Poxviruses

1
Poxviruses

• Chapter 14

2
Poxviruses

• Cowpox
• Smallpox (variola)
• Monkeypox
• Molluscum contagiosum
• Benign tumors (wart-like)

3
History of Smallpox

• Major poxvirus
• Smallpox killed over 500 million people in the
  20th century!
• Compared to
• 320 million deaths caused by wars, the Spanish
  flu and AIDS combined.

4

• Smallpox left people scarred by pockmarks and
  sometimes blind from corneal ulcerations.
• Famous victims and survivors of smallpox.

5
History of Smallpox

• First evidence of smallpox
• Egyptian pharaoh Ramses V mummified remains
• Written descriptions China (340 A.D.) and
  Southwestern Asia (910 A. D.)
• Smallpox carried by explorers and traders from
  the Old World to the New World where the
  population had no immunity to smallpox.

6
WHO Eradication Program

• In 1967, WHO initiated a program to eradicate
  small pox from the third world nations
• Strategy was to localize the reported cases of
  smallpox, their contacts and the people in the
  immediate area
• These individuals were then vaccinated
• WHO Smallpox Eradication Program - used smallpox
  recognition cards to search for cases in remote
  areas of India and Africa where the last pockets
  of smallpox existed.
• Also used rewards to prompt reporting of cases.
• This procedure was so successful that smallpox
  has essentially been wiped out and vaccination is
  no longer necessary to travel to these areas
  (1979)

7
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 minorcauses a mild form of disease
• 1 - 2 mortality rate

8
4 Types of Variola major Smallpox

• Ordinary - accounts for 89 of cases, 30
  mortality rate
• Vaccine modified - mild, occurred in 2.1 of
  previously vaccinated person, not lethal
• Flat or malignant pox - rare, very severe,
  represents 6.7 of cases, 90 mortality rate
• Hemorrhagic - rare, very severe, 2.4 of cases,
  96 mortality rate

9
Ordinary Smallpox
Figure 14.2a This man is suffering from an
ordinary Variola smallpox infection.
Courtesy of Barbara Rice/NIP/CDC
Figure 14.2b The usual centrifugal pattern of
smallpox maculopapular rash is revealed by this
1973 photograph of a Bangladesh boy.
Courtesy of James Hicks/CDC
10
Variola minor

• Less common, mild disease, 1 or less mortality
  rate.
• The last known person to have natural smallpox of
  any kind lived in Merka, Somalia in 1977

Figure 14.2c The last known person in the world
to have natural smallpox of any kind (Variola
minor).
Courtesy of the World Health Organization
11
Clinical Presentation of Smallpox

• Average incubation period is 12 - 14 days.
• Infected individual is not contagious during the
  incubation period.
• First symptoms
• Fever of 101 to 104oF
• Splitting headache
• Severe backache
• Vomiting (50 of individuals infected)
• Diarrhea (10 of individuals infected)
• Delirium and convulsions (7-15 of individuals
  infected, usually children)

12
Smallpox - Prodromal Phase

• 2-4 day period
• May be contagious during this phase
• Infected individuals too sick to carry on normal
  daily activities.
• As the fever declines (2nd or 3rd day), a macular
  rash appears on the tongue and in the mouth.

13
Smallpox - Prodromal Phase, cont.

• Rash becomes papular (raised) and vesicular
  (blistery).
• Contains large numbers of virus particles

Figure 7.CO Clinical manifestations of smallpox.
Reproduced from F. D. A. Fenner, et al, eds.
Smallpox and its eradication. World Health
Organization, 1988.
14

• As vesicles rupture, large numbers of virus
  particles are liberated into the saliva. Person
  is MOST contagious at this stage!
• Lesions line the respiratory tract.
• Some infected individuals may experience a sore
  throat.
• When the vesicles in the mouth start to break, a
  skin rash appears first on the face, spreading
  to the arms, legs, hands and feet within 24
  hours.
• Rash is described as being centrifugal.
• Important in the diagnosis of smallpox.

