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Smallpox
Jay Mung
Celia Rivera
Christopher Rinn
Smallpox 04.01.03 :: W4158 Microbiology
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Introduction
• Smallpox is a highly contagious human disease caused by the virus
variola. Named from the Latin word varius meaning “spotted”.
• There are two strains:
• Variola major, which has severe symptoms and a very high
mortality (20-40%).
– There are four types of variola major smallpox:
» Ordinary (the most frequent type, accounting for 90%
or more of cases);
» Modified (mild and occurring in previously
vaccinated persons);
» Flat; And hemorrhagic (both rare and very severe).
• Variola minor, which had less severe symptoms and lower
mortality (about 1%).
• It was named “smallpox” to differentiate it from the bubonic plague
a.K.A. “Great pox”.
• It wasn’t until the development of a smallpox vaccine, made from the
closely-related vaccinia virus, that eradication was achieved.
Smallpox 04.01.03 :: W4158 Microbiology
History of Smallpox
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• It is thought that smallpox
originated in Africa or china
more than 3000 years ago.
• From here it spread
throughout the middle east.
• There is evidence of a
smallpox outbreak in Egypt
around 1157BC.
• The Egyptian pharaoh
Ramses V’s mummy
contains pock marks.
• Linked to trade routes.
Smallpox 04.01.03 :: W4158 Microbiology
History of Smallpox
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• Smallpox reached Europe in
710 A.D.
• Transferred to America in
1520 by Hernando Cortez’s
assault on Techotitlan
(Mexico City). 3.5 million
Aztecs died in the following
2 years.
• British colonists gave
infected blankets to Native
Americans during the French
and Indian War. More than
50% of some tribes died.
• 18th century European cities
reached epidemic
proportions.
Smallpox 04.01.03 :: W4158 Microbiology
History of Smallpox
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• Early eradication dates back to
1500 BC. The Turks induced a
mild form of smallpox by
pricking the skin of patients,
inserting exudate from the
postulates of the disease.
Known as “variolation”
• During the 10 century in the
Szechuan region of China,
Taoist alchemists exposed
themselves to smallpox by
inhaling the scabs of those with
a mild infection. This was
known as “insufflation”.
Smallpox 04.01.03 :: W4158 Microbiology
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History of Smallpox
• Because of the devastation caused by the
disease, healers and physicians focused
attention on it and its prevention.
• The first step in prevention and treatment
is understanding the nature of the disease.
• Around 910 AD the Islamic physician
Rhazes differentiated between smallpox
and measles, which may seem similar at
the onset.
• Rhazes believed that once the disease
passed, the individual would be immune
to further diseases as the condition would
not reoccur.
Smallpox 04.01.03 :: W4158 Microbiology
History of Smallpox
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• General George Washington used
forced variolation to stop the spread
of smallpox in his troops.
• Washington was immune because he
survived smallpox in Barbados in
1751.
• However, a smallpox epidemic had
reduced his healthy troop strength to
half while the British troops, who
had been variolated, were already
immune to the spreading contagion.
• Washington proclaimed smallpox to
be his "most dangerous enemy";
and, by 1777 he had all his soldiers
variolated before beginning new
military operations.
Smallpox 04.01.03 :: W4158 Microbiology
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History of Smallpox
• During a smallpox epidemic in Gloucestershire,
England in 1788 Edward Jenner made an
interesting observation.
• Milkmaids who worked with cattle and
contracted the milder cowpox virus never
contracted smallpox.
• In 1796 Jenner inoculated 8 year old James
Phipps with fluid from the lesion of milkmaid
Sarah Nelmes.
• James Phipps complained for several days of
discomfort around his armpit; On the ninth day
he felt chills and a headache. He soon fully
recovered.
• 6 weeks later, Phipps proved to be protected
from the virus when Jenner exposed him to it.
Smallpox 04.01.03 :: W4158 Microbiology
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Smallpox 04.01.03 :: W4158 Microbiology
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Vaccine Discovered
• To avoid confusion, inoculation with smallpox (as practised in
Turkey) was termed "variolation" from "variola", the clinical term
for smallpox.
• Inoculation with cowpox (as practiced by Jenner) was termed
"vaccination" from "vaccinia".
• Initially, material for vaccination was obtained from genuine
cowpox, by extracting liquid from the sores of someone with
cowpox and inoculating this into a patient (as Jenner had done).
• Arm-to-arm vaccination, with pustules taken from someone
vaccinated a week earlier and inoculated into others (as also
practised by Jenner), followed and became the form of
immunisation in Britain.
