are not a consequence ol fortuitous adsorption incurred during the extraction procedure. Experiments utilizing l,irus inclusion hod) and virus tubule specitic Mabs in conjunction \+ith fuorcscence were used to determine if these structures are found in the soluble fraction of the cell. Comparison of fluorescence patterns from both fixed. unextracted and atracled cells indicated that most if not all \ irus inclusion bodies are associated ith the cytoskeleton whereas some virus tubules are present in the soluble phase. Such results support the findings from the extracted GCC studies. Virus inclusion bodies and virus tubules located \ithin extracted GCC are present as large antigenie ma\scs when compared to those expressed in thin sections. Probing of \ irus tubules in extracted cells revealed ;I major core protein VP3 and the non-structur~~l protein NSI (Fig. l4B) to be present at :I surface location. Similar probing experiments. using a biotin htrcpta\idin gstcm and lowicryl (K4M) failed to detect an\ associated protein: this is not surprising as the tubules arc' hollow and approximateI> 30 nm in diameter and thus in ultra-thin sections must present iin extrcmolq small antipenic morass. The advantage of using CCC in IEM is further exemplitied b> the probing ot' virus inclusion bodies. Post-embedding immunolubelling of ultra-thin lo icr~I sections indicated the internal presence of core proteins VP7 and VP3 and the non-structural protein NS2 (Hyatt and Eaton, 1988) . The presence of virus core proteins within virus inclusion bodies uas consistent with ;I role for this structure in the morphogenesis of core particles. Labclling of extracted BTV-infected cells. however. indicated the presence of only NS3 on the surface of thcsc inclusion bodies (Fig. 14A) . These results were contirmed with pre-embedding labellinp experiments involving the examination of subsequently gcncrated ultra-thin sections. Overall these approaches gencrated valuable data on the addition of viral proteins to developing particles (Gould c't rrl. 1988: Eaton ct rd.. 1989). One protein of particular interest in BTV morphogenesis is the outer coat protein VP?. This protein contains the major antigenic determinants responsible for serotypc specificity and virus neutralization (Huismans and Erasmus. I98 I ). WC were therefore interested in the site and mechanism of addition of this protein to the virus particle. VP2 was not located with the internal matrix of inclusion bodies (post-embedding procedures) or on the surface of this structure (CCC and pre-embedding procedures) (Gould 01 t/l., 198X ). This simple expcrimcnt con-Firmed that virus release was not dependent upon cell death and subsequent lysis. The negativestained GCC (Fig. 7C,D) showed individual BTV budding from the cell surface and aggregates in close proximity to disrupted areas of the host cell's plasma membrane. Examination of conventional and pre-embedded labelled ultra-thin sections also showed large virus aggregates partially embedded in the membrane. These results were interpreted as virus release as opposed to uptake (Eaton et al., 1989; Hyatt et al., 1989) . The data suggest that (a) BTV was released both by "budding" and penetration of the plasma membrane by individual and aggregates of virus particles and (b) the different modes of release could occur in a single infected cell at the same time. In the past. basic virological studies encompassing electron microscopy have largely depended on techniques involving virus purification, negativestaining and thin-sectioning. The same is true for virus diagnosis. In this review we have attempted to discuss the application of a technique using whole virus-infected cells to these areas of interest. The broad range of applications is attributed to several advantages inherent within the technique namely (a) rapid and simple preparative steps, (b) adsorption of large numbers of progeny viruses to the filmed grid substrate, (c) production of little or no contaminating debris and (d) preservation of virus and cellular morphology. Although the basic technique of growing virus-infected cells on filmed grids or equivalent substrates is not new, it is surprising that the full potential of this concept has not been fully appreciated, particularly in IEM where advanced quantitative studies can be easily performed. Another exciting area of use is in virus diagnosis. From the foregoing discussion and examples it is clear that the technique offers the potential for providing valuable additional diagnostic data, to that obtained by conventional procedures, within a short time period. Overall the technique, termed the grid-cell-culture technique. provides an attractive addendum to the more conventional techniques currently used in virus diagnosis and research. JPMR 1 I -c Cryo-electron microscopy of viruses Freeze-etching technique for the study of virus ultrastructure Bioapplication of colloidal gold in microbiological immunocytochemistry The application of scanning electron microscopy to the study of the cytoskeleton of cells in culture Cytopathologic changes and development of inclusion bodies in cultured cells infected with bluetongue vsirus Some morphological features of bluetongue virus A negative staining method for high resolution electron mrcroscopy of viruses Cytoskeletal elements of chick embryos fibroblasts revealed by detergent extraction C'ytuplasmic lihrlls 111 li\ing cultured cells. A light and electron microscopy aludy Pro/op/~r.sr,rrr, 64 Electron mxroxx~p~ 01 critlcal pmnt dried whole cultured cells. d. ,2/ic~ro.v~~. 104 Form and distrlhution Structural elements in adenovirus cores. Studies by means of freeze-fracturing and ultrathin sectioning Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis Virus metabolism and cellular architecture The use of freeze fracture, freeze etching and critical point drying for the study of enveloped viruses The actin cytoskeleton Vaccinia virus LS and NP-antibody labeling in whole infected African green monkey kidney BSC-I cells studied by high voltage electron microscopy High-voltage electron microscope study of the release of vaccinia virus from whole cells Structure of the bluetongue virus capsid Envelope structure of Semliki Forest virus reconstructed from cryo-electron micrographs Sind bis virus glycoproteins form a regular icosahedral surface lattice High voltage electron microscopy of whole cells infected with herpes simplex virus type I thank Terry Wise and Gary Crameri for their technical assistance and Mr. John White for supplying monoclonal antibodies.