15

• Skin vesicles mature into pustules by the 7th
  day.
• The 2nd week, the rash crusts over.
• Scabs fall off by days 22 - 27.
• In fatal cases, death occurs between the 10th and
  16th day of illness.
• The infected individual remains contagious from
  the onset of the rash, until all of the scabs
  come off.

16
Molluscum Contagiosum (MCV)

• Represents 1 of all skin infections
• Transmitted by direct contact, including sexual
  contact or more commonly through indirect
  contactfomites such as sharing towels from
  swimming pools.
• Rapidly transmitted among children at daycares
  and kindergartens.
• Becoming a significant opportunistic infection of
  AIDS patients.

Courtesy of UK Clinical Virology Network,
Gartnavel General Hospital
Figure 14.4 Molluscum contagiosum lesions.
17
Molluscum contagiosum, cont.

• Incubation period 2 - 8 weeks.
• Can serve as a marker for severe immune
  deficiency.
• Causes pin, pearl-like lesions (1-5 mm in
  diameter) on the face (especially eyelids), arms,
  and legs.
• Lesion has a dimple in the center.
• Infection is usually self-limiting in individuals
  with a competent immune system.
• Secondary bacterial infections are a complication
  of molluscum contagiosum.
• The smallpox vaccine does not prevent or protect
  persons from MCV infection.

18
Vaccinia Virus

• Used to vaccinate individuals against smallpox.
• Also protects against monkeypox infection.
• The exact origin of vaccinia virus is unknown.
• Genetically it is most similar to buffalopox.
• Prototype of poxviruses - can be grown easily in
  the laboratory.
• Causes a localized skin infection.
• May cause a severe and systemic disease in
  persons who are immune compromised or have
  pre-existing conditions such as eczema or atopic
  dermatitis.

19
Laboratory Diagnosis of Poxvirus Infections

• 1949, last natural case of smallpox in the U.S.
• Smallpox vaccination discontinued in 1972
• Very few physicians in the U.S. have seen an
  actual case of smallpox.
• Smallpox is a potential biological weapon.
• CDC has prepared information and instructions for
  physicians in case of a smallpox emergency.

20
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.

Figure 14.5 Rash distribution of smallpox vs.
chickenpox.
Adapted from World Health Organization, Diagnosis
of Smallpox Slide Set, http//www.who.int/emc/dise
ases/smallpox/slideset.
21
Preliminary Tests for Smallpox

• Electron microscopy and virus isolation on
  scrapings of pustules and scabs done by state
  health laboratories.
• ELISA assays
• PCR-RFLP analysis is the only way to accurately
  distinguish between Variola, monkeypox and
  vaccinia infections.

22
Cellular Pathogenesis

• Very little known about the pathogenesis and
  virulence of variola virus.
• The knowledge we have, predates modern advances
  in molecular virology and immunology.
• Variola virus is stored safely in 2 international
  repositories
• CDC, Atlanta, Georgia, U.S.
• State Research Center of Virology and
  Biotechnology (Vector), near Novosibirsk, Russia
• Must be worked with in a BSL-4 maximum
  containment facility

23
Poxvirus Characteristics

• Largest of all animal viruses.
• 350 270 nm on average
• Particles are brick-shaped
• Internally, poxvirus particles have a nucleoid
  and two lateral bodies surrounded by an outer
  membrane and lipoprotein envelope (visualized by
  electron microscopy)
• Functions of lateral bodies are not known
• dsDNA genomes with closed ends that are 130 - 230
  kilobases in length
• Contains its own DNA-dependent RNA polymerase
• Ends of the genomes have LTR sequences
• Clinical specimens have two forms
• M or Mulberry form found in vesicular fluid
• C or Capsule form associated with dried scabs

24
A Typical Poxvirus Virion
Figure 14-6a
25
Vaccinia Virus Replication

• Vaccinia is the prototype of poxviruses.
• Grows in a wide host range of cells in culture
  (e.g. monkey, rabbit, human, hamster, mouse etc.)
• Within 12-24 hours, it can be grown in high
  titers.
• 200 bp genome, linear, dsDNA
• 200 genes
• Ends contain LTRs
• Ends of the genome contain unessential genes