• By 1803 worldwide cowpox vaccination began .
• Variolation of a healthy person was banned in Britain in 1840,
under the terms of the Vaccination Act, which also meant anyone
who gave another smallpox was liable to a month`s imprisonment.
Smallpox 04.01.03 :: W4158 Microbiology
Smallpox Eradication
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Throughout the nineteenth century smallpox vaccination was inconsistent at
best.
– In 1869, Chicago`s Board of Health made vaccination a requirement for
children - but in 1872, there were 2,382 cases of smallpox with 655 deaths
in the city.
– In 1902, the Chicago Health Department developed a "vaccination creed",
which promoted the supposed "benefits" of vaccination, stating, `true
vaccination repeated until it "no longer takes" always prevents smallpox.
Nothing else does`. This "creed" was taken up by the US military.
Nationally, there were 21,064 cases with 894 deaths in 1900. Annual averages
for 1900-04 were 48,164 cases with 1,528 deaths.
In 1921, the number of cases had climbed to 102,791.
The last documented case of smallpox in the US was in 1949.
Sixty percent of the world’s population was still threatened by this disease
when, in 1967, the world health organization (WHO) launched an intensive plan
to eradicate smallpox.
It took a decade of intense international vaccination efforts, but the last natural
case of smallpox occurred in Somalia in 1977.
A year later, a laboratory worker in the United Kingdom became infected while
working with the vaccine.
Smallpox 04.01.03 :: W4158 Microbiology
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Smallpox Eradicated
• No other cases of smallpox have since been seen.
• In May 1980, the World Health Organisation declared the global
eradication of smallpox and recommended that all countries
cease vaccination.
• In 1986, the WHO first suggested all stocks of smallpox viruses
should be destroyed.
• For years, the US and Russia proposed - to sequence the virus and the smallpox virus was given a "reprieve" until December
1993.
• In May 1993, the WHO`s committee, which represented 156
nations, could not justify keeping the virus, and the committee`s
recommendation to destroy the virus was endorsed by several
groups including the American Society for Microbiology and
the Council.
• A small minority of American scientists expressed reservations.
The deadline was postponed.
Smallpox 04.01.03 :: W4158 Microbiology
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Destruction of Variola Virus stocks?
• In September 1994, the ten-member WHO Ad Hoc Committee on
Orthopox Virus Infections met, and unanimously agreed the potential cost
to humanity from biological warfare or inadvertent outbreaks of the
disease outweighed its supposed "benefits" to researchers.
• To the WHO Committee, the most compelling argument to get rid of the
smallpox strains was the potential for the virus to be used by terrorists for
biological warfare.
• Again, the decision to destroy the virus was tabled.
• Rescheduling of the “deadline” took place in 1995 and again in 1999.
• After resistance from the US and Russia, the deadline was extended yet
again - this time to be "not later than 2002".
• Finally, in January 2002, the United Nation`s health agency`s 32-member
executive board met and endorsed a recommendation by the WHO`s
Director General, Gro Harlem Brundtland, to drop the 2002 deadline and
to set no new target for the destruction of the stocks of smallpox virus.
• The world awaits the consequences........
Smallpox 04.01.03 :: W4158 Microbiology
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Victim
Smallpox was a fearful and devastating disease. If it didn't kill its
victims, it scarred and disfigured them for life.
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Molecular Bio
•Poxvirion is the largest of all virions.
•Poxviruses replicate in cytoplasm rather than in nucleus
•require various enzymes not found in DNA viruses
•viral DNA-dependent RNA polymerase
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Classification via RE
Causative agents traditionally classified by signs and symptoms
Situation changed by discovery of Muller et al (1978)
Smallpox 04.01.03 :: W4158 Microbiology
dendrograms
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Features of Viral DNA
•Single linear molecule
•Largest of all viral genomes
•Most DNA in genome unique
•Virulence genes near termini/more variable
•Genes for replication in central region of genome, highly conserved
•Sister strands form single stranded circular molecule when denatured
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Reactivation
Non-genetic reactivation of “killed virus” Berry & Dedrick (1936)
“killed virus” + non-virulent live-virus  infection.
