Taking a look at viruses under the microscope. Unlike other unicellular organisms like bacteria,
viruses are commonly referred to as particles rather than cells. These
particles (virion) are also not alive given that they are unable to grow or
multiply on their own. Their size also makes a majority of viruses impossible
to see under a light microscope.
* A virus is between 100 and 500 times smaller than
* Researchers suggest that all living organisms
(including unicellular organisms) are host to at least a single virus particle.
Viruses under the Microscope
Virions are very small ranging between 400nm (mimivirus,
poxviruses) to about 25nm (polio virus) given that the resolution of
conventional compound microscopes is limited to half the wavelength of
radiation that is typically used for imaging (200 nm) they only allow for users
to view the general morphology of "giant viruses".
Some of the
techniques that have been suggested for viewing viruses under the microscope include:
Fluorescence Microscopy Technique
This is a relatively new technique that is used
to track viral DNA in the cells of the host. For this technique, ethyl-modified
nucleosides are used to label the DNA of such viruses as adenovirus, herpes virus
and vaccinia virus that have infected the cells of the host. The cell samples
are then viewed under a confocal microscope.
The method therefore simply involves using
fluorochromes, which allows the organism to fluoresce when viewed. Essentially,
fluorochromes only fluoresce when they attach to the target organism/object. In
the event that the virus of interest is present in a sample, this allows for the attachment to be seen indicating the presence of the virus.
Total Internal Reflection Dark-Field Microscopy (TIRDFM)
TIRDFM is a label-free imaging technique that
can be used to view larger virus particles. As such, no labeling is required
for this method. This system can simply be achieved by using a perforated
mirror in place of a dichroic mirror that is commonly used in conventional
Sample preparation for TIRDFM is a relatively complex
procedure that involves the following steps:
Inoculation of Influenza
A/Puerto Rico/8/34 (H1N1) virus into the allantoic fluid of an 11 day embryonated
egg at 37 degrees centigrade for 3 days
Centrifugation of the
allantoic fluid solution at 2,500g for about 20 minutes at 4 degree
Pass the supernatant
through a membrane filter (pore size 0.45um)
Subject the resulting
solution to 10-60 percent sucrose density gradient centrifugation for about 2
hours at 120,000g and at 4 degree in a swing bucket rotor
Obtain 2mL aliquots of the
Analyze the fractions using
SDS polyacrylamide gels and centrifuge for 2 hours at 120,000 g at 4 degrees
Suspend the pellet in
phosphate buffered saline
When viewed under TIRDFM, it is possible to see
spots (blight spots) with a dark background.
A transmission electron microscope allows for
about 1000 times higher resolution compared to typical light microscopes. It allows for better observation of virions.
For a TEM, higher resolution
is achieved because the large wavelength light beam is replaced with an
electron beam with 0.005nm wavelength. Here, the electron beams are produced by
tungsten, LaB6 filaments or a field emission gun thereby replacing visible
light. The resultant monochromatic beam is then accelerated in vacuum through a
40-100kV voltage before going through magnet fields that work as lenses.
The sample to be viewed using transmission
electron microscope has to be carefully prepared for observation.
the following steps:
Apply 10um of the
suspension to a hydrophobic surface of a parafilm square in a Petri dish
Float a formvar-coated grid
on to the drop for about one minute (make sure the formvar side is in contact
with the liquid sample)
Float the grid in a drop of
1.5 percent phosphotungstic acid (2% ammonium molybdate or 1% aqueous uranyl
acetate can be used depending on the type of specimen being prepared) - these
serve as stains
When viewed under the transmission electron
microscope, virions can be seen as small particles inside the cell (in the
cells they have infected).
In order to get a better view of the structure
of a virus, researchers have been using a number of techniques including
electron tomography, immunoelectron microscopy and cryo-electron microscopy.
These have also been shown to be important techniques for showing how viruses attach to cells, how they assimilate during replication as well as their their
association with various cellular mechanisms during replication.
In cryo-electron microscopy, the sample is frozen using liquid nitrogen and observed
under a transmission electron microscope that is equipped with a cryo-stage.
However, the images may be reconstructed in order to obtain a three dimension
of the virus.
Basic Virus Characteristics
A majority of viruses are
very small (about 100 times smaller compared to bacteria)
They are dependent on the
host to reproduce
They have a
receptor-binding protein that allow them to attach to the host cell surface
Morphology of Virus Particles
When the structure of a virus is viewed under a powerful microscope, it may be icosahedral or helical. These particles are
composed of a protein coat that envelopes the genetic material and is typically
referred to as a nucleocapsid.
The shape and size of the capsid is largely
dependent on the family to which the virus belongs. They are made up of
subunits of proteins referred to as capsomeres (it is the arrangement of these
protein subunits that determines the shape of the capsid).
Although viruses are
typically very small, recent studies have discovered that some viruses are
large in size. A good example of this is the mimivirus that has been described
as a giant virus. With a diameter of about 700nm, the mimivirus has been shown
to be larger in comparison to some bacteria.
Virions carry their genome (DNA/RNA) inside the capsid.
Depending on the virus, viruses either carry DNA or RNA which is useful for the
production of new viruses. Whereas ordinary viruses carry between 2 and 100
genes inside their capsid, giant viruses like mimivirus may contain as many as
Unlike other types of unicellular organisms like bacteria, viruses
lack other types of essential organelles such as ribosome and mitochondria
among others that would make it possible for them to manufacture and store
important molecules like proteins for energy generation. As a result, they are
unable to exist (grow and reproduce) freely and can only do so when they infect
the cells of a host.
Once they infect the cells of the host, viruses will start
using the organelle of the said host to thrive. However, this is done at the expense
of the host's cells that are destroyed in the process. Here, infection of the
cell will result in new viral particles, which then go on to infect new
* At any given time, there are about 1031
viruses that exist
Life Cycle of a Virus
Because viruses are not alive, their life cycle
is largely dependent on the host.
It involves the following phases/steps:
Once the virus gets into the body, it uses
specific attachment proteins to bind receptors on the surface of the cell of
These attachment proteins are important and determine the type of
cell the virus will attach on. For this reason, some viruses are only capable of
infecting a few hosts as they can only infect a few cells.
*Example: Rabies virus use glycoprotein as the
attachment molecule. These molecules can attach to such receptors as
acetylcholine receptor present on neurons. This therefore makes it possible for
the virus to attach to and infect cells present on neurons.
Penetration is the process through which the
virus injects its genetic material in to the cell of the host. This process may
either take place through endocytosis or direct membrane fusion. In
endocytosis, the virus attaches onto the receptors at the coated pits. Here,
the pits pinch off and forms coated vesicles. These vesicles then fuse with
endocytic vesicles before fusing with lysosomes. However, the genome of the
virus fuses with the membrane in the presence of fusion proteins to avoid being
destroyed by lysosome enzymes.
Ultimately, the RNA is able to reach the
cytoplasm without being destroyed. On the other hand, the virus will use the plasma membrane as an enveloped virus having been induced by binding to the
receptor. This is different from endocytosis, which is induced by changes in
Uncoating and Targeting
While some viruses release their genome in the
cell after penetration (process known as uncoating), replication cycle starts
in the capsid with some viruses like retroviruses. This is made possible by the
conformational changes of the capsid
Gene expression and replication- During this phase, new
viral genomes and virion components are produced and the genome of the virus
gets into the DNA of the host.
Virus assembly and release - This is the last phase
that involves the assembly of new virions. Newly formed genomes are inserted
into a capsid forming new nucleocapsids that repeat the cycle.
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