Transcript Slide 1

Adenoviruses and Poxviruses
18 Muharram 1428/ 6th Feb 2007
SBM 2044
Medical Microbiology
Adenoviruses
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A valuable systems for biochemical and molecular
studies of eukaryotic cell processes.
Icosahedral symmetry; Linear ds DNA; no envelope;
capsids composed of 252 capsomeres. Hexon and
penton capsomeres are 2 main components. Hexon
is the major protein forming the 20 triangular faces
of the viral capsid.
Human adenovirus are divided into 6 groups (AF)
based on their physical, chemical, and biological
properties.
Adenovirus
Penton base carries a toxin-like activity  cytopathic effects and
rapid detachment of cells from the surface
Fibers contain type-specific antigens for serotyping and possess
haemagglutinating activity.
Adenoviruses
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Exist in all parts of the world, all year round.
Different serotypes may infect different
groups of people:
Eg. 1, 2, 5, 6 – 1st years of life;
4, 18 & 19 – not until adulthood;
3, 4 & 7 – common among military recruits.
Spread person-to-person by respiratory and
ocular secretions
Pathogenesis
Adenovirus Replication
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Replicate well only in cells of epithelial origin.
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A) Virus attachment:
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Adenovirus attaches to host cells via the fiber
structures. The receptor is usually CAR (coxsackieadenovirus receptor).
Internalisation of the virus takes place when the
penton base interacts with cellular integrins.
Virus then internalised into endosomes, and by
acidic pH into cytosol.
Next, uncoating commences in cytoplasm and
finishes in the nucleus, and simultaneously ends the
host-virus molecular interaction.
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Adenovirus Replication
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B) Early events:
Is the stage before the onset of viral DNA synthesis.
Aims: to induce the host cell to enter the S phase of
the cell cycle; to express viral functions that protect
the infected cell from host defense mechanisms;
and to synthesise viral genes for replication.
>20 early proteins are synthesised, many of which
are important for viral replication. Eg. E1A is vital
before the synthesis of other early regions. The E1B
encodes proteins that block apoptosis that occurs
due to E1A functions.
Adenovirus Replication
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C) Replication and Late Events:
Virus replicates in the nucleus.
Late genes mainly encode structural proteins.
D) Viral Assembly and Maturation:
Virion is made in the nucleus.
Capsomeres self-assemble into empty-shell
capsids in the nucleus, then only DNA enters
the capsids.
Adenovirus Replication
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E) Virus Effects on Host Cells:
Adenoviruses encode several gene products to counteract
antiviral agents from host. Virus prevent the activation of an
interferon-inducible kinase that phosphorylates and
inactivates eukaryotic initiation factor 2.
Adenovirus E3 inhibits cytolysis of infected cells by host
responses. E3 gp19-kDa protein blocks movement of the
MHC class I antigen to the cell surface, protecting the cell
from cytotoxic T lymphocytes-mediated lysis. Also blocks
induction of cytolysis by the cytokine TNF-α.
Cytopathology: marked rounding, enlargement, aggregation of
affected cells into grape-like clusters. Infected cells do not
lyse. Sometimes cells can be seen with rounded intranuclear
inclusions containing DNA (≈ CMV, but without multinucleated
giant cells or syncytia).
Gene Therapy
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Adenovirus as gene delivery vehicles or DNA
vaccination.
Advantage: because replication-defective
virus is able to lyse the endosome after
internalisation and release DNA into the
cytoplasm.
Prototype for adenoviral therapy is AV5
Adenovirus Infections
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Adenoviruses infect and replicate in epithelial cells
of resp T, eye, GIT, UT etc. Some may persists as
latent infections for years in adenoids and tonsils,
and are shed in the faeces several months after
infection.
Adenoviruses 1-7 are the common types. A single
serotype may produce more than one clinical illness.
Respiratory diseases with cough, nasal congestion
and sore throat are usually associated with Group C
viruses, commonly found in infants and children.
Adenovirus Infections
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Adenoviral pneumonia – by adenovirus 3, 7 and 21
– are thought to be the cause of childhood
pneumonia.
