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MicrobiologyPathobiology 445

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Title: MicrobiologyPathobiology 445


1
  • Microbiology/Pathobiology 445
  • Lecture 3, 3Apr01
  • Viral Structure and Diagnosis
  • James I. Mullins, Ph.D.
  • Professor and Chairman
  • Department of Microbiology
  • jmullins_at_u.washington.edu
  • Spring, 2001
  • Slides can be downloaded (ppt) from
    http//ubik.microbiol.washington.edu/Index.html

2
VIRUS STRUCTURE
Core - nucleic acid and tightly associated
proteins within the virion
Capsid - protein shell around NA or core
Definition Usage Viruses outside of cells are
usually metabolically inert. Virions consist of
either DNA of RNA (constituting the genome)
usually complexed with protein into a core,
surrounded by a protein coat called the capsid,
altogether called a nucleocapsid. The capsid is
composed of identical subunits called capsomeres.
It serves to protect and to ensure efficient
delivery of the nucleic acid genome to new cells.
Virally encoded peplomer spikes found protruding
from the envelope or at the surface of a naked
virion serve the critical function of receptor
recognition necessary for binding and entry into
susceptible cells. For many viruses, isolated
viral nucleic acid is by itself infectious,
albeit less so than when it is encapsidated.
3
Virions
  • One the function of the outer shells of a virion
    is to protect the fragile nucleic acid genome
    from physical, chemical, or enzymatic damage
  • The outer surface of the virus is also
    responsible for recognition of the first
    interaction with the host cell
  • Initially, this takes the form of binding of a
    specific virus-attachment protein to a cellular
    receptor molecule
  • The capsid also has a role to play in initiating
    infection by delivering the genome in a form in
    which it can interact with the host cell

4
Capsid Symmetry Virus Architecture
  • Since the approximate molecular weight of a
    nucleotide triplet is 1000 the average
    molecular weight of a single amino acid is 150, a
    nucleic acid can only encode a protein that is at
    most 15 of its own weight
  • Therefore, virus capsids must be made up of
    multiple protein molecules (subunit construction)

5
Helical animal viruses
  • The simplest way to arrange multiple, identical
    protein subunits is to use rotational symmetry
    to arrange the irregularly shaped proteins around
    the circumference of a circle to form a disk
  • Multiple disks can then be stacked on top of one
    another to form a cylinder, with the virus genome
    coated by the protein shell or contained in the
    hollow centre of the cylinder
  • Helical, naked (i.e. non-enveloped) animal
    viruses do not exist, but the reasons are not
    clear
  • This category includes many of the best known
    human pathogens, e.g. influenza virus, mumps
    measles viruses, Rabies virus
  • All helical animal viruses possess
    single-stranded, negative-sense RNA genomes

6
Rhabdovirus particle
7
Icosahedral Nucleocapsids Many viruses appear
spherical by electron microscopy, but they are
actually icosahedral, with the subunits of the
capsid located around the vertices or face of the
icosahedron. An icosahedron has 20 equilateral
triangles arranged around the face of a sphere.
Triangulating a dome into 20 is the best way of
producing a shell of equivalently bonded
identical structures. However, all known viruses
have more than 20 subunits, generally 60 x N
subunits
8
Enveloped Viruses
  • 'Naked' virus particles, i.e. those in which the
    capsid proteins are exposed to the external
    environment, are produced from infected cells at
    the end of the replicative cycle when the cell
    dies, breaks down lyses, releasing the virions.
  • Many viruses have devised strategies to exit from
    the infected cell without its total destruction.
  • All living cells are covered by a membrane
    composed of a lipid bilayer - the viability of
    the cell depends on the integrity of this
    membrane. Viruses leaving the cell must,
    therefore, allow this membrane to remain intact.
  • This is achieved by extrusion (budding) of the
    particle through the membrane, during which
    process the particle becomes coated in a lipid
    envelope derived from the host cell membrane
    with a similar composition.

9
Envelope proteins
  • If the virus particle became covered in a smooth,
    unbroken lipid bilayer, this would be its
    undoing.
  • Such a coating is effectively inert, although
    effective in preventing desiccation of or
    enzymatic damage to the particle, would not
    permit recognition of receptor molecules on the
    host cell.
  • Therefore, viruses modify their lipid envelopes
    by the synthesis of several classes of proteins
    which are associated in one of three ways with
    the envelope.

