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Applications of transmission electron microscopy to virus detection and

identification

F.F. Vale1, A.C. Correia

2, B. Matos

2, JF Moura Nunes

3 and A.P. Alves de Matos

2,4

1 Faculty of Engineering, Catholic University of Portugal. Estrada Octávio Pato, 2635-631 Rio de Mouro, Portugal. 2 Pathological Anatomy Department, Curry Cabral Hospital, Rua da Beneficência 8, 1069-166 Lisboa, Portugal. 3 Electron Microscopy, Portuguese Institute of Oncology, 1099-023 Lisbon, Portugal. 4 Center for Environmental and Marine Studies, University of Aveiro, Campus Universitário de Santiago, 3810-193

Aveiro.

The size of viruses is with a few exceptions (the mimiviruses, poxviruses and some iridoviruses) below the resolution of

the optical microscope. The 1000 times higher resolution of the transmission electron microscope (TEM), compared with

the light microscope, allows direct observation of small virus particles. The improvement of both TEM equipment and

technical procedures allows quick preparative methods and diagnosis of viruses at the family and genus level. Besides

routine diagnosis this approach may prove essential to the identification of emerging pathogens in animals and humans. A

review of the literature and a description of the main methods and applications of TEM to virus diagnosis and

characterization are presented.

Keywords Transmission electron microscopy; virus; diagnosis

1. Introduction

Virus diagnosis by electron microscopy is based on the visualization and morphological identification of virus particles

in samples of diseased tissues or organic fluids [1-3]. Before the advent of electron microscopy, viruses were only

known by their manifestations, especially their capability to induce disease in susceptible hosts. Because of their small

size they can not usually be imaged with a conventional light microscope, and their particulate nature is apparent only

indirectly through filtration and ultracentrifugation experiments [4, 5]. This fact set the stage for the rapidly growing

interest in viruses as soon as the electron microscope was invented [6]. The approach met with great success since

viruses, with their small size and particulate nature, were almost ideal samples for early transmission electron

microscope (TEM) observations. In fact, the development of the microscope by Ernst Ruska was heavily influenced by

the observations of viruses carried out by his brother Helmut during the early stage of development of the methodology

[5]. An historical review of the field was recently made by Roingeard [7].

The use of electrons for imaging allows the achievement of a resolution of about 0.2 nm that can be used to visualize

macromolecules, such as the capsid proteins or the nucleic acids of viruses. However, the need for high vacuum, thin

specimens, and the very high energy of the electron beam irradiating the specimen placed terrible constraints to the kind

of objects that could be visualized with the instrument [6]. Decades of technical advances that continue today, were

needed to observe the macromolecular world in a state of preservation close to the living one [8, 9].

The accumulated knowledge on virus ultrastructure formed the grounds for the first classification of viruses based on

virus intrinsic properties rather than on host characteristics and diseases produced by them [10].

As mentioned before, direct morphological observation of most virus particles is not possible with conventional light

microscopy combined with immunocytochemical methods, that can reveal the nature of the viral antigens and their

intracellular distribution, and constitutes a very powerful diagnostic method in virology [11]. Molecular or

immunological methods, such as PCR (Polymerase Chain Reaction) and ELISA (Enzyme Linked Immuno Sorbent

Assay), are very effective in detecting known viruses and can be used to scrutinize a large amount of samples.

Nontheless, for new or unknown pathogens that may occur in the context of bioterrorism attacks or as a result of the

manifestation of new pathogens, electron microscopy remains the only method that can provide a quick assessment of

all pathogens present in a sample, providing an “open view”, which is why electron microscopy is considered a “catch-

all method” [2, 3, 12, 13]. Electron microscopy can also be used to investigate viruses isolated in cell cultures, which is

useful in controlling the results of the virus isolation process, among other applications [3].

In this paper we aim to lay down a few useful guidelines for those wishing to use electron microscopy in the context

of virus diagnosis, on the basis of personal experience with human and veterinary samples and information from

relevant literature.

