Smallpox

1
Smallpox

• Charlotte McKinley
• Jessica Midence
• Izabella Messina

2

Smallpox

• Smallpox is a serious, contagious and sometimes
  fatal disease.
• There is no specific treatment for smallpox, and
  the only prevention is vaccination.
• The name smallpox is derived from the latin word
  spotted and refers to the raised bumps that
  appear on the face and body of an infected
  person.
• Smallpox is in the Orthopoxvirus genus of viruses.

3
Origin of Smallpox

• The name Variola was first used in the 6th
  century. Derived from the Latin word varius
  (spotted) or varus (pimple).
• Anglo-Saxons in the 10th century used the word
  poc or pocca (bag or pouch) to describe an
  exanthemous disease, possibly smallpox.
• In the 15th century, the English used the prefix
  small to distinguish variola the smallpox from
  syphilis, the great pox.

4
First Case of Smallpox

• There is no animal reservoir, and no human
  carriers.
• First certain evidence comes from the mummified
  remains of Ramses. (1157 B.C.)
• Written descriptions did not appear until the
  10th century in Southwestern Asia.

5
Smallpox Travel

• Smallpox was likely carried from Egyptian traders
  to India during the millennium B.C. where it
  became established as an endemic infection.
• Epidemics of the disease can be found in the
  bible, and in ancient Greek and Roman literature.
• From Asia and Africa smallpox spread with
  increasing frequency into less populous areas,
  and then into Europe.

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Smallpox in the Ancient World

• Peloponnesian Wars (430 B.C.)
• Thucydides recorded smallpox symptoms.
• A person aboard a ship from North Africa came to
  Athens infected with smallpox.
• Described as violent heats, unnatural, putrid
  odors, stomach distress, and the body covered
  with small pustules and ulcers.
• Also noted that those who survived became immune.

7
Smallpox in the New World

• In the early 16th century smallpox began to
  imported into the western hemisphere.
• The Spanish inadvertently owe success in
  conquering the Aztec and Incas in Mexico to
  smallpox.
• Smallpox arrived in North America via Canada, and
  Mexico.

8
Smallpox as Biological Warfare

• Lord Jeffrey Amherst, Commanding General of
  British Forces in North America during the French
  and Indian War. (1754-1763)
• Used blankets (smallpox blankets) coated with
  smallpox dust as germ warfare to wipe out the
  Native American population.

9
History of Variolation and Vaccine

• Known that survivors became immune to the
  disease.
• As a result, physicians intentionally infected
  healthy persons with smallpox organisms.
• Variolation is the act of taking samples (pus
  from pustules or ground scabs) from patients
  whose disease had been benign, and introducing it
  into others through the nose or skin.

10
Survival Rates with Variolation

• Two to Three percent of variolated persons died
  of smallpox, became the source of a new epidemic,
  or developed other illnesses from the lymph of
  the donor such as tuberculosis or syphilis.
• The case fatality rates were still ten times
  lower in those that were variolated compared to
  those with naturally occurring smallpox.
• Side effects of variolation were the appearance
  of smallpox itself, but it would disappear after
  a week or so.

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Variolation in the New World

• Reached the New World in 1721.
• Used to stop the epidemic in Boston.
• In 1766 American Soldiers under George Washington
  were unable to take Quebec from the English
  because of smallpox.
• Smallpox was apparently one of the main causes of
  the preservation of Canada in the British Empire.
• In 1777 Washington had all of his soldiers
  variolated beginning with new military operations.

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Cows, Milkmaids, and the Pox

• In rural areas of Europe it was known the
  milkmaids became immune to smallpox after
  developing smallpox
• 1774, farmer Benjamin Jesty was the first to
  vaccinate his wife and kids with material taken
  from the utters of cows.
• 1791, school teacher Peter Plett vaccinated his
  students with material from the utters of cows.

13
Edward Jenner
14
Edward Jenner

• Studied to become a physician in England.
• In May of 1776 dairymaid Sarah Nelmes consulted
  Jenner about a rash on her hand.
• He took this opportunity to test the protective
  properties of cowpox against smallpox.
• Determined that cowpox can be passed from person
  to person as well as from cow to person.
• The next step was to see if the cowpox would
  protect the patient from smallpox.

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Edward Jenner

• Jenner published the data collected in 1798.
• However, Jenners technique did not catch on as
  anticipated.
• After more and more people were becoming immune
  to smallpox vis a vis cowpox it became accepted
  as the way of the future.
• In 1840 variolation was forbidden by an Act of
  Parliament.
• In England vaccination with cowpox became
  compulsory in 1853.

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Edward Jenner

• Jenner spent his time supplying cowpox material
  to others around the world.
• In recognition of his work the British government
  awarded Jenner ten thousand pounds in 1802, and
  twenty thousand pounds in 1807.
• Jenner was honored for his technique, and vaccine
  became the universally used term to indicate
  introducing material under the skin to produce a
  protection against disease.

17
Eradication

• In 1801 Jenner said, The annihilation of the
  smallpox, the most dreadful scourge of the human
  species, must be the final result of this
  practice.
• Compulsory vaccinations began in the following
  years
• 1807 in Bavaria
• 1810 in Denmark
• 1835 in Prussia
• 1853 in Britain

18
Eradication

• Even after vaccination outbreaks still continued
  because the virus was imported by travelers where
  there were still endemics.
• Not until after WWI did most of Europe become
  smallpox free, and not until after WWII was
  transmission stopped throughout Europe and North
  America.
• In less developed countries smallpox continued
  largely unabated until the middle of the 20th
  century.

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Eradication

• 1940s large scale preparations of a stable
  freeze dried vaccine was perfected by Collier.
• 1950 Pan American Sanitary Organization decided
  to undertake a hemisphere wide eradication
  program.
• 1958 Union of Soviet Socialist Republics
  proposed to the WHO that a global smallpox
  eradication program be undertaken.

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Eradication

• The campaign was based on a two fold strategy.
• 1) Mass vaccination campaigns in each country
  using a vaccine of ensured potency and stability
  that would reach at least 80 of the population.
• 2)Development of a system to detect and contain
  cases and outbreaks.

21
Eradication

• 26 October 1977 the last naturally occurring case
  of smallpox was recorded in Merka Somalia.
• In 1978 two cases were reported. These were both
  from people working in labs with smallpox in
  England.