10
Matrix proteins
  • These are internal virion proteins whose function
    is effectively to link the internal nucleocapsid
    assembly to the envelope.
  • Such proteins are not usually glycosylated are
    often very abundant.
  • For example, in retroviruses they comprise
    approximately 30 of the total weight of the
    virion

11
Envelope and matrix proteins
12
Diagnosis of Viral Infection
13
  • Acute infection is diagnosed by the detection of
    HAV-IgM in serum by EIA.
  • Past Infection, i.e., immunity, is diagnosed by
    the detection of HAV-IgG by EIA.

14
  • A battery of serological tests are used for the
    diagnosis of acute and chronic hepatitis B
    infection
  • HBsAg - used as a general marker of infection
  • anti-HBc IgM - marker of acute infection
  • anti-HBcIgG - past or chronic infection
  • HBeAg - indicates active replication of virus and
    therefore infectiveness.
  • Anti-HBe - virus no longer replicating. However,
    the patient can still be positive for HBsAg which
    is made by integrated HBV.
  • HBV-DNA -indicates active replication of the
    virus, more accurate than HBeAg especially in
    cases of escape mutants. Used mainly for
    monitoring response to therapy.

15
Diagnosis of Viral Infection
  • 1. Direct Examination
  • 2. Indirect Examination (Virus Isolation)
  • 3. Serology

16
Direct Examination
  • 1. Antigen Detection immunofluorescence,
    ELISA etc.
  • 2. Electron Microscopy morphology of
    virus particles
  • immune electron
    microscopy
  • 3. Light Microscopy histological
    appearance
  • inclusion bodies
  • 4. Molecular Methods hybridization with
    specific nucleic acid probes
    polymerase chain reaction (PCR)

17
Immunofluorescense
18
Electronmicrographs
19
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 is coated with specific anti-sera. Virus
    particles present in the sample will be absorbed
    onto the grid by the antibody.

20
Molecular Methods
  • Methods based on the detection of viral genome
    are also commonly known as molecular methods. It
    is often said that molecular methods is the
    future direction of viral diagnosis, and it is
    certain that the role of molecular methods will
    increase rapidly in the near future

21
Classical Molecular Techniques
  • Dot-blot, Southern blot, in-situ hydridization
    are examples of classical techniques. They depend
    on the use of specific DNA/RNA probes for
    hybridization.
  • The specificity of the reaction depends on the
    conditions used for hybridization. However, the
    sensitivity of these techniques is not better
    than conventional viral diagnostic methods.
  • However, since they are usually more tedious and
    expensive than conventional techniques, they
    never found widespread acceptance.

22
Hybridization Techniques
23
The Polymerase Chain Reaction (PCR)
24
Polymerase Chain Reaction (1)
  • PCR allows the in vitro amplification of specific
    target DNA sequences by a factor of 106 and is
    thus an extremely sensitive technique.
  • 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
    thermostable Taq polymerase. Repeated cycles
    (usually 25 to 40) of denaturation of the
    template DNA (at 94oC), annealing of primers to
    their complementary sequences (50oC), and
    primer extension (72oC) result in the exponential
    production of the specific target fragment.
  • Further sensitivity and specificity may be
    obtained by the nested PCR.
  • Detection and identification of the PCR product
    is usually carried out by agarose gel
    electrophoresis, hybridization with a specific
    oligonucleotide probe, restriction enzyme
    analysis, or DNA sequencing.

25
Polymerase Chain Reaction (2)
  • Advantages of PCR
  • Extremely high sensitivity, may detect down to
    one viral genome per sample volume
  • Easy to set up
  • Fast turnaround time
  • Disadvantages of PCR
  • Extremely liable to contamination
  • High degree of operator skill required
  • Not easy to set up a quantitative assay.
  • A positive result may be difficult to interpret,
    especially with latent viruses such as CMV, where
    any seropositive person will have virus present
    in their blood irrespective whether they have
    disease or not.
  • These problems are being addressed by the arrival
    of commercial closed systems which requires
    minimum handling. The use of synthetic internal
    competitive targets in these commercial assays
    has facilitated the accurate quantification of
    results. However, these assays are very
    expensive.

