1. introduction to virology
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Introduction to Virology
Oumer Ali (MD, MSc)
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Nature of Viruses
Virus particles are called virions.
are composed of either RNA or DNA that is encased
in a protein coat called a capsid.
are either naked or enveloped, depending on
whether the capsid is surrounded by a lipoprotein
envelope.
replicate only in living cells and therefore areobligate intracellular parasites.
cannot be observed with a light microscope
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Differences between cells and viruses
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The viral genome
• may be single-stranded or double-stranded, linear or
circular, and segmented or nonsegmented.
• Single-stranded viral RNA genomes are further
subdivided into those of positive polarity , and those of
a negative polarity or are antisense (that is,
complementary to messenger RNA which cannot
therefore be used directly as a template for protein
synthesis
•
is used as one criterion for viral classification.• is associated with viral-specific enzymes, other
proteins within the virion, or both
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The viral capsid
is composed of structural units called
capsomers, which are aggregates of
viral-specific polypeptides.
has a symmetry that is classified as
• helical
• icosahedral (a 20-sided polygon),
is used as a criterion for viral
classification.
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Figure Nucleocapsid of a helical virus.
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The viral capsid
serves four functions:
– As protection of the viral genome
– As the site of receptors necessary for naked viruses
to initiate infection – As the stimulus for antibody production
– As the site of antigenic determinants important in
some serologic tests
The viral nucleocapsid:
– refers to the capsid and enclosed viral genome.
– is identical to the virion in naked viruses
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The viral envelope
The viral envelope
– surrounds the nucleocapsid of enveloped
viruses.
– is composed of viral-specific glycoproteins and
host-cell derived lipids and lipoproteins.
– contains molecules that are necessary for
enveloped viruses to initiate infection,
– act as a stimulus for antibody production,
– and serve as antigens in serologic tests.
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Characteristics Used to Define Virus Families,
Genera, and Species
Viruses are divided into related groups, or
families, and, sometimes into subfamilies
based on:
1) type and structure of the viral nucleic acid,
2) the strategy used in its replication,
3) type of symmetry of the virus capsid (helical
versus icosahedral), and
4) presence or absence of a lipid envelope.
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Characteristics Used to Define Virus
Families, Genera, and Species
Within a virus family, differences in additional specific
properties, such as host range, serologic reactions,
amino acid sequences of viral proteins, degree of nucleic
acid homology, among others, form the basis for division
into genera (singular = genus) and species (Figure ).
Species of the same virus isolated from different
geographic locations may differ from each other in
nucleotide sequence. In this case, they are referred to as
strains of the same species.
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Figure Classification of viruses:
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Viral replication
occurs only in living cells.
involves many host-cell enzymes and
functions.
may be incomplete in some cells (abortive
infection)
may lead to the death of the host cell (virulent
viruses)
or may occur without apparent damage to the
host cell (moderate viruses).
is similar for all viruses in a specific family.
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Replication process
has a sequential pattern that includes thefollowing steps:
Attachment
Penetration
Uncoating of the viral genome
Synthesis of early proteins involved ingenome replication
Synthesis of late proteins (structuralcomponents of the virion)
Assembly
Release
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Attachment
involves the interaction of viral receptors
and specific host-cell receptor sites.
plays an important role in viral pathogenesis,
determining viral cell tropism.
may be inhibited by antibodies (neutralizing
antibodies) against viral receptors or cellular receptor sites.
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Penetration
can occur by a cellular mechanism called
receptor-mediated endocytosis
can involve the fusion of the virus envelope
with the plasma membrane of the host cell.
Uncoating
refers to the separation of the capsid from
the viral genome.
results in the loss of virion infectivity
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Budding
is the process by which enveloped virusesobtain their envelope.
is preceded by the insertion of virus-specific
glycoproteins into the membranes of a hostcell.
occurs most frequently at the plasma
membrane, but also occurs at other membranes.
confers infectivity to enveloped viruses
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Diagnostic Methods in Virology
Direct Examination
Indirect Examination (Virus Isolation)
Serology
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1. Electron Microscopy morphology / immune
electron microscopy
2. Light microscopy histological appearance - e.g.
inclusion bodies
3. Antigen detection immunofluorescence, ELISA etc.
4. Molecular techniques for the direct detection of
viral genomes
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Electron Microscopy
106 virus particles per ml required for visualization, 50,000 -
60,000 magnification normally used.
Viruses may be detected in the following specimens.
Faeces Rotavirus, Adenovirus
Vesicle Fluid HSV
VZV
Skin scrapings papillomavirus,
molluscum contagiosum
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Electron Microscopy
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Problems with Electron Microscopy
Expensive equipment
Expensive maintenance
Require experienced observer
Sensitivity often low
Requires at least 105 to 106 / ml particles in
preparation
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Immune Electron Microscopy
The sensitivity and specificity of EM may be enhanced by
immune electron microscopy. There are two variants:-
Classical Immune electron microscopy (IEM) - the sample is
treated with specific anti-sera before being put up for EM.
Viral particles present will be agglutinated and thus congregate
together by the antibody.
Solid phase immune electron microscopy (SPIEM) - the grid iscoated with specific anti-sera. Virus particles present in the
sample will be absorbed onto the grid by the antibody – easily
observed
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Light Microscopy
• Histological changes in infected cells
• Viral inclusion bodies are basically collections
of replicating virus particles either in the
nucleus or cytoplasm.