2. Overview of the transmission electron microscope

The resolution of a compound microscope is limited to half of the wavelength of the radiation used for imaging. This

limits the light microscope to a maximum resolution of 200 nm, when using visible light with a wavelength of 400-

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800nm. The limited resolution of the light microscope only allows the observation of the general morphology of

bacterial cells and extra-large viruses. The internal structure is not clearly observed by light microscopes. Bacteria

dimensions range from 100 to 200 nm for the smallest bacteria (e.g. some members of Mycoplasma, which equals the

large-sized poxvirus), to 7 µm in diameter for the largest sized specimens (e.g. cyanobacterium Oscillatoria, with the

same size as a red blood cell), including medium sized bacteria with 1.1 µm by 2.0 to 6.0 long (e.g. Escherichia coli). In

summary, bacteria can be observed using a light microscope, but subcellular bacterial structures, bacterial appendages,

and most viruses are not usually visible since their largest dimension is smaller than 0.2 µm [14].

A huge improvement in resolution is achieved with TEM by replacing the large wavelength light beam with an

electron beam that has a wavelength of approximately 0.005 nm. When using TEM, electron beams generated either by

a tungsten or LaB6 filament, or by a field emission gun, substitute visible light. The monochromatic beam of electrons

is accelerated under vacuum through a voltage of 40 to 100 kV for biological work, and goes through magnetic fields

that work as lenses. These electromagnetic fields are generated by solenoids that can focus the electron beam. The

overall optics is very similar to the light microscope [6, 15].

Electron beams are notoriously unable to penetrate through matter, and therefore only the thinnest possible

specimens kept under high vacuum are suitable for imaging with this type of microscope. This posed enormous

constraints to the development of the field and a complex array of technical achievements in producing very thin

samples had to mature before relevant data could be extracted from electron microscopic images [16].

The images of biological samples produced by the electron microscope are inherently of very low contrast, since

organic matter is made of relatively light atoms of carbon, oxygen and hydrogen, that have low electron scattering

power. Staining methods requiring the introduction of heavy atoms into the specimen have to be employed in order to

reveal ultrastructural detail [17]. Positive staining, shadow casting, and negative staining procedures were developed for

visualization of thin sections and particulate samples. In particular, the negative staining method introduced by Brenner

and Horne provides a suitable and very fast way of revealing virus particles in TEM, and is thus extensively used for

diagnosis [18].

Another difficulty arises from the nature of the electron beam that is invisible to the human eye. Hence, auxiliary

devices that convert the electron image into a visible light image are needed.

For a well corrected optics, the resolution limit of the microscope is dependent on the wavelength of the radiation

beam. But it is difficult to correct spherical aberration in electron lenses. Energy spread in the electron beam, which can

be caused by the specimen also introduces chromatic aberration [15]. These defects limit the electron microscope

resolution to about 0.2 nm although the small wavelength of the electron beam could in theory allow much better

resolutions. Recently, correction of spherical aberration and chromatic aberrations has been achieved, improving

significantly electron optics [19].

The maximum useful magnification a microscope is able to achieve is set by the resolution, as the goal is to make

visible to the human eye the smallest object that the microscope can image (the size of the resolution limit). Since the

human eye can distinguish objects down to 0.2 mm, the useful magnification for a microscope with a resolution of 0.2

µm (light microscope), is about 0.2 mm/0.2 µm or 1,000x. There is really no restriction to the magnification that can be

achieved, except for the fact that higher magnifications are not needed. In practical terms magnifications of 30000 to

50.000 are suitable for viral diagnostic observations. The resolution of 0.2 nm of the TEM allows for a useful

magnification of 1,000,000x, a factor 1000x higher than for the light microscope [14].

3. Role of the electron microscope in virus identification

3.1 Sample collection

A successful virus diagnosis by electron microscopy depends heavily on the collection methods and on specimen

preparation [20, 21].

Collection is usually made by personnel not specifically trained in electron microscopy, and more general methods

used in other applications, such as molecular biology, are unsuitable for TEM or can even damage the specimen beyond

any hope of recovery [22]. Freezing the samples or fixing in formalin are common mistakes frequently found in the

practice of TEM.

Biological fluids can be easily and quickly studied by negative staining. Fresh fluid from lesions or dried fluid onto a

glass microscope slide are suitable specimens, while swabs offer significantly lower success rates [23].

Solid specimens can be embedded and sectioned with the methods mastered by any biological TEM lab. This takes

considerable more time than negative staining. For these methods, very small pieces of fresh tissue (less than 1 mm3)

must be immersed in glutaraldehyde fixative immediately after collection, as autolytic and anoxic changes that can

obscure the true pathological alterations of the tissue settle within minutes of the removal of the sample [17].

Knowledge of the ultrastructure of the virus particles gathered in these studies, together with the nature of its nucleic

acid formed the basis of an early classification of viruses that is still valid today [10, 24]. Morphological traits allow for


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their identification up to the family or genus level [25]. Further information on the virus type must be obtained by

means of serologic or molecular methods.

