Transcript Poxviruses

Poxviridae
Molecular Virology
Introduction
• Poxciruses have been known - the
characteristic "pocks" produced by
variola virus (Smallpox) .
• The Pharaoh Ramses V (left) died of
smallpox in 1157 B.C
• The disease reached Europe in 710 A.D.
and was transferred to America.
• In the cities of 18th century Europe,
smallpox reached plague proportions and
was a feared scourge - highly infectious
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Introduction
• In 1967, WHO initiated a program to
eradicate small pox from the third world
nations .
• Smallpox has now been eradicated - the
last naturally occurring outbreak of
smallpox was in Somalia on 26th
October 1977.
• The last known person to have natural
smallpox of any kind lived .
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Taxonomy
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Taxonomy
Family Poxviridae
Subfamily
Chordopoxvirinae
Entomopoxvirinae
insect poxvirus
Genus
Members
Avipoxvirus
Fowlpox virus
Capripoxvirus
Sheeppox virus
Leporipoxvirus
Myxoma virus
Molluscipoxvirus
Molluscum contagiosum virus
Orthopoxvirus
Vaccinia virus；Variola virus；
Monkeypox virus ；Cowpox virus
Parapoxvirus
Orf virus (contagious pustular dermatitis virus)
Pseudocowpox virus (milker’s nodule virus)
Suipoxvirus
Swinepox virus
Yatapoxvirus
Yaba monkey tumor virus
Entomopoxvirus A
Melolontha melolontha entomopoxvirus
Entomopoxvirus B
Amsacta moorei entomopoxvirus
Entomopoxvirus C
Chironomus luridus entomopoxvirus
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Poxviruses Pathogenic for Humans
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Morphology
Poxviruses are the largest regular-shaped viruses.
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Oval or "brick-shaped" particles 200-400nm long - can be visualized by the best
light microscopes .
Morphology
• The external surface is ridged in parallel rows,
sometimes arranged helically.
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Morphology
• The particles are extremely complex, containing many
proteins (more than 100) and detailed structure is not
known.
• Biconcave (dumbbell-shaped), with two "lateral bodies"
(function unknown).
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Morphology
Properties
The outer surface is composed of lipid and protein which surrounds the core
The core is composed of a tightly compressed nucleoprotein.
They have a double-stranded DNA (130–375 kb) genome contained in a helical
nucleocapsid structure seen as a dumbbell shape.
Outside of which are several virus-encoded proteins in structures referred to as
'tubules'( mesh-like coat. )
This particle is referred to as an intracellular infectious virion.
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Morphology
• IMV - intracellular mature virions intracellular
particles only have an inner membrane
• EEV - extracellular enveloped virions
The extracellular forms contain 2 membranes
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Genome
• Linear, d/s DNA of 130-300kbp
(cross-linked ends).
• No know origins
• Replication in cell cytoplasma
• Encodes all transcription and
replication enzymes needed for
viral gemone
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Harrison S C et al. PNAS 2004;101:11178-11192
Genome
• Ends of genome consist :
1. a terminal hairpin loop (no free ends)
2. with several tandem repeat sequences
3. this arrangement is found at the ends of chromosomes
from a number of different organisms
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Harrison S C et al. PNAS 2004;101:11178-11192
Genome
• The ends of the genome form direct repeats called
inverted terminal repeats (ITRs).
• Most of the essential genes are located in the central
part of the genome
• There are ~250 genes in the genome.
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Genome
• Strand displacement DNA synthesis. No RNA primer
• Complete mRNA synthesis system
(RNA polymerase/capping enzyme/polyadenylation system)
• Encode functions proteins
DNA synthesis/nucleotide scavenging/immune escape mechanisms
• Encode immune modulation factors
interferon/complement/inflammatory responses
• Encode many proteins
initially stimulate host cell growth
lead to cell lysis
viral spread
(Epidermal growth factor homologue)
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Harrison S C et al. PNAS 2004;101:11178-11192
Replication
1.Entry
Intracellular mature
virion (IMV) particles
bind to unknown
receptor(s) and fuse with
the cell membrane.