Not to be confused with bacterial transformation:
Smallpox 04.01.03 :: W4158 Microbiology
Illustration of virion
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Naked vs. enveloped
Virions naturally released from cell are enclosed in envelope
Virions released by cellular disruption are without envelope
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Viral Replication I
Adsorption and penetration of host cells
•Enveloped virions
•AKA Externally enveloped (EEV)
•adsorbed more efficiently via endocytosis
•Most virulent / easily spread
•Naked virions
•AKA intracellular mature virus (IMV)
•outer membrane fuses with plasma membrane at cell surface
•Naked virions used for vaccines
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Viral Replication II
•nucleoprotein core complex passes out of the virion
•transcription system is produced
•Virion formation
•crescent/cupules w/ bilayer membrane and spicules form
•more proteins produced for mature virion
•DNA replication outside of nucleus
Smallpox 04.01.03 :: W4158 Microbiology
Viral Replication III
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•Most mature progeny virions remain cell-associated
•released when cell undergoes necrosis
•released from intact cells via budding
•infects contiguous cells.
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Changes on Cellular Level I
•1st –2nd hours noticeable toxic changes occur in cells
•4th hour viral factories appear in cytoplasm,
•16th hour aggregation of cells into hyperplastic foci / rupture
Smallpox 04.01.03 :: W4158 Microbiology
Changes on Cellular Level II
ectodermal cells of chick embryo
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skin of patient with hemorrhagic smallpox
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Proteins for Replication I
RAP94
•RNA-polymerase associated protein
•confers specificity for early promoters
(shortens viral genome RNA polymerase )
•poly-A polymerase
•Capping enzyme
•topoisomerase
•to early transcription factor
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Proteins for Replication II
•encoded by orthopoxviruses:
•DNA polymerase
•nucleoside triphosphatase
•DNA glycosylase
•thymidine kinase
•thymidylate kinase
•ribonucleotide reductase
•dUTPase
•Ribonuceoside reductase
•small catalytic and a large regulatory unit
•converts ribonucleoside diphosphates (NDPs) to
deoxyribonucleoside diphosphates (dNDPs).
•70-80% structural homology with their eukaryotic counterparts
Smallpox 04.01.03 :: W4158 Microbiology
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Proteins for Entering Cell
A33R, A34R, and A36R.
•Mediates formation of the actin-composed microvilli
•actin tails responsible for CEV’s efficiency in spread
•allow CEV to be targeted to neighboring cells
•CEVs mediate efficient cell-cell spread between neighboring cells
(whereas EEVs mediate long range transmission)
haemagglutinin and envelope glycoprotiens
•Antigens promote cell fusion  cell-to-cell spread of virions.
Smallpox 04.01.03 :: W4158 Microbiology
Proteins for Evading Host Immune Response I
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•Variola produces defense proteins: protection from host immune system
•Virokines
•resemble cytokines, secreted immune response regulatory proteins
• secreted by infected cells
•Viroreceptors
•cellular receptors altered by virus
•lose membrane anchoring sequences
•bind ligands to form free-floating complexes in extracellular fluid
•intracellular proteins
•interfere with host’s normal signaling and enzymatic pathways.
Smallpox 04.01.03 :: W4158 Microbiology
Proteins for Evading Host Immune Response II
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SmallPox Inhibitor of Complement Enzymes – (SPICE)
•complement system destroys pathogens
•viruses
•virus-infected cells
•SPICE prevents assembly of C3/C5  mediate immune response, by
inactivating complement components
•creates environment around variola-infected cells protected from
complement-attack  viral replication factories.
•located in the terminal region of the variola genome.
Smallpox 04.01.03 :: W4158 Microbiology
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Why is smallpox so lethal in Humans?
•DNA replication begins one to two hours after infection
•results in 10,000 copies of genome per cell
•Half are packed into virions for further rounds of infection upon
cell lysis.
•Variola virus
•uniquely suited to evading the human immune response
•Whereas, Vaccinia
•causes no disease in non-immunocompromised individuals
•structural similarities cause it to be a good vaccine
•produce antibodies specific to vaccinia and variola virulence
factors.
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Why is smallpox so dangerous?
• Smallpox is a Category A bioterrorist threat.
• It is extremely infectious:
– The infective dose is only a few virions, which can be
transmitted from the mouth of an infected person that is simply
speaking or coughing.
• Virus causes a painful and disfiguring illness.
• Smallpox has a very high mortality rate of around 30%.
• There is no specific treatment available.
Smallpox 04.01.03 :: W4158 Microbiology
Pathogenesis and Symptoms I
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Initial Symptoms/ Prodrome Phase (Duration: 2 to 4 days)
• Infection occurs after the implantation of the virus onto the
oropharyngeal or respiratory mucosa. After the virus begins to
multiply in the local lymph nodes an asymptomatic viremia develops
on the 3rd or 4th day. This is followed by multiplication of the virus in
the bone marrow, spleen and lymph nodes.