3.2 Handling of infectious specimens

Specimens for TEM diagnosis are concentrated suspensions of virus particles, which can be highly infectious. This is

particularly important when dealing with human samples. All manipulations must therefore be done under adequate

biosafety level laboratories. Diagnosis of exotic viruses such as the hemorrhagic fever agents Marburg and Ebola or

viruses producing encephalitis like the West Nile virus or the Dengue virus, must be performed in specialized centers

with high biosafety level facilities [2].

A particularly important point is that viruses are not completely inactivated even after being subjected to the vacuum

and electron beam of the electron microscope. Therefore, the sample and specimen holder must be disposed off and

decontaminated, respectively. If inactivation of the sample is needed, the virus particles can be treated with

glutaraldehyde fixative before or after adsorption to the grid without significantly changing their morphology [12].

3.3 Concentration methods

The minimum virus concentration necessary for successful detection by TEM is about 106 particles per ml. This is a

rather concentrated suspension and viruses are often present in diseased tissues and organic fluids at considerably lower

concentrations [12]. Another problem is the presence of other structures like bacteria and cell debris that may make

virus identification difficult, like in faecal samples, for instance [3]. In these cases, viruses must be separated from

contaminating debris and concentrated to get a workable preparation for electron microscopy.

Clearing the preparation from debris usually involves centrifugation at 1000-2000g for 5 to 10 minutes. The

supernatant is then submitted to ultracentrifugation at 100000g for thirty minutes to one hour to generate a pellet of

concentrated virus particles, which is ressuspended in a small drop (10-15 µl) of bidistilled water [3]. Alternatively, the

centrifugation can be done directly onto the grid, using a bench ultracentrifuge with a specially designed rotor

(Beckman Airfuge® with a EM-90 rotor), a procedure that greatly improves the sensitivity of the method [26].

3.4 Immunomethods

Concentration of the virus preparation may also be achieved by immunological means. The virus suspension may be

treated with antiserum against a known virus that may come from the patient himself. If the appropriate virus antigen is

present, clumps of virus particles are formed and easily visible in the electron microscope image. In the presence of an

excess of antibody, particles may become covered with a layer of antibody, which is visible by electron microscopy,

instead of forming clumps. Alternatively, the antibody can be adsorbed to a membrane-covered electron microscope

grid. Virus particles attach specifically to the treated membrane, and contaminants can be washed away, allowing for an

increased sensitivity of the method [3].

The use of specific antibodies also allows a more precise identification of the virus particles. If this is required, the

viruses adsorbed to the membrane-covered grid or thin sections of virus infected tissues, can be treated with gold-

labelled antibody. Colloidal gold particles with a definite size (5-15 nm) are attached to the virus antigens and can be

ready visualized in the electron microscope [2].

3.5 Specimen preparation for TEM observation

For negative staining, a drop of about 10 µl of the virus suspension to be studied is applied to the hydrophobic surface

of a parafilm square in a Petri dish. A formvar-coated grid is floated onto this drop for one minute, with the formvar

side of the grid in contact with the liquid. The excess liquid is removed from the grid by touching its border with a cut

piece of filter paper. The grid is immediately floated in a drop of 1.5% phosphotungstic acid, 2% ammonium molybdate

or 1% aqueous uranyl acetate, depending on the specimen. For a better assessment of the samples, two or three grids

should be prepared, each stained with one of these stains. After staining for one minute, the excess stain is removed

with filter paper and the grid left to dry for a few minutes, before insertion into the microscope column [3].

If solid samples are to be studied, such as organ fragments from biopsies or autopsy specimens, it may be necessary

to make thin sections of the pathologically altered tissues. This calls for a complex sequence of fixation in appropriate

fixatives, epoxy embedding, thin sectioning and staining, which is usually mastered by well trained personnel in

electron microscopy laboratories. Detailed accounts of the methods can be found elsewhere [16, 17]. If these methods

become necessary, an electron microscopy facility should be contacted for obtaining the appropriate primary fixative

and instructions for sample collection and submission to further processing. Common mistakes, such as freezing the

sample or leaving it unfixed for even “short” periods of time can ruin the sample and are to be avoided at all cost.