Extracellular
enveloped virion (EEV)
particles bind to
unknown receptor(s) and
are endocytosed into the
cell.
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Replication
2. Initial Uncoating
The viral core particle
(CORE) is released into
the cytoplasm
It containing:
1.viral genome,
2.viral DNA-dependent
RNA polymerase
3. other enzymes
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Replication
3. Early Transcription
Early genes are
immediately transcribed.
It including
immunomodulatory
proteins, enzymes, and
replication and
transcription factors
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Replication
4.Translocation
The viral core particle
translocates to the
outside of the cell
nucleus.
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Replication
5. Secondary Uncoating
The viral nucleoprotein (NP)
complex, which contains
the viral genome, is
released.
The viral genome is
replicated 、transcription
and translation of
intermediate genes .
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Replication
6. Late Transcription
The viral late genes
(coding for structural
proteins, enzymes, and
transcription factors) are
transcribed and translated.
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Replication
7. Assembly
Concatemeric
intermediates are
resolved into linear
double-stranded DNA
And packaged with late
viral proteins into
immature virions (IV).
IVs mature into IMVs via
an undescribed
mechanism which may
include processing of the
IV through the Golgi
apparatus.
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Replication
8. Release
The IMVs are transported to
the periphery of the cell
where they are released in
one of three ways.
1. IMVs released via cell lysis
remain IMVs
2. IMVs can bud through to
the cell surface, picking up
a viral envelope from the
cell plasma membrane.
3. IMV can bud through the
plasma membrane picking
up an envelope and
becoming an EEV.
cell-associated enveloped virus (CEV)
intracellular enveloped virus (IEV).
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intracellular mature virus (IMV)
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Variola (smallpox)
Clinical Features of Human Poxviruses
• Smallpox is caused by two strains of the
same virus:
– Variola major - more common, causes a
severe form of the disease
• Mortality rate of 15 – 30%
– Variola minor - causes a mild form of disease
• 1 - 2% mortality rate
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Transmission
• Person  Person
– Primarily droplet, or aerosol
– smallpox in droplet nuclei can live between a
few hours and a few days in the environment
– No animal reservoir or vector
• Very contagious
– Persons are very sick before contagious
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Pathogenesis of Variola
Virus entry & multiply in
respiratory epithelium
Fever, Headache, Backache, SICK!
NOT
Infectious Blood circulation
(12-14 d ) primary viremia
NOT
Infectious
(2-4 d)
Liver, spleen
2nd viremia
Capillary vessel
VERY
Infectious
Dermis
macules -> papules -> vesicles -> pustule-> crust
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Multiplies in mouth and Dermis (deep layer of skin)  Rash
Case Definition of Smallpox
• Illness with acute
onset of fever greater
than or equal to 101oF.
• Followed by rash of
firm, deep seeded
pustules.
• Centrifugal pattern of
lesions compared to
chickenpox.
Rash distribution of smallpox vs. chickenpox.
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Adapted from World Health Organization, Diagnosis of Smallpox Slide Set,
http://www.who.int/emc/diseases/smallpox/slideset.
The stages of lesions in poxvirus infections
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skin rash of Variola
papules
vesicles
pustules
crusts
(Emond RTD., A colour atlas of infectious disease 1974)
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Smallpox Lesions:
Start on Face, Arms, Mouth
Palms and Soles
Deeply Embedded into Skin
Similar Stage of Development
Occur in Very Sick Persons
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Laboratory Diagnosis
• Biopsy
Intracytoplasmic inclusion bodies (varying size and duration)
• Direct examination
Electron microscopy
Giemsa staining of infected cell scrape
• Virus isolation
Embryonated egg: chorioallantoic membrane (Pock lesions)
Cell culture : primary monkey kidney cells
• Serology
hemagglutination inhibition test (only the Orthopoxvirus)
neutralization test
ELISA
IFA
• PCR-RFLP analysis / Real-Time PCR / Sequencing
The only way to accurately and rapid distinguish between Variola,
monkeypox and vaccinia infections.