22
Eradication

• 1980 WHO formally declared that smallpox was
  dead.
• The eradication of smallpox was one of the most
  important branches of modern medicine.
• Jenner has been acknowledged as the father of
  immunology,

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TAXONOMY

• FAMILY POXVIRIDAE
• SUBFAMILY CHORDOPOXVIRINAE (infect vertebrates)
• GENERA ORTHOPOXVIRUS (variola, vaccinia,
  cowpox, monkeypox)
• AVIPOXVIRUS (fowlpox)
• CAPRIPOXVIRUS (sheep-pox)
• LEPORIPOXVIRUS (myxoma)
• PARAPOXVIRUS (milkers
  nodule)
• SULPOXVIRUS (swinepox)
• 2. SUBFAMILY ENTOMOPOXVIRINAE (infect
  arthropods)

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Species of the Genus Orthopoxvirus Species of the Genus Orthopoxvirus Species of the Genus Orthopoxvirus Species of the Genus Orthopoxvirus
Species Animals Infected Host Range Geographic Range
Variola Human Narrow Formerly worldwide
Vaccinia Human,a cow, pig, buffalo, rabbit, etc. Broad Worldwideb
Cowpox Rodent,a cow, human, cat, etc. Broad Europe
Monkeypox Squirrel,a monkey, ape, human Broad Western and central Africa
Ectromelia Mouse, mole Narrow Europe
Camelpox Camel Narrow Africa and Asia
Taterapox Gerbil Narrow Western Africa
Volepox Vole ? United States
Raccoonpox Raccoon ? United States
Skunkpox Skunk ? United States
Uasin Gishu Horse Medium Eastern Africa
aPrimary host. bSecondary to vaccination no known natural host. aPrimary host. bSecondary to vaccination no known natural host. aPrimary host. bSecondary to vaccination no known natural host. aPrimary host. bSecondary to vaccination no known natural host.
http//books.nap.edu/html/variola_virus/ch2.htmlT
opOfPage
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CHARACTERISTICS SHARED BY SPECIES OF
ORTHOPOXVIRUS

• - The largest and most complex viruses
• Virons particles can be seen with a light
  microscope
• - They contain a linear genome of a single
• double-stranded DNA
• They replicate in the cytoplasm of the host
  cell,
• therefore they must provide their own mRNA and
  
• DNA synthetic machinery
• (including DNA-dependent RNA polymerase)
• - Inclucison bodies type B and type A
• Virions have a brick-like shape and are present
  in
• 2 forms, both are infectious
• 1. EEV (Extracellular Enveloped Virus)
• 2. IMV (Intracellular Mature Virus)
• Serological cross-reactivity
• - Produce a hemagglutininin antigen (HA)

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Fenner,F. et al. Smallpox and Its Eradiction.
Genevea, SwitzerlandWHO. 19981460
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Fenner,F. et al. Smallpox and Its Eradiction.
Genevea, SwitzerlandWHO. 19981460
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MORPHOLOGY OF THE VIRION
- have an brick-like shape dimensions
400x200nm - four major elements 1. core ( 9
nm thick membrane, biconcave disk, a
tightly compressed nucleoprotein) 2. lateral
bodies ( unknown function) 3. outer membrane (
a protein shell 12nm thick, the surface
consists of irregularly arranged tubules) 4.
envelope ( an inconstant element,
proteins are glycosylated and acylated)

- Virons are present in two infectious forms
1. EEV (Extracellular Enveloped Virus)-
released from cells spontaneously, by
exocytoses are enclosed within a lipoprotein
envelope, which contains the haemagglutinin and
other specific polypeptides - CEV (Cell
Associated Enveloped Virus) 2. IMV
(Intracellular Mature Virus) released by
cellular disruption, lacks envelope, naked
virus
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Vaccinia Virus Electron micrographs
A. Non- enveloped virion (surface of outer
membrane with tubular elements) C. Thin section
of non-enveloped virion (biconcave core) B.
Enveloped virion, found in extracellular
medium D. Viral core, released after treatment of
virions with Nonidet
Fenner,F. et al. Smallpox and Its Eradiction.
Genevea, SwitzerlandWHO. 19981460
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VIRAL REPLICATION CELL CYCLE
Fenner,F. et al. Smallpox and Its Eradiction.
Genevea, SwitzerlandWHO. 19981460
31
VIRAL REPLICATION CELL CYCLE
http//www.rkm.com.au/VIRUS/SMALLPOX/smallpox-life
-cycle.html
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CELLULAR CHANGES
Viral factory visualized in stained cells as
the B-type inclusion body, is first seen at
2.5h cupules first appear at 4 hours and some
are completed as immature particles
6-8hours. From 8 hours onwards mature particles
appear.
Toxicchanges may occur in the inf.cells, which
in monolayer cultures become rounded and
retract from each other.
Fenner,F. et al. Smallpox and Its Eradiction.
Genevea, SwitzerlandWHO. 19981460
33
Cytoplasmic inclusion bodies in infected cells
B-type (Guarnieri bodies)- sites of viral
replication
produced by all orhopoxviruses A-type
strongly eosinophilic, found in cells infected
with cowpox,ectromelia and
raccoonpox virus appear late in the infection
and are not associated with viral
replication (may contain mature virions)

Fenner,F. et al. Smallpox and Its Eradiction.
Genevea, SwitzerlandWHO. 19981460
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STRUCTURE OF THE VIRAL GENOME
- Schemat of vaccinia virus DNA
- Contains a single linear molecule of a double
stranded DNA About 200 kbp long
guaninecytosine content 36 - when denatured the
two sister strands form a large single-stranded
circular molecule, being attached at each end
of the genome by covalent links - for the most
part, the DNA sequences in the central part of
the genome are unique, but the terminal fragments
(inverted terminal repeats) cross-hybridize with
each other and with the termini of other species
of orthopoxvirus
- The ITRs include an AT-rich, incompletely
base-paired, hairpin loop that connects the two
DNA strands set of short tandemly repeated
sequences. The ITRs are variable in length
owing to deletions, repetitions, and
transpositions
- Inverted repeats in vaccinia are 10 kbp long
in variola are 725
bp - Variola vs. Vaccinia genomes are highly
conserved with gt95 nucleotide identity, however
towards the termini the sequences diverge
Poxviruses that have been inactivated that dont
damage their DNA can be reactivated
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VIRAL PROTEINS