26
Vaccinia Virus Replication

• More than 40 different poxvirus genomes have been
  sequenced.

http//www.poxvirus.org
Figure 14-7
27
Vaccinia Virus Replication

• 2 forms of virus particles in cell culture
• Extracellular Enveloped Virion (EEV), contains
  two membranes (Golgi and plasma membrane)
• Intracellular Mature Virion (IMV) contains one
  membrane (derived from Golgi)
• IMVs are released upon cell lysis.
• Each infectious form is thought to enter cells by
  different mechanisms.
• During uncoating, the outer membrane of EEV is
  removed and the particle enters the cell, where
  it loses the 2nd membrane and the viral core
  passes into the cytoplasm.

28
Vaccinia Virus Replicates Solely in the Cytoplasm
of the Cell

• Unique hallmark of vaccinia/poxviruses
• Replicates genome in the cytoplasm.
• Can replicate in cells without nucleus
• Bring everything they need to replicate on their
  own
• All other dsDNA viruses replicate genomes in the
  nucleus.
• Gene expression (transcription) is sequential
• 3 classes of mRNAs
• Early
• Intermediate
• Late

29
Vaccinia Gene Expression is Sequential and
Temporally Regulated

• Early mRNAs encode enzymes and factors need for
  transcription of the intermediate class of mRNAs.
• Intermediate mRNAs encode enzymes and factors
  required for late gene expression.
• Late mRNAs expression occurs after DNA
  replication.
• Encodes factors and enzymes packaged into the
  virion.
• Structural proteins

30
Vaccinia Assembly, Maturation and Release

• After the late gene products are synthesized,
  infectious particles are assembled.
• Assembly involves interactions with the
  cytoskeleton (e.g. actin-binding proteins and
  microtubules).
• The particles are wrapped with a Golgi-derived
  membrane and transported to the periphery of the
  cell.
• EEV picks up an additional plasma membrane as it
  is released outside of the cell.

31
Life Cycle of Vaccinia Virus
Figure 14-8
Adapted from G. McFadden, Nat. Rev. Microbiol. 3
(2005) 201-213.
32
Smallpox Eradication

• Why was smallpox (variola) a good candidate for
  eradication?
• 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.

33
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.

34
Mass-Vaccination Strategy

• WHO goal to vaccinate 100 of the human
  population.
• Over time, goal changed to containment
  vaccination or surveillance vaccination around
  newly discovered cases or outbreaks.
• This strategy evolved after countries like
  Nigeria and India experienced outbreaks in
  regions where religious groups refused
  vaccination.
• Use of smallpox recognition cards, containment
  books, rewards, rumor registers and the
  bifurcated needle.

35
Smallpox Vaccination Today

• Who is vaccinated today?
• Laboratory workers who work with poxviruses
• Healthcare workers (volunteer compliance)
• Members of the military
• Bifurcated needle, deltoid of arm
• Primary inoculation - 3 pricks
• Secondary inoculation - 15 pricks

36
Typical Smallpox Reaction
Figure 14-14
Courtesy of John D. Millar/CDC
37
Smallpox Vaccine Complications

• Based on 1960s stats, 1,000 of every 1 million
  vaccinees have a serious reaction to the vaccine
  (0.1)
• Serious side effects occur in individuals
• with skin conditions such as eczema and atopic
  dermatitis
• with weakened immune systems
• with other his risk factors such as high blood
  pressure, diabetes, and high cholesterol
• Safer vaccines are needed in case of bioterrorism
  event that would require mass-vaccination.

38
U.S. Bioterrorism and Biowarfare

• 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).
• Following the Civil War, professional grave
  robber William Cunningham delivered a corpse of a
  recently buried smallpox victim to the anatomy
  dissecting room at the Medical College of Ohio,
  Cincinnati. Several students were infected.

39
Bioterrorism is Inevitable

• 1947 NYC Eugene LeBar returned from Mexico where
  he had contracted smallpox.
• Within 3 weeks, 5.35 million NYC residents
  vaccinated.
• What would happen if a terrorist infected
  himself/herself deliberately and entered the
  United States today?