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Poxvirus infected cells
Cowpox in primary monkey kidney cells, cytoplasmic
inclusions with halo ( Guaneiri body)
(Versteeg J., A Color Atlas of Virology,1985)
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Pocks on chorioallantoicnic
membrane
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Other Poxvirus Infections
– Poxvirus infections also occur in animals
– Transmission to humans requires contact with
infected animals
– Infections of humans are usually mild
– Can result in pox and scars but little other
damage
– Edward Jenner used cowpox to immunize
individuals against smallpox
Edward Jenner (1749-1823)
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Edward Jenner (1749-1823)
• Physician in England, credited with cowpox vaccination
• Experimented with the folklore that milkmaids who
contracted cowpox “did not take the smallpox.”
• 1796, Jenner variolated the 8-year-old son of a local farmer
with fluid from the cowpox pustules from the hand of a local
dairymaid.
– A few months later, the boy was injected with smallpox
and failed to develop the disease.
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Smallpox Eradication
• Why was smallpox (variola) a good candidate
for eradication?
–
–
–
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Variola has a narrow host range.
There are no carriers.
There are no animal reservoirs.
A highly effective an inexpensive freeze-dried
vaccine was available.
– Surveillance of the disease was easy (centrifugal
rash).
– The WHO created a program to eradicate it.
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Bioterrorism or Re-emergence?
• Although smallpox has been eradicated, there are
concerns about the potential use of variola virus as a
weapon of terror.
• Use of variola as a biological weapon has a long history.
• Variola as germ warfare against Native American
Indians, French and Indian Wars (1754 - 763).
• Consequently, destruction of the last (official) remaining
smallpox stocks held in Russia and USA has now been
postponed indefinitely.
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Bioterrorism or Re-emergence?
• The possibility of an orthopoxvirus such as variola,
monkeypox, camelpox or taterapox virus emerging or
re-emerging as a threat to human health increases .
• Finally, it is unclear whether all, only a few, or just one of
the differences between the genomes of viruses such as
camelpox and smallpox.
• Genetic modification of camelpox to delete genes that
are present in camelpox but absent in smallpox might be
highly dangerous.
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Application
Expression Vectors
• Use of homologous recombination in infected
cells to introduce foreign DNA coupled to VV
promoter into virus genome.
• Indicator systems such as beta-galactosidase to
give recombinant plaques a blue color.
• Problems for human vaccines:
The proportion of the human population which has
already been vaccinated - lifelong protection may result
in poor response to recombinant vaccines (?).
Dangerous in immunocompromised hosts.
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Application
Expression Vectors
• The possible solution to this may be to use
Avipoxvirus vectors, as 'suicide vectors'
- undergo abortive replication in
mammalian cells:
• can express high levels of foreign proteins
• no danger of pathogenesis
• no natural immunity in humans
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Expression Vectors
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敬請指教 歡迎討論
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Monkeypox
• Monkeypox occurs mainly in the rain forests of central and west
Africa. The disease was first discovered in laboratory monkeys in
1958, but the virus also occurs in a number of African rodent
species. In 1970, monkeypox was reported in humans for the first
time.
• In humans, monkeypox is similar to smallpox, although it is often
milder. Unlike smallpox, monkeypox causes lymph nodes to swell
(lymphadenopathy). The incubation period for monkeypox is about
12 days (range 7 to 17 days). The illness begins with fever,
headache, muscle aches, backache, swollen lymph nodes, a
general feeling of discomfort, and exhaustion. Within 1 to 3 days
(sometimes longer) after the appearance of fever, the patient
develops a papular rash (i.e., raised bumps), often first on the face
but sometimes initially on other parts of the body. The lesions
usually develop through several stages before crusting and falling
off. The illness typically lasts for 2 to 4 weeks. Human monkeypox is
believed to have a fatality rate of 1% to 10%.
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Monkeypox
• In June 2003 there was an outbreak of
monkeypox in the USA resulting from
importation of Gambian giant rats into the
U.S. as pets. Import restrictions were
subsequenbtly imposed on six specific
genera of African rodents to try to prevent
a repeat of this outbreak.
• Is human monkeypox infection emergent
disease or has it always been there but
masked by smallpox? And now that we
have eradicated smallpox, will
monkeypox evolve to take it's place?
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