26
Other Newer Molecular Techniques
  • Branched DNA (bDNA) is essentially a sensitive
    hydridization technique which involves linear
    amplification. Whereas exponential amplification
    occurs in PCR.
  • Therefore, the sensitivity of bDNA lies between
    classical amplification techniques and PCR. Other
    Newer molecular techniques depend on some form of
    amplification.
  • Commercial proprietary techniques such as LCR and
    NASBA depend on exponential amplification of the
    signal or the target.
  • Therefore, these techniques are as susceptible to
    contamination as PCR and share the same
    advantages and disadvantages.
  • PCR and related techniques are bound to play an
    increasingly important role in the diagnosis of
    viral infections.
  • DNA chips are another promising technology where
    it would be possible to detect a large number of
    viruses, their pathogenic potential, and their
    drug sensitivity at the same time.

27
Comparison between PCR and other nucleic acid
Amplification Techniques
28
Indirect Examination
  • 1. Cell Culture cytopathic effect (CPE)
  • haemabsorption
  • immunofluorescence
  • 2. Eggs pocks on CAM
  • haemagglutination
  • inclusion bodies
  • 3. Animals disease or death

29
Virus Isolation
  • Cell Cultures are most widely used for virus
    isolation, there are 3 types of cell cultures
  • 1. Primary cells - e.g., Monkey Kidney
  • 2. Semi-continuous cells - Human embryonic kidney
    and skin fibroblasts
  • 3. Continuous cells - HeLa, Vero, Hep2, CEM
  • Primary cell culture are widely acknowledged as
    the best cell culture systems available since
    they support the widest range of viruses.
    However, they are very expensive and it is often
    difficult to obtain a reliable supply. Continuous
    cells are the most easy to handle but the range
    of viruses supported is often limited.

30
Cell Culture Methods
  • Cell culture began early in the twentieth century
    with whole-organ cultures, then progressed to
    methods involving individual cells, either
  • primary cell cultures (somatic cells from an
    experimental animal or taken from a human patient
    which can be maintained for a short period in
    culture), or
  • immortalized cell lines, which, given appropriate
    conditions, continue to grow in culture
    indefinitely.

31
Cytopathic Effect (1)
Cytopathic effect of enterovirus 71 and HSV in
cell culture note the ballooning of cells.
(Virology Laboratory, Yale-New Haven Hospital,
Linda Stannard, University of Cape Town)
32
Cytopathic Effect (2)
Syncytium formation in cell culture caused by RSV
(top), and measles virus (bottom). (courtesy of
Linda Stannard, University of Cape Town, S.A.)
33
Plaque Assays
34
Haemadsorption
Syncytial formation caused by mumps virus and
haemadsorption of erythrocytes onto the surface
of the cell sheet. (courtesy of Linda Stannard,
University of Cape Town, S.A.)
35
Serological Methods
36
Complement Fixation Test
Complement Fixation Test in Microtiter Plate.
Rows 1 and 2 exhibit complement fixation obtained
with acute and convalescent phase serum
specimens, respectively. (2-fold serum dilutions
were used) The observed 4-fold increase is
significant and indicates recent infection.
37
ELISA for HIV antibody
  • Microplate ELISA for HIV antibody colored wells
    indicate reactivity

38
Western Blot
  • HIV-1 Western Blot
  • Lane1 Positive Control
  • Lane 2 Negative Control
  • Sample A Negative
  • Sample B Indeterminate
  • Sample C Positive

39
Animal host systems still have their uses in
virology
  • To study viruses which cannot be propagated in
    vitro, e.g. HBV
  • To study the pathogenesis of virus infections,
    e.g. Coxsackieviruses
  • To test vaccine safety, e.g. oral Poliovirus
    vaccine.
  • Nevertheless, they are increasingly being
    discarded because
  • Breeding maintenance of animals infected with
    viruses is expensive
  • Whole animals are complex systems, in which it is
    sometimes difficult to interpret
  • Results obtained are not always reproducible, due
    to host variation
  • Unnecessary or wasteful use of experimental
    animals is morally repugnant
  • They are rapidly being overtaken by cell culture
    molecular biology
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