• Encodes about 200 proteins
• The central part of the genome encodes for
  structural and functional proteins
• Virulence genes are found near the inverted
  repeats,
• -numerous virus-encoded enzymes,are packaged
  within the virus core,
• including
• multisubunit DNA-dependent RNA
  polymerase
• RNA polymerase associated
  protein of 94kd (RAP94)
• a transcription factor (VETF)
• capping and methylating enzymes
• poly(A) polymerase
• These components are used to synthesize
  translatable mRNA
• -Importatnt proteins for replication
• topoisomerase
• thymidine kinase allows the incorporation
  of Thymidine into DNA
• thymidylate kinase catalyzes the reversible
  phosphorylotransfer between ATP and TMP
• ribonuceoside reductase converts
  ribonucleoside diphosphates ( NDPs ) into
  deoxyNDPs
• dUTPase minimize the misincorporation of
  Uracil into DNA

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non enzymatic -VIRAL PROTEINS

• Membrane proteins
• A33R, A34R, A36R N-glycosylated, phosphorylated
• fromation of
  actin tail and microvilli, which facilitate
• viral
  dissemination
• A36R required for kinesin
  recruitment and is involved in
• 
  microtubule-based motility of IEVs
• A56R Hemagglutinin, N- and O- glycosylated ,
  promote cell fusion and
• cell to cell viral spread
• A27L required for the formation of IEV, fusion
  protein,
• microtubule dependent movment,
• normal sized plaques , has additional
  role in the viral assembly
• A28L fusion protein A28 deficient virions with
  normal amounts of A27 and
• A17 (binding partner) are unable to
  induce cell fusion
• 2. Core proteins

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VIRAL IMMUNOMODULATORY STRATEGIES

• Poxviruses encode multiple classes of
  immunomodulatory proteins to inhibit
• diverse processes as
• apoptosis
• the production of interferon
• the production of chemokines
  and inflammatory cytokines
• the activity of complement,
  NK, CTLs, antibodies

2. The inhibitory proteins, produced by virus,
fall into three main classes - Virokines
resemble host cytokines
secreted from infected cells to block hosts
receptors vIL-10, vIL-18 -
Viroreceptors mimic host cellular receptors
altered cellular
receptors that have lost their transmembrane
sequnces and consequently
are secreted from infected cells
to sequester ligands
vINF-Rs, vTNFRs - Intracellular
proteins target host signal transduction
pathways
inhibit inner antiviral pathways
apoptosis vFLIPs, serpins

proinflammatory cascades - TNF

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VIRAL IMMUNOMODULATORY PROTEINS
j
Johnston,J.B.et al.Poxvirus Immunomodulatory
StrategisCurrent Perspectives. Journal of
Virology (2003), 77 p.6093-6100
39
VIRAL IMMUNOMODULATORY PROTEINS
Complement Regulatory Proteins - VCP
Vaccinia virus Complement control Protein,
consists of short consensus
repeats found in hosts complement regulatory
proteins. Inhibits the classic
and alternative pathways of complement through
binding and inactivating both C4b
and C3b

• SPICE- the smallpox inhibitor of complement
  enzymes
• molecularly engineered homologue of VCP
  (Rosengrad et al.Univ. of Penn.)
• Demostrated the functional advantage of variola
  complement regulatory protein
• Over the vaccinia homologue
• - More human complement specific than VCP
• 100-fold more potent at inactivating C4bC3b
• SPICE serves to inhibit the formation of the
  C3/C5 convertases necessary for
• Complement-mediated viral clearance

SPICE- provides the first evidence that variola
proteins are particulary adept at overcoming
human immunity, and the decreased function of VCP
suggests one reason why the vaccinia virus
vaccine was associated with relatively low
mortality. Disabling SPICE may be useful
therapeuticaly
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Smallpox

• Clinical Presentations, Transmission, Treatment,
  Vaccination

41
Transmission of Smallpox

• Humans are the only natural host of smallpox and
  it is not known to be transmitted by insects or
  animals (no animal reservoir)
• Transmission generally occurs from direct and
  fairly prolonged face-to-face contact (in order
  for infected spit particles to pass from one
  person to another)
• Infected aerosols and air droplets spread in
  face-to-face contact with an infected person
  after fever has begun, esp. if symptoms include
  coughing
• Smallpox can also be spread through direct
  contact with infected bodily fluids or
  contaminated objects (ie. Bedding and clothing)
• In rare instances, smallpox can spread through
  the air of an enclosed area
• Variola major renders infected people bedridden
  so spreading to the community is reduced
• In variola minor, however, the symptoms are so
  mild that patients remain ambulatory during the
  infection phase and can spread the virus more
  widely

42
Pathogenesis of Smallpox

• The portal of entry for smallpox is the
  respiratory tract or inoculation on the skin
• Excretions from the mouth and nose, rather than
  scabs, are the most important source of
  infectious virus
• Studies have shown that primary infection in the
  nose or mouth do not produce a primary lesion
  that ulcerates and releases virions onto the
  surface
• Four models have been studied to learn about the
  spread of the infection through the body
  mousepox in mice, rabbitpox in rabbits, and
  monkeypox and smallpox in monkeys and apes
• During incubation the virus proceeds through
  infection, replication, and liberation (usually
  accompanied by cell necrosis) first at the site
  of inoculation and then to the regional lymph
  nodes, then deeper lymph nodes and possibly into
  the bloodstream

43
Pathogenesis of Smallpox

• When tests were performed on humans to determine
  the pathogenesis, the virus was only rarely
  collected from the serum of infected persons,
  even though viraemia had definitely occurred
• Therefore it was determined that viraemia in
  ordinary smallpox was restricted to the
  pre-eruptive and early eruptive stages of the
  disease
• In hemorrhagic smallpox, however, virus was
  readily found in the blood and titers were high
  thus hemorrhagic smallpox can be associated with
  overwhelming infection and the continued release
  of virus into the bloodstream
• The primary event for production of lesions of
  the rash in orthopoxvirus infections is
  localization (in the small blood vessels of the
  dermis) of virus particles that circulate in the
  bloodstream
• Vasodilation leads to greater density of lesions

