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Full summary infectious diseases
- Instelling
- Radboud Universiteit Nijmegen (RU)
Full summary for the course infectious diseases (NWI-BB097) given in 2023 - Radboud University, Nijmegen
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Infectious DiseasesIntroduction to viruses and Diagnostics
What are Viruses?
Viruses are obligate intracellular parasites or symbionts that possess their own genomes encoding
information required for virus reproduction and, hence, a degree of autonomy from the host genetic
system, but do not encode a complete translation system or a complete membrane apparatus.
This means that they are small, obligatory intracellular infectious
agents. The virus only replicates in the host and not in medium. They
are fully dependent on a host for energy metabolism, lipid biogenesis
and protein synthesis. They do not grow or divide but are produced
de novo from the assembly of pre-formed components. They must
make mRNA that can be read by host ribosomes.
Viruses were discovered based on diseases in plants, animals and human hosts in the past.
Nowadays, we use metagenomic approaches (based on gene sequence) to discover new viruses.
Viruses infect all living organisms.
General Replication Cycle
Generally, the virus attaches to the cell membrane via a
receptor and penetrates the membrane. Inside the cell, it will
uncoat, thereby releasing its viral RNA. Uncoating is followed
by replication, in which viral mRNA is synthesized and viral
proteins are translated. When there is enough viral mRNA
present, the cell will assemble new virions using capsids and
release the virions either via cytolysis (no envelope) or via
budding (with envelope).
Virus Diversity and Classification
Viruses have a wide diversity in genetic make-up, replication
strategies, virion morphology, host specificity, disease,… Virus taxonomy addresses the classification
of viruses. However, within a virus species, you can also have different variations of virus sequences.
There are seven genome types according to
the Baltimore classification. In dsRNA, the +
strand can be used for translation. Ss + RNA
strands can also immediately be used as the
template for proteins. However, - strands
need to be transcribed by viral RNA
polymerase into + strands before it can be
translated by host ribosomes.
One can deduce the basic steps in the viral
life cycle from the nature of the viral
genome, but the pathogenesis cannot be
explained by genome organization.
,Viruses can also be classified on their physical characteristics: nature and sequence of nucleic acid in
the virion, symmetry of the capsid, presence/absence of a lipid membrane, dimensions of the virion
and capsid, …
In virus taxonomy, the realm is the highest class, followed by order (-virales), family (-viridae), genus
(-virus), species and virus. A virus within the same family/genus share an overall replication strategy.
However, they are not necessarily transmitted the same way nor do they cause the same diseases.
Viruses from different families can cause similar symptoms.
,Detection of Viruses
Detection of viruses is important in order to diagnose patients with a suspected virus infection, for
fundamental virology/immunology research, epidemiological research and surveillance to prevent
epidemics.
Viruses can be detected via direct and indirect (serology) methods. In direct methods, the whole
microorganism, their structural components or their products are detected. In indirect methods, the
antibodies targeting the microorganisms are detected.
Direct Methods
Direct methods include detection of virus particles using culture based techniques (e.g. plaque assay)
or microscopy (e.g. electron microscopy); detection of viral antigen using antigen ELISA, fluorescence
microscopy or lateral flow assays; detection of genetic material using PCR or sequencing.
Virus Detection by Microscopy
Electron microscopy can be used to visualize virus particles in a sample. However, it is not used in
routine diagnostics due to the high level of expertise and expenses needed. On top of this, one
cannot know if they are truly looking at a virion. It is used in fundamental research instead.
Virus Detection by Cell Culture
Virus infection can result in structural changes
in a monolayer of susceptible cells due to their
cytopathic effect (CPE). Different viruses can
lead to different types of CPE (e.g. zika virus:
cells die; cytomegalovirus: owls eye). Some
types of CPE are typical for a specific virus,
allowing identification of the virus.
There are two methods relying on CPE: plaque assay and end point dilution. These techniques are
primarily used in scientific research due to them taking a long time.
A plaque is a local area in which cells have died due to a virus infection. It is the result of a single
initial virus infection. The sample is diluted until the amount of plaques is count-able and then
calculated back to the total concentration in the original sample.
In end point dilution, 10-fold serial dilutions of the virus are added to cells in a multiple-well plate. It
is then incubated and the amount of infected wells (%) is plotted against the virus dilution. From
that, one can determine the dilution needed to infect 50% of the wells.
Virus Detection by Genetic Material
One of the most widely used techniques to detect viruses and other pathogens is a (quantitative)
PCR. Another method is (next generation) sequencing, which is mostly used in research currently.
Culture based PCR
Pros Detection of infectious particles High sensitivity and specificity
Gives the chance to find the unexpected Relatively short time from sample to result
(e.g. when not sure if individual is infected)
Cons More laborious Relatively expensive
Long waiting time from sample to result High sensitivity also detects low virus levels
Virus identification is often not possible (e.g. during recovery and no longer
contagious)
Analysis is biased towards expected viruses.
, Virus Detection via Viral Antigen
Antigen ELISA, fluorescence microscopy and lateral flow assays
rely on the detection of fluorescently labelled antibodies.
Lateral flow assays have a control line and a test line. The control
line will bind to the pre-loaded antibodies and the test line will
bind to the antigen from the sample.
Agglutination can be used to detect viral antigen. The beads are
covered with specific antibodies that will bind to the antigen of
the pathogens.
Immunoassay ELISA can be used for both the antigen (direct) and antibody (indirect). In an ELISA for
antigen, the antibody is bound to a solid phase and an unknown antigen is added. Then, a second,
labelled antibody is added, which will bind to the antigen.
Indirect Methods
Indirect methods include antibody ELISA and agglutination tests.
Agglutination can also be used to detect antibodies. It tests whether immune complexes are formed
between antibodies present in the sample and the latex beads covered with a specific antigen.
In antibody ELISA, you first bind the antigen to solid phase and then add an unknown antibody. Then
you add a secondary, labelled antibody to the sample and measure the label. The secondary antibody
will bind to the unknown primary antibody. The initial antibody response is IgM mediated, which
declines after a few weeks. There is an isotype switch to IgG after a few weeks. IgG is detectable
many months/years, sometimes even for life.
Tutorial
CPE is not often used in laboratory diagnostics anymore due to the long waiting time and the lack of
specificity, making identification of the infecting virus difficult.
A semisolid agarose matrix on top of the cell monolayer during a plaque assay prevents virus
particles from freely floating through the cell culture dish.
A lower CT value means more DNA in the sample.
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