• Though not sensitive or specific , they are
useful adjunct in the diagnosis
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Inclusion bodies
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Inclusion bodies
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Identification of virus
Effect on cell culture
CPE
Inclusion body formation
Giant cell formation
Neutralization with specific serum
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Cytopathic Effect
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Effect of enterovirus 71 and HSV in cell culture: note the
ballooning of cells. (Virology Laboratory, Yale-New
Haven Hospital, Linda Standard, University of Cape
Town) Cytopathic
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Serology
Serologic diagnosis is based
on a greater-than-fourfold rise in IgG antibody
concentration when acute- and convalescent-phaseserum samples are analyzed at the same time.
A simultaneous fall in IgM antibody confirms recent
primary viral infection
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Serology
Criteria for diagnosing Primary Infection
• 4 fold or more increase in titer of IgG or total antibody
between acute and convalescent sera
• Presence of IgM
• Seroconversion
• A single high titer of IgG (or total antibody) - very unreliable
Criteria for diagnosing Re-infection
•4 fold or more increase in titer of IgG or total antibodybetween acute and convalescent sera
• Absence or slight increase in IgM
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Usefulness of Serological Results
How useful a serological result is depends on the individual virus.
1) For viruses such as rubella and hepatitis A, the onset of clinicalsymptoms coincide with the development of antibodies. The detection
of IgM or rising titers of IgG in the serum of the patient would indicate
active disease.
2) Many viruses often produce clinical disease before the appearance of
antibodies such as respiratory and diarrhea viruses. So in this case,
any serological diagnosis would be retrospective and therefore will not
be that useful.
3) There are also viruses which produce clinical disease months or years
after Seroconversion e.g. HIV and rabies. In the case of these viruses,
the mere presence of antibody is sufficient to make a definitive
diagnosis.
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Problems with Serology
Long period of time required for diagnosis for
paired acute and convalescent sera.
Mild local infections such as HSV genitalis may not
produce a detectable humoral immune response.
Extensive antigenic cross-reactivity between related
viruses e.g. HSV and VZV, Japanese B encephalitis
and Dengue, may lead to false positive results.
immunocompromised patients often give a reduced
or absent humoral immune response.34
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Problems with Serology
Patients with infectious mononucleosis and those with
connective tissue diseases such as SLE may react non-
specifically giving a false positive result.
Patients given blood or blood products may give a
false positive result due to the transfer of antibody.
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Typical Serological Profile After Acute
Infection
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Note that during reinfection, IgM may be absent or
present at a low level transiently
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ELISA
Direct EL ISA
Direct ELISA can be regarded as the simplest form
of the ELISA
Antigen is attached to the solid phase by passive
adsorption.
After washing, enzyme-labeled antibodies are
added.
After an incubation period and washing, a substrate
system is added and color is allowed to develop
Not commonly used
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Direct ELISA
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ELISA
Ind irect ELISA
The indirect system is similar to the direct system in thatantigen is directly attached to the solid phase
and targeted by added antibodies (detecting antibodies).
However, these added antibodies are not labeled
with enzyme but are themselves targeted by antibodieslinked to enzyme.
Such antibodies are produced against theimmunoglobulins of the species in which the detectinganti-bodies are produced and are termed antispeciesconjugates
After an incubation period and washing, a substratesystem is added and color is allowed to develop
Ind irect ELISA is widely used in diagnosis
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INDIRECT ELISA
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Microplate ELISA for HIV antibody: coloured wells indicate reactivity
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Western blots
Western blots can confirm the presence of
antibody to multiple specific viral proteins
simultaneously.
The proteins are separated by size and
transferred to an inert membrane, where they
are incubated with serum antibodies.
Western blots are inherently difficult to
quantitate
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Western blots
HIV-1 Western Blot Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive
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IMMUNOFLOURESCENCE
Flourescent dies can be covalently
linked to antibody molecules and made
visible by UV light in the flourescentmicroscope
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IMMUNOFLOURESCENCE
The direct fluorescent antibody (DFA) test
The DFA test is used to detect antigen in
the patient.
The labeled antibody directly interacts with
unknown antigen
The sample could be any tissue suspected
of infection with a specific organism.
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IMMUNOFLOURESCENCE
The indirect fluorescent antibody (IFA) test
The IFA test is used to detect antibody in the patient
Known antigen is attached to the slide
The patients serum (unlabelled) is added
Flourescent labeled antihuman antibody added &
examined by UV microscopy
If the test Ag is fluorescent following these steps,then the patient had antibody against this antigen
in their serum.
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POLYMERASE CHAIN REACTION
Detection of this amplified product (amplicon) indicates that the
sample is positive for the target virus .
It is based on an enzymatic reaction involving the use of
synthetic oligonucleotides flanking the target nucleic
sequence of interest.
These oligonucleotides act as primers for the thermo stable Taq
polymerase. Repeated cycles (usually 25 to 40)
denaturation of the template DNA (at 94oC)
annealing of primers to their complementary
sequences (50oC
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PCR -principles
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PCR ti
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PCR reactions Thermostable DNA polymerase is used for the PCR.
– This enzyme, which is isolated from thermophilic
bacteria, like for example Thermus aquaticus ,
– does not denature at high temperature and remains
active through the entire PCR reaction.
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PCR ti
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PCR reactions To perform PCR we just need to mix in the reaction tube:
1. DNA containing the gene to be amplified
2. Primers complementary to the flanking regions of the gene
3. Thermostable DNA polymerase
4. Nucleoside triphosphates (dATP, dGTP, dCTP, dTTP) and
then place the reaction mixture in the PCR machine that can
rapidly change the temperature of the reaction mixture
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Molecular methods
The advantage of molecular techniquesare
their sensitivity
specificitySafety
These technique do not require
isolation of the infectious agent and canbe performed on chemically fixed orinactivated samples or extracts.
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