44
Pathogenesis of Smallpox

• After introduction of smallpox into the
  oropharyngeal cavities it spreads to the regional
  lymph nodes
• Asymptomatic viremia occurs on the 3rd or 4th day
  after infection, with further dissemination of
  the virus to spleen, bone marrow, and other lymph
  nodes.
• The virus localizes in small blood vessels of the
  dermis and oropharyngeal mucosa, leading to
  initial onset of the enanthem and exanthem, at
  which point (about day 14) the patient becomes
  infectious
• The spleen, lymph nodes, kidneys, liver, bone
  marrow, and other viscera also may contain large
  amounts of virus
• Secondary viremia occurs by the 8th to 12th day
  after initial infection this is followed by
  onset of fever and toxemia.
• Smallpox is disseminated through the body in the
  lymph and bloodstream as cell-associated
  particles
• Enveloped forms of the virus are more important
  than non-enveloped in the dissemination of the
  virions over a distance

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Pathogenesis of Smallpox

• The development and evolution of skin lesions is
  an extremely valuable clue to the diagnosis and
  involves the following steps
• Dilatation of the capillaries in the papillary
  layer of the dermis occurs initially, followed by
  swelling of the endothelial cells in the vessel
  walls perivascular cuffing with lymphocytes,
  plasma cells, and macrophages can be seen.
• Lesions then develop in the epidermis, where the
  cells become swollen and vacuolated
  characteristic B-type inclusion bodies can be
  found in the cytoplasm.
• The cells increase in size and the cell membranes
  rupture, leading to vesicular lesions.
• Pustulation results from the migration of
  polymorphonuclear cells into the vesicle.
• The contents of the pustule gradually become
  desiccated, leading to crusting or scabbing of
  the lesions.
• Re-epithilialization and scarring occur as the
  lesions heal.

46
Effects on Other Organs

• A striking feature of smallpox reports is that
  there is an absence of specific lesions anywhere
  except in the skin and mucous membranes
• The endothelial cells lining the sinusoids of
  liver were often swollen and occasionally
  proliferating or necrotic (in more severe cases)
• Reticulum cell hyperplasia occurred in bone
  marrow and spleen
• Spleen was usually engorged with very numerous
  large lymphoid cells

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Pathogenesis of Smallpox

• When studies were done on rabbits with rabbitpox
  to determine the cause of death the rabbits had
  extreme hypertension, leading to a shock-like
  syndrome, decreased urinary output and a rise in
  blood-urea and plasma potassium levels
• Death seemed to be caused by lethal
  concentrations of potassium ion, which was
  possibly from the hypertension

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Clinical Presentations

• Smallpox is also called Variola
• 4 orthopoxviruses are known to infect humans
  variola, vaccinia, cowpox, and monkeypox
• Variola virus is strictly a human virus, although
  primates and other animals can be infected under
  lab conditions
• Infection begins when the virus comes into
  contact with oropharyngeal (mouth and throat) or
  respiratory mucosa virus multiplication then
  occurs in regional lymph nodes
• There are 2 clinical forms of Smallpox Variola
  Major and Variola Minor
• Variola major is severe and the most common form
  with more extensive rash and higher fever with a
  death rate of about 30
• Variola minor has less common presentation and
  much less severe with death rate of 1 or less

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• Variola Major has 4 clinical presentations based
  on the nature and evolution of the lesions those
  4 types are
• Ordinary most frequent (more than 90 of cases
  in both vaccinated and unvaccinated persons)
  corresponds to classical description of smallpox
• Modified milder and may occur in previously
  vaccinated people rarely fatal
• Flat and Hemorrhagic very severe but uncommon

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Stages of Smallpox

• Incubation Period
• Lasts on average 12-14 days but can range from
  7-17 days
• Person is not contagious and exhibits no symptoms
• Prodrome Phase
• This is when initial symptoms develop and is also
  called the pre-eruptive stage
• Begins abruptly with fever, malaise, headache,
  head and body aches, prostration, and often
  nausea and vomiting
• Body Temperature rises to at least 101F and is
  often higher
• Note that this severe febrile prodrome right
  before the onset of the rash is characteristic of
  smallpox and can be used to differentiate it from
  other rash illnesses
• This phase lasts 2-4 days and is sometimes
  contagious

52
Stages of Smallpox Rash Phase

• When the first visible lesions appear the fever
  may start to go down
• This is the most contagious period (the time when
  the saliva has the most virus in it)
• Lasts 4 days
• Rash emerges as small red spots on tongue and in
  mouth (about 24 hours before the appearance of
  rash on the skin)
• Lesions in the mouth and pharynx enlarge and
  ulcerate quickly, releasing large amount of virus
  into the saliva
• These spots develop into sores that break open
  and spread large amounts of virus into mouth and
  throat
• The rash starts on the face as a few macules,
  known as herald spots, and spreads to the arms
  and the legs then to the hands and feet
• The rash usually spreads to all parts of the body
  in just 24 hours
• By the 3rd day of the rash the rash becomes
  raised bumps or papules
• By the 4th day the bumps become vesicular, fill
  with a thick, opaque fluid and often have
  depression in center that looks like a
  bellybutton (this umbilication is a major
  distinguishing characteristic of smallpox
  especially from chickenpox)

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Stages of Smallpox Rash Phase

• The fever will rise again at this time and remain
  high until scabs form over the bumps
• By the 6th or 7th day the lesions have become
  pustules they are sharply raised, typically
  round, tense, firm to the touch (some describe
  them as feeling like BBs under the skin)
• B/w 7 and 10 days the pustules mature and reach
  their max size, and the umbilications remain
  throughout the whole time
• Although lesions are dense around the nose and
  mouth, the majority of lesions are discrete (in
  some cases the lesions become confluent)
• At around 10 days of the rash the pustules begin
  to form a crust
• At about 14 days most of the lesions scab over
  and some begin to separate
• About 3 weeks after the onset scabs have
  separates and the site of each lesion is
  depigmented and eventually becomes pitted scars
  because of deeper skin layer involvement

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Stages of Smallpox Rash Phase