Acute respiratory disease syndrome among military
recruits, which caused by types 4 and 7.
Others: keratoconjunctivitis by types 8, 19 & 37;
and enteritis in children by types 40 & 41 . Types 11
and 21 may cause acute haemorrhagic cytitis in
children esp boys. Patients receiving transplants
are immunocompromised are susceptible to
pneumonia usually caused by Types 1-7 eg.
Children receiving liver tx → adenovirus hepatitis.
Laboratory diagnosis
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Infected cells can be cultured, and tissues are
looked for rounding and clustering of swollen cells.
Adenovirus also cause increase glycolysis in cells,
hence the growth medium becomes more acidic.
Viruses can be detected by immunofluorescence
against antihexon Ab.
PCR of samples using primers from a conserved
viral sequence (eg. Hexon VA1) for all serotypes.
Examination of faecal extracts by electron M, ELISA
or by latex agglutination. Since adenovirus can be
excreted for prolonged periods, the presence of
virus does not necessarily mean it is associated with
disease.
Treatment and Prevention
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Most infections are mild and require no therapy. But
serious adenovirus illness can be managed by
treating symptoms and complications.
Careful hand washing environment surface by
sodium hypochlorite (bleach).
Vaccine: 1971 – US focuses on military with live
adenovirus types 4 & 7, enclosed in gelatine-coated
capsules which allows the virus to bypass resp T
and be released in intestine instead.
Poxviruses
Poxviruses
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Largest and most complex of viruses; characterised by
extensive rash and high fever.
Include variola virus, etiologic agent of smallpox. 300-500
million deaths in 20th century, in 1967 alone, 15 million
contracted with the disease, and 2 million deaths. 1980 –
apparently eradicated. But some strains are still kept for
research purpose. Smallpox as biological weapon.
Complex structure, brick and oral-shaped. The extracellular
forms contain 2 membranes (EEV - extracellular enveloped
virions), intracellular particles only have an inner membrane
(IMV - intracellular mature virions).
Ds DNA, linear, 130-375 kbp. Virions contain >100
polypeptides.
Adenovirus vs. Host
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Poxviruses are unique – replication in cytoplasm,
suggest that poxviruses encode viral genes for
many cellular proteins.
Viral-encoded host modifier genes:
1. polypeptide of early genes of vaccinia V is closely
related to epidermal GF and to transform GF-α.
2. genes resemble mammalian genes for proteins
that would inhibit host defense mechanism eg. TNFReceptor, IFN-γ Receptor, IL-1-Receptor and
complement binding protein. All this disrupts
complement and ck network hence enhanced virus
replication.
Poxvirus Infections in Humans
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Control by variolation – inoculating someone
with mild forms of smallpox to produce
immunity.
Smallpox vaccination used vaccinia virus.
Unlike variola, vaccinia has a broad host
range including mice. Some strains cause
serious disease in animals.
Pathogenesis
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Variola virus entered the mucous membranes
of the upper resp T. Followed by: 1. primary
multiplication in lymphoid tissue; 2. transient
viraemia; 3. secondary multiplication; 4.
secondary and intense viraemia; 5. clinical
disease.
6-9 days after infection, lesions in the mouth
ulcerated and discharge virus (early).
Later, pustules broke down and discharged
virus into the environment.
Smallpox
Pathogenesis
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All the layers of the skin were involved, and
there was actual necrosis of the dermis.
Hence, scarring after variola infections.
Vaccinia virus causes localised pustular
lesions only at the site of inoculation.
Incubation period is 10-14 days. Pustules
formed crusts that fell off after 2 weeks.
Replication I
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Receptors are not known, but probably >1 on different cell types.
For Vaccinia, probably the EGF receptor (epidermal growth
factor).
Penetration is complex and may also involve >1 mechanism.
Uncoating occurs in two stages, removal of the outer membrane
as the particle enters the cell and in the cytoplasm, the particle
(minus its outer membrane) is further uncoated and the core
passes into the cytoplasm.
