Viral Pathogens and Impostors: Who's Who in the Electron Microscope
Sara E. Miller, Ph. D.
Department of Pathology
Duke University Medical Center
Durham, NC 27710 U. S. A.
[email protected]
Introduction
Electron microscopy (EM) is invaluable in describing new viruses and in diagnosing viral
illnesses. Ability to visualize whatever unknown organism may be present in a sample is
advantageous, compared to searching for a mysterious agent using a biochemical test with
specific markers. All molecular tests that identify a given virus require the use of a probe, i.e.,
antibodies with secondary visual markers, polymerase chain reaction (PCR), and polyacrylamide
gels of nucleic acids and proteins all require reagents (specific antibody, PCR probes, and
nucleic acid or protein standards). Selecting the correct reagent when the pathogen is unknown
can be time consuming and expensive, and in some cases, no reagents are available.
Additionally, mutant genomes may not react in PCRs, and there can be antibody cross-reactions
or no reaction with immunological reagents.
EM has further advantage in that it does not require live viruses as does cell culture. Viruses
may die during transport, and some cannot be grown in routine tissue culture systems. Finally,
EM is relatively fast (0.5-1 hr for viruses in fluids, and 3-24 hr for infected tissues).
Discussion
Electron microscopy is an invaluable adjunct in viral diagnosis if viral concentration in fluids is
high enough (105-106 particles/ml) and in tissues if viral infection is not too small or focal.
Procedures, results, and interpretations are listed in outline form below.
Methods
Viral specimen preparation and examination techniques
Negative staining for viruses in fluids
0.5 – 2 % aqueous uranyl acetate, ammonium molybdate, or phosphotungstate
Examine fluids at 40,000-60,000x for 20-30 min.
Thin sectioning for viruses in tissue
Any routine EM tissue preparation procedure that includes aldehyde fixation,
osmication, possibly uranyl acetate en bloc staining (not required),
dehydration, and embedment in epoxy resin.
Examine tissues between 20,000-40,000x.
Problems and pitfalls
EM is less sensitive than PCR; viruses may be too dilute in fluids.
Focal infections can be missed in tissues.
Look-a-likes and masqueraders can mimic viruses.
Bacteria and other organisms can produce confusing structures.
Organisms “on” or „in” sections may resemble or obscure viruses (if “on” sections, they
are contamination in reagents/washes; if “in” sections, they could be
contamination or actual pathogens).
Bacteriophages in water may look like pathogens.
Source of the sample should be considered (An endoscope can carry down oral flora;
stored specimens and water sources can harbor bacteria that can produce
bacteriophages.
Suboptimal specimens (Viruses may or may not be recognizable.)
Frozen
Wax embedded
Stained section on a glass slide
Dried on side of container
Formalin fixed
Immuno fixed (Michel‟s fixative)
Unfixed and water- or buffer-stored
Ways to get around problems
Ultracentrifugation to concentrate viruses in fluids
Antibody concentration (Kapikian & Dienstag)
Confocal microscopy of tissue slabs to select areas of pathology (Miller et al., 1997)
Sampling multiple tissue locations
If the only tissue available is in a paraffin block: de-paraffinize in xylene, stain with Os,
and embed.
If all the tissue has been sectioned and stained on a glass slide, embed in situ, and glue
slab onto a blank block.
If fixed in light microscopy fixative, transfer to glutaraldehyde.
Where to look in tissues: inflammation, nucleated cells, necrosis edge (not center),
unusual ultrastructure for the tissue type
Thaw frozen specimens in glultaraldehyde.
Results
Morphological types of human viruses
Naked (non-enveloped) icosahedral (spherical like a baseball)
RNA or DNA
3 size ranges
Large (70-90 nm, e.g., adenovirus, rotavirus)
Medium (40-55 nm, e.g., polyomavirus, papillomavirus)
Small (20-35 nm, e.g., parvovirus, picornavirus, calicivirus)
“Capsid” or “nucleocapsid” in this case is the same thing as the complete “virion”
Enveloped (nucleocapsid surrounded by protein membrane; usually pleomorphic like a
raisin or an underinflated beach ball)
RNA or DNA
Nucleocapsids
Icosahedral (20-120 nm, e.g., herpesvirus)
Filamentous 18 x ≤1500 nm, e.g., paramyxovirus)
Complex (dumbbell-shaped with lateral bodies, poxvirus)
Complete virions (nucleocapsid surrounded by envelope) (40-400 nm)
Small (40-70 nm, e.g., flavivirus, alphavirus)
Medium (100-150 nm, e.g., coronavirus, orthomyxovirus, bunyavirus)
Large (100-400 nm, e.g., paramyxovirus, poxvirus)
Globular (e.g., paramyxovirus)
Filamentous (e.g., Ebola virus)
Brick-shaped (e.g., vaccinia a poxvirus)
Things that can look like viruses but are not
In negative stains of fluid samples
Resemble spherical virions or nucleocapsids
Lipid droplets
Protein droplets
Membrane vesicles
Ribosomes
Tailless Bacteriophages (are viruses, but non pathogenic)
Resemble filamentous nucleocapsids
Crystalline structures such as salt precipitates
Flagella and pili; fibers from clothing, gauze, food, and filters
Strands or liquid boundaries in sputum and urine
Resemble spiked enveloped viruses
Inside-out mitochondrial membranes
Resemble poxviruses
Melanosomes
Resemble viral capsid material
Bacteriophage neck and tail proteins
Bacterial cell wall debris
In thin sections of infected tissue
Resemble spherical virions or nucleocapsids
Ribosomes
Microtubules cut in cross section (look like parvovirus)
Glycogen
Clathrin coated vesicles
Membrane vesicles (caveolae, pinocytotic vesicles, lysosomes,
phagosomes, peroxisomes, Golgi vesicles, synaptic vesicles,
microvesicles, cell debris from lysed cells)
Neurosecretory vesicles
Nuclear granules
Nuclear bodies
Nuclear pores
Mitochondrial dense granules
Microvilli and collagen in cross section
Resemble filamentous nucleocapsids
Microtubules
Intermediate filaments
Amyloid
Cryoglobulin
Tubuloreticular inclusions (TRI) (seen in lupus, HIV infection, interferon
production or therapy
Clumped filamentous chromatin
Resemble spiked enveloped viruses
Clathrin-coated vesicles
Resemble poxvirus
Melanosomes
Conclusions
Electron microscopy is frequently used for identifying viruses because it does not require an a
priori notion of which agent might be present for reagent selection and because it can yield rapid
results. However, in negative stains of liquid specimens, artifacts and cell debris may resemble
viruses. Also, in thin sections, normal cell organelles and/or artifacts resulting from non-viral
causes, including delayed or improper fixation, can be confusing. Knowledge of proper
specimen preparation methods, virus ultrastructure, potential confusing elements, and normal
cell organelles is essential to reliable virus identification.
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Online:
The Big Picture Book of Viruses
http://www.virology.net/big_virology/
All the Viruses on the WWW
http://www.virology.net/garryfavweb11.html
Animal Viruses with Emphasis on Pathogens of Humans
http://textbookofbacteriology.net/themicrobialworld/AnimalViruses.html