• The rash of smallpox has a centrifugal
  distribution, meaning it is most dense on the
  face, and more dense on the extremities than on
  the truck
• The palms of the hands and soles of the feet are
  involved in the majority of cases
• These characteristics are important in
  differentiating smallpox from other causes of
  rash illness
• Another differentiating characteristic of
  smallpox is that the lesions are all in the same
  stage of development on that part of the body
  (unlike chickenpox) these stages of development
  are macules, papules, vesicles, and crusted
  lesions

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That was ordinary smallpox, now for the 3 other
types

• Modified prodromal illness still occurs but is
  less severe than the ordinary type, usually a
  fever but not as high, skin lesions tend to
  evolve more quickly, are more superficial and may
  not show the uniform characteristic of more
  typical smallpox
• Flat (or malignant) smallpox sever with a high
  fatality rate in flat-type the lesions remain
  more or less flush with the skin and dont become
  raised, accounted for 5-10 of cases in India,
  the prodrome symptoms are severe and last 3-4
  days, fever remains elevated throughout the
  course of the illness and severe toxic symptoms,
  skin lesions progress very slowly lesions
  contain very little fluid and are not
  umbilicated the lesions are soft and velvety to
  the touch, most cases are fatal
• Hemorrhagic smallpox sever and uncommon form of
  smallpox that is almost always fatal, involves
  extensive bleeding into the skin, mucous
  membranes and GI tract, in the large Indian
  series it occurred in 2 of cases, the prodrome
  has prolonged fever with little or no remission,
  intense headache, restlessness, extreme
  prostration and toxicity death often occurs
  between the 5th and 7th day of illness when only
  a few maculopapular cutaneous lesions are present

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When is a person contagious?

• A person with smallpox is sometimes contagious
  with the onset of fever (the prodrome phase) but
  a person is most contagious with the onset of the
  rash
• Luckily, by the time a person gets the rash they
  are so sick they cant likely move around the
  community
• An infected person is contagious until the last
  smallpox scab falls off

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Clinical Diagnosis

• There are 3 major criteria for diagnosing if a
  rash is indeed smallpox
• Prodrome that begins 1-4 days before rash onset
  and includes fever over 101F, and at least one of
  the following symptoms prostrations, headache,
  backache, chills, vomiting, abdominal pains
• Presence of classic smallpox lesions firm,
  round, deep-seated vesicles or pustules
• Lesions on the palms of the hands and/or soles of
  the feet
• There are 5 minor criteria looked at for
  diagnosis of smallpox
• Lesions are centrifugal distribution
• First lesions appear on the oral mucosa, face, or
  forearms
• Patient appears toxic
• Rash has slow evolution, each stage lasting 1-2
  days
• There are lesions on palms of the hands and/or
  soles of feet

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Common conditions confused with Smallpox

• Varicella (primary infection with varicella
  zoster virus)
• Disseminated herpes zoster
• Impetigo
• Drug eruptions
• Contact dermatitis
• Erythema multiforme minor
• Eyrthema multiforme (includes Steven Johnsons
  Syndrome)
• Enteroviral infection esp. Hand, foot and mouth
  disease
• Disseminated herpes simplex
• Scabies and insect bites
• Molluscum contagrosum

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Laboratory Diagnosis of Smallpox

• Culture on Egg chorioallantoic membrane (CA)
  classical method poxvirus grow on CA
• Direct examination of vesicle or pustular
  material aggregations of virus may be seen in
  certain cytoplasm upon staining
• Tissue culture growth in cultured cells
• EM negative staining is used to visualize
  characteristic large brick shape of poxvirus
• Relatively rapid
• Can distinguish orthopox viruses from other viral
  agents
• Cannot differentiate between variola and vaccinia
  viruses
• May not be as sensitive as PCR-Based methods

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Laboratory Diagnosis of Smallpox

• PCR Based method In North America a positive
  test is considered diagnostic for vaccinia virus
  unless medical or epidemiologic evidence suggests
  otherwise
• With slight modifications to the fluorescently
  labeled probe, this assay can also be used to
  detect variola virus
• Family specific primers are used first, then
  subgroup-specific primers are used if the former
  is not successful in producing PCR product
• DNA Probes Assays using immobilized
  oligonucleotides in a microarray have been
  developed to identify and discriminate among
  orthopoxviruses
• In situ hybridization of formalin-fixed tissues
• Serology Classical methods such as complement
  fixation and gel precipitation commonly were used
  in the past experimental enzyme-linked
  immunoassays are currently being evaluated
• Strain identification A restriction fragment
  length polymorphism assay (RFLP) has been
  developed by CDC using polymorphisms found on 45
  variola strains from 1939 to the 1970s

66
Laboratory Diagnosis

• Lab diagnosis of smallpox is made by examination
  of material from a skin lesions
• For a patient who meets criteria for moderate
  risk, the most important laboratory procedure is
  rapid diagnostic testing for varicella zoster
  virus, or VZV
• Most common method for detecting VZV is direct
  fluorescent antibody, or DFA
• This method detects VZV directly in cells using
  anti-VZV antibody conjugated to fluorescein dye
• This technique is very sensitive and specific but
  is critically dependent on careful collection of
  material from lesion
• Electron microscopy, detection of VZV DNA by
  polymerase chain reaction testing of vesicular
  fluid or scabs, standard PCR, and cytology smears
  are other rapid methods for detections of VZV
• Ultimately, smallpox is a disease which can be
  easily diagnosed by trained health workers
  without the need for laboratory support

67
Outcomes of Infection

• Those who survive usually have scars
• If eye involvement then blindness could occur
• Recovery results in long lasting immunity to
  reinfection with variola virus no evidence of
  chronic or recurrent infection with variola virus
• In fatal cases death usually occurs b/w the 10th
  and 16th days of illness
• The cause of death from smallpox is not exactly
  clear since the infection involves multiple
  organs perhaps uncontrolled immune response as
  well as overwhelming viremia and soluble variola
  antigens

68
Treatment of Smallpox

• Vaccine is administered up to 4 days after
  exposure to the virus and before the rash
  appears, provides protective immunity and can
  prevent infection or ameliorate the severity of
  the disease
• There is really no effective treatment, other
  than the management of the symptoms
• Adequate fluid intake (difficult)
• Alleviation of pain and fever
• Keeping skin lesions clean to prevent bacterial
  infection
• Some compounds, such as Cidofovir, are under
  investigation as chemotherapeutic agents