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4. Application of EM to the study of several viruses

For an example of a TEM application to the study of viruses Fig.1 shows virus particles recovered by cell culture from

tissues Triturus marmoratus, from a high mortality episode that occurred in a lagoon at the Peneda-Gerês National Park,

Portugal. The same virus had previously been observed in ulcers from diseased animals [27]. Among the many possible

causes for such an event, TEM was able to point the right direction with little effort and with no need for exhaustive

testing of specific suspected pathogens. The viruses have the size and overall morphology of Ranaviruses, and this was

subsequently confirmed by PCR. This case illustrates the advantages of TEM in virus identification, making it probably

the most common method for detecting new viruses [3]. Isolation of viruses in cell cultures is also extensively used in

research and diagnostic virology labs. The infected cells and isolated agents can conveniently be assessed by TEM, in

particular when atypical cytopathic effects occur.

Fig. 1 Thin section of ranaviruses in cell cultures. Skin ulcers of dying Triturus marmoratus from a lagoon in Peneda Gerês

Natural Park, showed virus-like particles suggestive of ranaviruses. Cell culture isolation combined with TEM and PCR studies

subsequently confirmed the presence of the viruses.

4.1 General virus particle morphology

The virus nucleocapsid contains the genetic material and a protective protein coat. Nucleocapsids can be icosahedral or

helical, and can be naked or enveloped in a protective lipoprotein coat. When present, the lipoprotein coat usually

shows projections that are important for virus identification. The poxviruses shown below have a more complex

arrangement [10, 28].

4.2 DNA viruses

The morphology of poxviruses observed using negative staining and thin sections are show in Fig. 2a and 2b. They are

brick-shaped with a size of about 300 nm. Poxviruses are agents of a variety of diseases, including smallpox that,

although considered extinct, may be reintroduced in the context of a bioterrorist attack. Electron microscopy would be

the preferred method for early detection of such a threat [3]. Monkeypox can also be transmitted to humans and poses

some concern as well. Other Poxviruses diseases of cattle and domestic animals can be diagnosed this way. Orfvirus is a

parapox virus that causes Muluscum contagiosum that also affects humans that handle the animals (Fig. 2b).

Differential diagnosis in skin lesions induced by other viruses like Herpes (Chickenpox) or papilloma can be done by

TEM [2].

Herpesviruses are usually found in skin lesions, but they are able to induce a wide spectrum of diseases in human and

animals, including respiratory infections, hepatitis in birds – Pacheco’s disease [29] – and encephalitis. Electron

microscopy is useful in the diagnosis of these specimens [30].

Adenoviruses mainly cause mild respiratory infections but are also responsible for canine hepatitis, which can be

diagnosed by searching for the virus in faeces (Fig. 3). Bacteriophages are frequently present in faecal material and

should not be confused with pathogenic animal viruses (Fig 4).

Polyomaviruses, although widespread in mice, monkeys and humans, are not usually associated with disease, but

they can induce tumours and cell transformation in vitro. The viruses can become active in immunossupressed patients

and be excreted in urine [28] (Fig. 5).

Parvoviruses are the smallest viruses known with 20-25nm in diameter. The human parvovirus B19 was discovered

in serum by electron microscopy and shown to be associated with aplastic anemia and fifth disease [3]. Parvoviruses

also induce feline panleukopenia and canine parvovirosis and the virus can be found in faeces of infected animals (Fig

6) [31, 32].


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Fig. 2 Poxviruses. Negative staining of swinepox virus particles (a). Thin section of Molluscum cotagiosum virus particles obtained

from the TEM reprocessing of a paraffin block in a human case of the disease (b).

4.3 RNA viruses

A large variety of viruses can be detected in the faeces by electron microscopy. Most, but not all (see above the

parvoviruses and adenoviruses), are RNA viruses that, for the most part, were first discovered with this same method.

Caliciviruses (Fig. 7) are main agents of viral gastroenteritis worldwide, being transmitted trough contaminated water or

food products. Norovirus, assumes particular importance in developed countries, where treated water efficiently

removes pathogenic bacteria, but does not equally removes viral particles. Other viruses associated with gastroenteritis

are rotaviruses, astroviruses and coronaviruses. These viruses are of particular importance during childhood, causing

several outbreaks of gastrointestinal disease [2].

Paramixoviruses cause mumps, measles and respiratory disease in humans. They can cause Newcastle disease in

avian flocks and respiratory disease in other animals [28, 33].

