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
Rhabdovirusparticle
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
HybridizationTechniques
23
The PolymeraseChain 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