69
Vaccination

• In 1796, Edward Jenner demonstrated that immunity
  to smallpox could be produced by inoculating a
  human with material from a lesion on the udder of
  a cow (cowpox) Jenner called this material
  vaccine from vacca which is Latin for cow
• At some time during the nineteenth century, the
  virus used for smallpox ceased to be cowpox and
  was changed to vaccinia
• Vaccinia is in the same family has cowpox and
  smallpox but genetically different
• In the early 1950s (150 years after Jenners
  vaccination came out) an estimated 50 million
  cases of smallpox occurred in the world each
  year, which feel to around 10-15 million by 1967
  b/c of vaccination

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Vaccination

• The smallpox vaccine is actual live vaccinia
  virus, unlike other vaccines which use dead
  virus for this reason the vaccination site must
  be cared for to prevent spread
• Smallpox vaccine is administered using a
  bifurcated needle, not an injection, unlike any
  other vaccine
• The bifurcated needle is dipped into the vaccine
  and then used to prick the skin 15 times in about
  3 seconds in a 5mm radius area
• It is administered into the superficial layer of
  the skin

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Vaccination

• If vaccination is successful a red, itchy bump
  develops at the vaccine site in 3-4 days this is
  caused by the vaccinia virus replicating in the
  basal cells of the epidermis producing a papule
  surrounded by erythema
• In the first week the bump becomes a blister,
  fills with pus, and begins to drain
• A person is considered protected with the
  development of a pustule like this at the
  vaccination site
• During the second week the blister begins to dry
  and a scab forms the scab then falls off leaving
  a scar
• Most people experience the side effects of a sore
  arm, fever, and body aches and axillary
  lymphadenopathy (3-5 days after vaccination)
• 1st time vaccinators have a stronger reaction
  than those who are re-vaccinated
• Because the virus is live it can be spread to
  other parts of the body or to other people so
  great care must be given to the vaccination site
  to prevent this

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Vaccination

• The vaccine provides a high level of immunity for
  3-5 years and decreasing immunity thereafter
• If a person is re-vaccinated the immunity lasts
  even longer
• Studies show that even 30 years after a
  vaccination, while a person may not be protected
  against smallpox they have a less severe disease
• The vaccine has been effective in preventing
  smallpox in 95 of people vaccinated
• Evidence for a brisk cell-mediated immune
  response has also been detected
• It is believed that healing of the vaccinia
  infection is associated with intact cell-mediated
  or T-cell and cytokine immune competence, and
  that viremia is defended by an intact antibody or
  B-cell immune competence
• If the vaccine is given in 1-2 days after
  exposure to smallpox it is effective in
  preventing smallpox or mitigating the symptoms of
  those who have been exposed
• The fatality rate among people vaccinated less
  than 10 years before exposure was 1.3 it was 7
  among those vaccinated 11-20 years prior, and 11
  among those vaccinated 20 or more years prior to
  infection 52 of unvaccinated people died

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Vaccination

• The antibodies induced by the vaccinia vaccine
  cross protect for other Orthopoxviruses
  (including monkeypox, cowpox, and smallpox)
• Three different smallpox vaccines are available
  (or will soon be) in the U.S.
• All 3 contain the New York City Board of Health
  strain of live vaccinia virus
• Dryvax is the currently licensed vaccine it was
  produced by Wyeth Lederle in the early 1980s from
  calph lymph containing live vaccinia virus the
  vaccine is provided as a freeze dried powder in a
  multi-dose vial
• Dryvax is produced by infecting a calf with
  vaccinia and then collecting the lymph from the
  virus filled pustules on the calfs udder
• Dryvax contains the antibiotics polymyxin B,
  streptomycin, tetracycline and neomycin
• A second vaccine produced years ago by Aventis
  Pasteur would be available in the case of an
  emergency

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Vaccination

• New smallpox vaccines also contain live New York
  City Board of Health vaccinia virus, but are
  produced using cell culture technology rather
  than live animals these vaccines may also be
  distributed as a freeze dried powder but do not
  contain antibiotic
• In October 2002 Acambis-Baxter Laboratories began
  preparing Tissue culture cell vaccines
• The two types of cells being used to cultivate
  the vaccinia virus are Vero monkey kidney cells,
  and human fibroblast cell line (MRC5)
• It is thought that these cell cultured vaccines
  will have less side-effects than the calph-lymph
  vaccine
• The other vaccines currently being developed
  Modified vaccinia Ankara (MVA) vaccine, Japanese
  strain LC16m8 vaccine
• Current supply there are 14.8 million doses of
  DryVax available and 85 million doses of the
  Aventis product, and when the Acambis vaccines
  are available there will be an additional 209
  million doses available

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Who Should Not Get the Vaccine?

• The vaccine is contraindicated for
• Persons who have experienced a serious allergic
  reaction to a prior dose of vaccine or to a
  vaccine component
• Persons with significant immunosuppression from
  any cause (HIV, transplant, receiving treatment
  for cancer) or anyone with an immunosuppressed
  person in their household
• Pregnant women and persons with a pregnant person
  in the household
• Breastfeeding women
• Children under 12 months in fact the Advisory
  Committee on Immunization Practices (ACIP)
  advises against non-emergency vaccination in
  children under 18
• Persons with any heart problems, stroke or
  transient ischemic attack, high blood pressure,
  high cholesterol, or diabetes
• Persons with any sort of skin condition
• Persons with inflammatory eye diseases requiring
  steroid therapy

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4 Main Complications of Vaccination

• Ecxema vaccinatum
• Occurred in vaccinated persons or unvaccinated
  contacts who were suffering from or had a history
  of eczema
• An eruption occurred at the sites on the body
  that were at the time affected by eczema or had
  been
• Symptoms severe eruptions became intensely
  inflamed
• Occurred in 74 persons with no deaths 60 cases
  from contact with vaccinated persons with 1 death
• Progressive vaccinia (vaccinia necrosum)
• Occurred in persons who suffered from an immune
  deficiency or with humoral or immune globulin
  deficiencies
• Local lesion at the vaccination site failed to
  heal all lesions spread progressively until the
  patient died, usu. 2-5 months later
• Occurred in 11 person, with 4 deaths
• Generalized vaccinia
• Occurred in otherwise healthy individuals
• Characterized by development, from 6-9 days after
  vaccination, of a generalized rash, sometimes
  covering whole body
• Prognosis good
• Occurred in 143 persons with no deaths
• Postvaccinial encephalitis
• Most serious complication
• Occurred in 2 forms 1. seen in infants under 2
  years old, had violent onset, characterized by
  convulsions 2. seen in children older than 2
  years had abrupt onset, with fever, vomiting,
  headache, and malaise
• Fatality rate was about 35, with death usually
  occurring within a week
• Occurred in 16 persons with 4 deaths