• The first symptoms include fever, head aches, malaise, and sometimes
vomiting. High fevers occur in the range of 101 to 104 degrees
Fahrenheit. At this time people are usually too sick to carry on their
normal activities.
Early Rash (Duration: ~4 days)
• When the second viremia begins, the virus which was contained in
leukocytes, localizes in small blood vessels of the dermis and beneath
the oral and pharyngeal mucosa. This causes subsequent infection of
the adjacent cells.
• A rash first appears in the mouth and pharynx. These lesions ulcerate
quickly releasing large amounts of the virus into the saliva. Patients
are most contagious during the first week because the virus titers in the
saliva are high.
Smallpox 04.01.03 :: W4158 Microbiology
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Pathogenesis and Symptoms II
• Soon after the sores arise in the mouth, the rash appears on this skin,
beginning with the face and spreading to the arms, legs, hands and
feet. The rash usually spreads to all parts of the body within 24 hours.
As the rash appears, the fever usually subsides.
• By the 4th day of the rash, the bumps are raised and filled with a thick,
opaque fluid with a depression in the middle. Fever may begin to rise
again until scabs form.
Pustular Rash (Duration ~5 days)
• The bumps become pustules, which are characteristically round, tense
and deeply embedded in the dermis.
Pustules and Scabs (Duration ~5 days)
• By the end of the second week after the rash appears, most of the
pustules have scabbed over.
Resolving Scabs (Duration ~6 days)
• The scabs fall off and characteristic pitted scarring gradually develops.
The scars are most apparent on the face as a result of the destruction of
sebaceous glands followed by shrinking of granulation tissue and
fibrosis.
• Individual is contagious to others until all of the scabs have fallen off.
Smallpox 04.01.03 :: W4158 Microbiology
Pathogenesis and Symptoms III
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Quote from P. Tiessier, Academy of Medicine in France, January
23, 1923.
“As the result of intensive inflammatory swelling, the festinum
becomes hideous, with bulging face and bright red cheeks,
swollen and closed eyelids, turned-out lips, with fungal
infection (where the edges of the lips become swollen and bleed
easily) and bleeding. From the half open mouth a dribble of
saliva constantly and abundantly runs and is fetid; with blocked
nasal passages a pussy liquid oozes (purulent and bloody) and
equally fetid; the tongue is swollen and ulcerous, the pharynx is
covered with ulcers, which means that every effort to swallow
gives rise to great pain. The poor patient whose body is covered
in pus and gangrenous lesions in places, from which a nauseous
and repugnant odour emanates, still suffers from an agonizing
dyspepsia, from an unquenchable thirst, and from complete
insomnia.”
Smallpox 04.01.03 :: W4158 Microbiology
Pathogenesis and Symptoms IV
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At least 90% of all smallpox cases are clinically characteristic and easily
diagnosed. There are two forms of smallpox which are difficult to
recognize: Hemorrhagic and Malignant.
Hemorrhagic
• Cases are uniformly fatal and occur among all ages and sexes, but
pregnant women appear to be more susceptible. The incubation period
is shorter and is characterized by severely prostrating prodromal
illness: high fever, head, back, and abdominal pain. Erythema
develops, followed by hemorrhages into the skin and mucous
membranes. Death usually occurs by the 5th or 6th day after onset of
the rash.
Malignant
• The sudden onset and prostrating prodromal symptoms are similar to
the Hemorrhagic cases. Lesions develop slowly, but never progress to
the pustular stage. Instead they remain soft, flat and velvety. This
form is usually fatal, but if the patient does survive, the lesions usually
disappear without forming scabs, or in more severe cases, large
amounts of epidermis might peel off.
Smallpox 04.01.03 :: W4158 Microbiology
Symptoms Images
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Smallpox 04.01.03 :: W4158 Microbiology
Symptoms Images II
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Smallpox 04.01.03 :: W4158 Microbiology
Symptoms Images III
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Smallpox 04.01.03 :: W4158 Microbiology
Symptoms Images IV
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Transmission
• Generally, direct and relatively prolonged face-to-face contact is
required to spread smallpox from one person to another.
• Smallpox can also be spread through direct contact with infected
body fluids or contaminated objects (i.e. clothing and bedding).
• Patients spread smallpox, mainly to family members and
friends.
Smallpox 04.01.03 :: W4158 Microbiology