Common respiratory and enteric infectious caused by reoviruses can also be analysed by TEM (Fig. 8). Other

examples of emerging diseases, for which agent ultrastructure was analyzed by TEM, are Severe Acute Respiratory

Syndrome (SARS), which is caused by a coronavirus [34], influenza A, caused by new strain of influenza virus, such as

H5N1 [35] and H1N1 [36], or hemorrhagic fever caused by Ebola [37] and Marburg virus [38]. TEM permitted in these

cases the elucidation and characterization of the viral agent [1].

Fig. 3 Negative staining of adenoviruses in faeces from a

case of canine hepatitis.

Fig. 4 Bacteriophages commonly observed in faeces

material by negative staining.


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Fig. 7 Caliciviruses in a pellet of ultracentrifugated liver homogenate obtained from the necropsy of a rabbit during an epidemic of

hemorrhagic disease. The pellet was incubated with serum from a convalescent animal. The aggregation of virus particles suggests

the etiological role of the Calicivirus.

Fig. 8 Reoviruses in swine faeces observed by negative staining.

5. Advantages and limitation of TEM in (re)emerging virus detection

For a long time during the 20th century, infectious diseases were perceived as controlled, due to the

development/utilization of sanitization, water treatment, domestic hygiene, antibiotics, control of vector organism and

vaccination. Since the 1980s, there has been an emergence of unrecognised infectious diseases and a re(emergence) of

pathogens that were thought to be under control. Emerging virus are pathogenic agents that are recently present in a

Fig. 5 Thin section of polyomaviruses in a pellet of cells,

obtained from the urine of a kidney transplant patient.

Fig. 6 Negative staining of canine parvoviruses in

faeces.


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community or, that existed before in a specific population and are experiencing an increase of incidence or geographic

spread. A re-emerging pathogenic agent was previously present in a specific population and experienced a decrease of

incidence and reappears in the population, causing an increase of the incidence rate. The emergence of infectious

diseases is due to anthropogenic, social, and environmental changes. Modifications in medical care (nosocomial

infectious) and medical technology (blood transfusion, organ transplantation, re-used syringes, contamination of

vaccines, etc.) are important as anthropogenic causes. Social causes include general social changes, such as worldwide

urbanization, intravenous drug abuse, changing sexual practices, accelerated human mobility and refugee populations,

intensive food production and commercial trading. Finally, climate changes refer to global warming and ecosystem

disturbances, such as deforestation, eutrophication of waterways, reduction in the population of predator of disease

vector organisms and changes in the habitat or migratory routes of vector organisms [39].

For instance, gastrointestinal viruses are the leading cause of death among children. Most of these fatalities occur in

Africa, where health services are deficient and misdiagnosis is common, such as in the case of misdiagnosis of viral

diseases as malaria cases [40]. However, the aetiology of gastroenteritis is currently poorly investigated even in

developed countries. The routine detection methods used evolved over time. Initially, diagnosis was done by electron

microscopy, but this was replaced by molecular detection methods, using reverse transcription-PCR and confirmation of

positive samples by sequencing. There are cases of gastroenteritis that are negative for all RT-PCR techniques, for

which other methods must be applied, such as new molecular methods, electron microscopy or cell culture isolation. An

investigation of the aetiology of outbreaks in the Netherlands leaves 12.2% of these unexplained [41], in these the use

of TEM is still useful for at least determining the viral origin of the outbreak.

In addition to the capability of detection and identification of virus, electron microscopy can also help differentiate

between similar viruses, such as the Ebola and Marburg viruses. Both are from the filovirus family, but Marburg virions

are shorter than Ebola virions, and the spikes are also different [3, 42].

Electron microscopy has several advantages in emerging virus diagnosis. It allows an “open view” of whatever is

present (“catch-all method”), while molecular methods require previous knowledge of the virus to be tested. Electron

microscopy has also the advantage of being able to identify dual infectious (caused by more than one virus), which

could be missed by molecular or antigen tests. Indeed, no prior virus selection is necessary. Electron microscopy

permits a rapid diagnostic, since negative staining can be made in a few minutes. Viruses can be identified to the

group/family level on a morphological basis. Hence, even in the age of molecular diagnostics, the use of electron

microscopy is still a mainstay in detecting new and unusual outbreaks of unknown viruses and those that were not

considered for analysis by the clinician [3, 11].

Viruses stored in various solutions for long periods of time may not be viable for culture detection or molecular

testing, but may be useful for electron microscopy, since this technique does not require live or intact viruses for

identification [3]. However, if virus particles are in low concentration, or too altered morphologically due to sample

degradation, or as a result of inappropriate preservation procedures, the use of electron microscopy may be not useful,

which is a clear limitation of the technique.