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Eczema Vaccinia
Progressive Vaccinia
Generalized Vaccinia
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Progressive Vaccinia
Developed Erythema Multiforme 1 mo. After vaccine
Secondary herpes infection
Contact Vaccinia
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Treatments for Complications from Vaccination

• Vaccinia Immune Globulin (VIG) is a sterile
  solution of the immunoglubulin fraction of plasma
  from person who was vaccinated with smallpox
  vaccine
• VIG was produces in the 1960s and it contained a
  high titer of anti-vaccinia neutralizing antibody
• VIG has shown to work in the treatment of
  smallpox vaccine adverse reactions caused by
  continued replication of vaccinia virus, such as
  the above mentioned complications
• VIG is available from CDC under an
  investigational new drug protocol
• Cidofovir is an antiviral medication (not
  previously used in humans) used to treat vaccinia
  infection it is considered a second line therapy
  for smallpox vaccine adverse reactions
• A new intravenous formulation of VIG is being
  produces to support treatment of adverse events
  that may result from smallpox vaccination
• VIG is not recommended for mild instances of
  inadvertent inoculation, mild generalized
  vaccinia, erythema multiform, post-vaccination
  encephalities, and isolated vaccinia keratitis

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Vaccinations Now

• Routine vaccination in the U.S ended in 1972 for
  children and 1976 for healthcare workers
• On December 13, 2002, President Bush announced
  the following US policy
• Smallpox vaccinations are required for military
  personnel according to the Department of Health
  and Human Services (DHHS), approximately 500,000
  military personnel will be vaccinated.
• Smallpox vaccinations are recommended for
  smallpox response teams comprised of public
  health staff and healthcare workers likely to be
  involved in the initial care of any patients with
  smallpox.
• Smallpox vaccinations also are being offered to
  other healthcare workers and to first-responders
  (including police officers, firefighters, and
  emergency medical technicians).
• Smallpox vaccinations likely will be made
  available to the general public on a voluntary
  basis once large stockpiles of the vaccine are
  licensed.
• Small Response Teams Department of Health and
  Human Services will work with state and local
  governments to form volunteer smallpox response
  teams who can provide critical services in the
  event of a smallpox outbreak

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Vaccinations Now

• On April 30, 2003, the President signed into law
  the Smallpox Emergency Personnel Protection Act
  of 2003
• The law establishes a no-fault program to provide
  benefits and compensation to certain individuals
  (ie, healthcare workers and emergency responders)
  who are injured as a result of administration of
  smallpox vaccination or other smallpox
  countermeasures

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Vaccinations Now

• In a report released in August 2003, the
  Institute of Medicine (IOM) Committee on Smallpox
  Vaccination Program Implementation made several
  recommendations regarding offering smallpox
  vaccination to the general public
• Specifically, the Committee recommended that CDC
  proceed by
• Conducting brief quantitative surveys to
  determine public interest and desire for smallpox
  vaccine these surveys should include public and
  private health agencies as well as the general
  public, in order to understand the potential
  scope of public interest.
• Determining the budgetary and other requirements
  that would meet the demand noted.
• Identifying, monitoring, and referring people to
  existing or planned smallpox vaccine clinical
  research trials or other well-structured clinical
  programs that meet the basic requirements of
  medical and public health ethics, including
  assurances for safety of vaccinees and their
  contacts, acceptable balance between risk and
  benefit, and acceptable distribution of scarce
  public health resources to meet all preparedness
  as well as other public health goals The
  committee encourages CDC to consider utilizing a
  pilot program or some other means of evaluating
  the initial experiences with this effort.

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• During the smallpox eradication campaign (1970s)
  and during smallpox outbreaks in the past, a
  "ring vaccination" (used in 1967) strategy has
  been followed
• This approach is incorporated into the current
  CDC Smallpox Plan
• Ring vaccination essentially involves creating a
  circle of vaccinated persons around each case to
  interrupt the chain of transmission.
• The strategy involves the following steps
• Rapid identification and isolation of all
  smallpox cases
• Identification and vaccination of contacts of
  smallpox cases
• Monitoring contacts for development of fever and
  isolating them if fever occurs
• Vaccination of household members of contacts if
  no contraindications to vaccination exist
• In addition to ring vaccination, rapid voluntary
  vaccination of a large population may be required
  to
• Supplement priority surveillance and containment
  control strategies in areas with smallpox cases
• Reduce the "at-risk" population for additional
  intentional releases of smallpox virus if the
  probability of such occurrences is considered
  significant
• Address heightened public or political concerns
  regarding access to voluntary vaccination

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Management of an Outbreak

• Surveillance of smallpox infection is probably
  easier than for any other infectious disease
  because of the distinctive rash, which is wholly
  characteristic
• Containment involves efficient detection of cases
  and identification and vaccination of contacts
• Patients diagnosed with smallpox should be
  physically isolated
• All specimen collectors, care givers and
  attendants coming into close contact with
  patients should be vaccinated as soon as smallpox
  is diagnosed as the cause of an outbreak
• Medical care givers, attendants, and mortuary
  workers, even if vaccinated, should wear gloves,
  caps, gowns, and surgical masks
• Contaminated clothing and bedding, if not
  incinerated, should be autoclaved or washed in
  hot water containing bleach
• Fumigation of premises may be done with
  formaldehyde

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Isolation Precautions

• Airborne and Contact Precautions in addition to
  Standard Precautions should be implemented for
  patients with suspected smallpox.
• Airborne Precautions
• Place the patient in a private room with negative
  air-pressure ventilation (minimum 6 air
  exchanges/hr).
• Use external air exhaust or high-efficiency
  particulate air (HEPA) filters if the air is
  recirculated.
• Keep the door to the room closed.
• Contact Precautions
• Place the patient in a private room if available.
  