Gene expression is carried out (exclusively?) by viral enzymes
associated with the core and is divided into 2 phases:
 Early genes: ~50% genome, expressed before genome
replication
 Late genes: expressed after genome replication; late promoters
are dependent on DNA replication for activity.
Replication II
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Poxvirus gene expression has been studied in detail because of the
interest in the use of Vaccinia virus as a vector for expression of
heterologous genes.
Genome replication is believed to involve self-priming, leading to the
formation of high m.w. concatemers (isolated from infected cells) which
are subsequently cleaved and repaired to make virus genomes. The
many virus-encoded enzymes involved in replication (e.g. thymidine
kinase - tk) offer potential targets for chemotheraputic agents.
Assembly occurs in the cytoskeleton; the events involved in putting
together such a complex particle are not understood, but probably
involve interactions with the cytoskeleton (e.g. actin-binding proteins).
Inclusions are formed in the cytoplasm which mature into virus
particles. Actin 'comet tails' form which shoot IEV through the cytoplasm
to the cell surface, and possibly into adjacent cells - these have been
timed moving at 3µm/min. This may provide an alternative mechanism
for cell to cell spread (c.f. EEV).
Overall, replication of this large, complex virus is rather quick ~12h.
Laboratory diagnosis
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1) Specimen lesions poxviruses are stable
and remain viable for weeks without
refrigeration.
-Direct examination – rapid (1hr)
identification, large enough to be seen by
light microscopy.
2) Differentiate among poxviruses by
inoculation of vesicular fluid onto
chorioallantoic membrane of chick embryos.
Look for lesions on membrane – variola
pocks are much smaller
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Pock on chick chorion-allantois membrane induced
by India-3a strain. 1- epithelium, 2 - connective
tissue, 3 - blood vessel. Arrows show congested
blood vessel near epithelium. Thick arrow shows
border of the pock.
Laboratory diagnosis
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- look for growth in cell cultures. Only
orthopoxviruses grow well in human and nonhuman cells.
3) PCR test specific virus.
Differential diagnosis is important: smallpox
may be confused with drug rashes, varicella.
Rx: vaccinia immunoglobulin vaccinia virus –
limited to CDC; methisazone –
chemotherapeutic agent and prophylaxis.
Transmission
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Highly contagious, very stable in extracellular
environ; by respiratory spread; dried virus in
crusts from skin lesions could survive in
clothes and other materials.
Highly contagious once fever had begun ~1st
week rash. Resp droplets infections come
earlier than skin lesions.
Smallpox amenable to total eradication
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No known non-human reservoir
1 stable serotype
Had made 1 effective vaccine
Subclinical infection cases did not occur
No chronic asymptomatic
Severe patients get quick attention from medical
authorities, hence interrupt cycle.
Vaccination: vaccinia virus infected on calves 
vaccine prepared from vesicular lesions in skin 
vaccine contains 40% glycerol, 0.4% phenol.
Reactions
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3-4d: papule increases in size 
5-6d: vesiculation
9d : maximum size  pustular. Dessication
up to 2 wks, whereby depressed pink scar
turns white.
Person is fully protected after vesicular or
pustular surrounding a central lesion (scab or
ulcer)~7d. If not, vaccination again.
Other Poxviruses
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Monkeypox – also orthopoxvirus, rare zoonosis;
acquired by direct contact with wild animals in
Africa.
Pronounced lymphadenopathy-feature not seen in
smallpox or chickenpox
Several outbreaks: 1996-97 Zaire, 2003 US.
Cowpox – also orthopoxvirus, less sever and
milder lesions confined to teats and udders.
Direct milking with hands
Buffalopox
Orf virus – occupational disease, mainly seen in
sheep and goats.
Tumours associated with poxviruses
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Molluscum contagiosum:
Is a benign epidermal tumour
Had not transmitted to animals
Has not been grown in tissue cultures
Studied in human lesion by EM, Molluscipoxvirus
causes small pink wart-like tumous, rarely on palms,
soles or mucous membranes.
Tanapox and Yaba Monkey Tumour:
Tanapox is a fairly common skin infection in Africa,
Kenya and Democratic Republic of Congo.
Yatapoxvirus – morphologically similar to orthopox