The emergence of viral infectious diseases affecting humans and animals will continue both in developed and

developing countries and is very difficult to predict. Surveillance systems and the use of a large range of diagnostic

methodologies can contribute to control outbreaks. The use of immunological methods, such as production of antibodies

against specific virus, and the use of molecular biology tools like PCR imply some previous knowledge of the virus.

The identification of new unknown virus depends on the initial characterization of viruses by TEM. The study of

samples using TEM may achieve quick virus identification, or provide enough information on the pathological process

to point to the right direction. Additionally, as electron microscopy is a rapid procedure, it can be of major importance

in the surveillance of viruses that can be used as biological weapons [3]. Indeed, electron microscopy can be used to

detect viral agents, such as Ebola virus and smallpox virus. However, before becoming involved in the monitoring of

virus bioterrorism samples, laboratories must consider several issues, including their expertise in virus identification,

capacity for handling biohazards, and the health and immune status of the laboratory staff [43]. The identification on the

basis of virus morphology requires well trained and experienced microscopists, which are hardly present in every virus

diagnosis laboratory. The use of tele-microscopy, i.e., the control of the microscope and diagnosis by a spatially remote

expert, can guaranty an additional quality assurance level in the morphologic diagnostic [39, 40].

6. Conclusions

Electron microscopy was the gold standard for virus diagnosis for decades (from the 30s to 80s of the 20th century), but

the interest in the technique decreased with the implementation of molecular biology techniques. Indeed, electron

microscopy was successfully used in both infected cell lines and “dirty” clinical samples, such as plasma, urine, faeces

and vesicle fluids [1, 7]. Because electron microscopy is not a high throughput technique, many molecular and

immunologic methods have been developed. Immunological methods have almost unlimited throughput and are very

specific, which can contribute to the failure of identification of viruses with different antigenic determinants. Similarly

molecular techniques, such as PCR rely on previous knowledge of genomic content so that primers can be designed.

Mutations, which are very common in viruses, may lead to false negative results, because the primers may not recognise


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the complementary mutated sequence [12]. Nonetheless, electron microscopy is still the gold standard for the

identification of unknown viruses, in situations where no probes or primers are available, which is often the case of

emergent viruses.

Electron microscopy was is and will be always necessary for new virus characterization. This especially applies to

veterinary virology, in which several agents are continuously being identified with this technique [1]. The future of

electron microscopy goes through rapid diagnosis of viruses, the exclusion of presence of pathogenic virus in

bioterrorism samples, and in quality control of produced vaccines. Considering that its use in diagnosis, although

declining, is of prime importance for both diagnosis and research, it is important to prepare a new generation of

microscopists with the appropriate technical skills [2]. The recourse to tele-microscopy should also be considered, so

that excellence centres in electron microscopy can concentrate their expertise and better interact with the diagnostic labs

[44, 45]. Besides being applied in diagnostic virology, electron microscopy can be used to understand the mechanisms

of virus attachment and replication within the host cell. This information can be useful in the design of novel antiviral

drugs and vaccines. The study of viral effects on cells and tissues enriches the knowledge of which cells and organs are

involved in disease. Ultrastructural studies contribute for both the design of novel drugs against viruses, but also help

elucidating the function of several viral elements [3, 7]. Surely electron microscopy will still be needed for the

identification of new unknown virus. The “open view” approach of electron microscopy permits rapid and “catch-all”

detection of viruses, if these are present in a high concentration, and makes it especially useful in emergency situations.

The nature of the samples to be analyzed can be tremendously diverse, from body fluids or biopsies performed after or

before cell culture, or environment samples [12].

Finally, an additional advantage of electron microscopy is its promoting value. It is reassuring to have the picture of

the screened virus and “a picture is worth a thousand words”. Among the disadvantages of electron microscopy usage

there are the need for training microscopists, the capital expenses in instrument acquisition and maintenance, need for

an appropriate room for its operation, the unsuitability of use for all viruses, the intensive labour, the imprecision in

distinguishing between many similar viruses, and the low throughput [1]. All methods have advantages and

disadvantages and their applications should be careful planned. The combined use of several methods should not be

discarded. For example, results obtained by TEM can be confirmed at the species level by directly testing the virus

adhered to grids by PCR [46], joining and taking advantage of two distinct techniques.

Acknowledgements The English review by Patrícia Fonseca is gratefully acknowledged.

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