• If a private room is not available, place the
  patient in a room with a patient who has active
  infection with the same organism (ie, cohort
  patients with smallpox).
• Wear gloves when entering the room, change gloves
  after having contact with infectious material,
  remove gloves before leaving the room, and
  immediately wash hands using an antimicrobial
  agent.
• Wear a gown when entering the room if clothing
  will have significant patient contact remove the
  gown before leaving the room.
• Move and transport the patient for essential
  purposes only. If transport is necessary, a mask
  should be placed on the patient.
• When possible, dedicate the use of noncritical
  patient-care equipment.

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Autopsy and Burial Procedures

• According to WHO "Cadavers should be cremated, in
  a properly designed facility, whenever possible
  and all persons coming in contact with them
  should be vaccinated or at least placed on daily
  fever watch. Body bags treated with hypochlorite
  bleach can also be used."
• Consideration should be given to using sealed
  systems when burying patients who have died of
  smallpox. An example of the type of system that
  can be used to seal remains prior to placing them
  in a casket for burial is the BioSeal Facility
  System, produced by Barrier Products
• This system utilizes a poly-aluminum
  foilextruded laminate material that when used
  with a heat sealer will provide Level 1
  containment for all gases, fluids, vapors, and
  odors associated with the transport and storage
  of human and animal remains.

90
BIOTERRORISM

• The Center for Disease Control and Prevention
  (CDC) divided biological
• agents that are critical biothreat agents into 3
  categories ( A, B,C ) based upon
• their risk for causing mass casualties in the
  event of bioterrorist atack.
• Variola ( smallpox) wass classified as a
  category A agent
• poses a risk to national security (it can be
  easily disseminated or transmitted
• person to person)
• causes high mortality, with potential for
  major public health impact
• might cause public panic and social
  disruption
• demands special action for public health
  preparedness
• - Variola requires Lab. Biosafty Level 4

91
BIOTERRORISM

• Smallpox is a disease that followed humanity for
  thousands of years until 30 years ago.
• It was possible to eradicate smallpox, because
  an effective live vaccine from
• crossreacting virus vaccinia was developed.
• The global vaccination program was financed and
  managed by the World Health
• Organization (WHO) and the official eradiction
  announcement was made in 1980.
• Over 20 years have passed since general
  vaccinations stopped and very few people
• are protected against the disease today.
• General vaccination, due to the number of
  complications that can be expected,
• is probably not possible with the present
  vaccines, except in an emergency.
• There is ongoing research in order to develop
  new vaccines.

Tengell et al. Smallpox-eradicated, but growing
terror threat. Clin Microbiol Infect (2002), 8
504-509
92
BIOTERRORISM

• It was formerly thought that smallpox was not
  very suitable for biological warfare,
• because it was too infectious. An attack would
  entail a high risk of a worldwide
• epidemic, striking even the populations of the
  attackers.
• Terrorists generally lack the means for
  vaccinating large groups of people and
• have less opportunity to run large scale
  virus cultures. Thus, it has been
• considered far more probable that they would
  prefer bacterial resource such as
• anthrax, which entails less risk of
  uncontrollable epidemics.
• Terrorist movement or the military command of a
  state could draw a conclusion
• that an attack with variola in the USA could
  allow the epidemic to remain local
• because of the expected vigorous
  countermeasures. If the risk of a worldwide
• epidemic seems small, an attack might be an
  attempt.
• Additionally, we have now seen that terrorist
  can be extremely fanatical.
• The attacks on September 11th, 2001
  demonstrated the great ruthlessness and
• considerable resources of some terrorists.

Atlas R.F. Bioterrorism From Threat to Reality.
Annu Rev.Microbiol.(2002)56 167-85
93
BIOTERRORISM

• Why is variola virus considered an ideal
  bioterrorist weapon
• It is highly transmissible by the aerosol route
  from infected to susceptible
• people
• The civilian populations of most countries
  contain a high proportion of
• susceptible persons
• Smallpox is associated with high morbidity and
  about 30 mortality
• Initially, diagnosis of a disease that has not
  been seen for 20 years would
• be difficult
• At present, other than the vaccine, which may be
  effective in the first few
• days post-infection, there is no proven drug
  treatment available for
• clinical smallpox
• - It induces panic

Mahy,B.W.J. An overview on the use of a viral
pathogen as a bioterrorism agent why
smallpox? (2003) Antiviral Research 57 1-5
94
BIOTERRORISM

• Vaccinia virus, if released as an aerosol and not
  exposed to UV lights may
• persist for as long as 24 hours. It is belived
  that variola virus would exhibit
• similar properties.
• -Vaccinia virus, if released as an aerosol, is
  almost completely destroyed within
• 6 hours in an atmosphere of high temperature
  (31C 33 C) and
• humidity (80). In cooler temperatures (10 C-
  11 C) and humidity (20), nearly
• two thirds of a vaccinia aerosol survives for
  as long as 24 hours. It is belived that
• variola would behave similarly.
• Virus in scabs is more durable. At a temperature
  of 35 C and 65 humidity ,
• the virus can persist for 3 weeks. At cooler
  temp. (26 C) the virus has survived
• for 8 weeks at high humidity and 12 weeks at a
  relative humidity less than 10
• Dutch investigators demonstrated that it was
  possible to isolate variola virus from
• scabs that had been sitting on a shelf for 13
  years.
• Vaccinia / Variola viruses are sensitive to
  disinfectants like hypochlorite (bleach)
• and ammonia are effective for cleaning
  surfaces

Henderson,D.A. et al. Smallpox as Biological
Weapon. (1999) JAMA, 281 2127-2137
95
BIOTERRORISM

• Bill Patrick, a former bioweaponeer who worked
  at Fort Detrick, Md. ,
• before the American offensive biological weapons
  program was dismanteled
• in 1969, says
• - Bioweaponeers in the US program had begun
  weaponizing smallpox before
• the US biological weapons offensive effort was
  halted.
• We made a beautiful powder for smallpox. We
  used chemical to protect it
• during dissemination and aerozolation
• A gram of powdered virus ( equivalent of a
  quarter of a teaspoon of baking
• powder) would infect 100 people