Download Pox virus

POXVIRUSES
Genus Orthopoxvirus













buffalopox virus {buffalo, cattle, human}
camelpox virus {camel} (CMLV)
cowpox virus {rodents, felines, bovines, human}
ectromelia virus {mousepox, reservoir unknown}
monkeypox virus {rodents, primates, human}
rabbitpox virus {colonized rabbit only}
raccoonpoxvirus {North America}
skunkpox virus {North American striped skunk}
taterapox virus {African gerbil}
vaccinia virus {no natural reservoir}
Uasin Gishu disease {Central African horses}
variola virus {human; eradicated from nature}
volepox virus {California pinon mouse and voles}
 Variola (smallpox) virus is most closely related to camelpox
 Both are believed to have evolved from a comment ancestor
 probably a rodent poxvirus,
 5,000-10,000 years BC:
Virus Structure
 most complex of the viruses that infect
animals
 dry weight vaccinia contains
 90% protein,
 100 proteins
 functional enzymes,
 polymerases, kinases, ligases
etc.,
 structural proteins
 5% lipid
 3.2% DNA.
Virus Structure
 very large,




brick-shaped
viruses
300 x 200 nm
size of chlamydia
visible under light
microscope
complex internal
structure
Virus Structure
 "core"
 biconcave = dumb bell
shaped
 tightly compressed
nucleoprotein
Virus Structure
 Core
 linear double-stranded DNA genome
 terminal hairpin loop
advantage?
 several tandem (i.e. direct) repeat sequences
 ends of the genome form direct repeats
 inverted terminal repeats (ITRs).
 Core DNA
 most essential genes located
in the central part of the
genome
 non-essential genes are
located at the ends.
 general
 130-300kbp
 Vaccinia
190,000 nucleotide
base pairs
 completely
sequenced

Virus Structure
Virus Structure
 CORE
 Enzymes
 Host RNA polymerase is in the
cell nucleus
 Pox replicates in cytoplasm
 poxviruses use a virally-coded
DNA-dependent RNA
polymerase
 needed immediately upon
infection,
o in virions
 flanked by 2 "lateral bodies"
 function unknown
 surface of virus covered
with filamentous protein
“
ball of knitting wool".
 surface tubules
 envelope
 intracellular particles only have an
inner membrane
 IMV - intracellular mature
virions
 not host membrane
 extracellular forms contain 2
membranes
 EEV - extracellular enveloped
virions
 second derived from Golgi or ER
Virus
Structure
Replication
 general
 cytoplasm of host cell.
 other DNA viruses in nucleus
 Use host enzymes for DNA synthesis
 no host cell enzymes for DNA synthesis.
 all of the enzymes necessary for DNA
synthesis in virion
 can Replicate DNA but not mature in
enucleated cells
Replication
 attachment - vaccinia
 host cell receptors for epidermal growth factor
(EGF).
 VGF for vaccinia growth factor.
 penetration
 direct penetration of the core.
 enters cells via clathrin-coated
pits
 require an acid pH for fusion to
occur
 CAN’T fuse directly with the
plasma membrane.
 taken up by invagination of
clathrin coated pits into
endosomes.
Replication
 penetration
 endosomes become acidified,
 latent fusion activity of
the virus proteins
becomes activated
 virion membrane fuses
with the endosome
membrane.
 delivery of the internal
components of the virus
to the cytoplasm
Replication
uncoating
 two stages,
 Removal of the outer membrane as enters the cell
 particle (minus its outer membrane) is further
uncoated
 Within minutes of entry,
 viral mRNA transcripts
 early' genes.
 ~50% genome
 protein products complete the uncoating
 nucleocapsid released into the cytoplasm.
protein and NA synthesis
 viral factories,
 bounded by virally synthesized membranes
 form the envelope of released mature virus.
 proteins
 early
 VGF
 secreted
 causes non-infected cells to
divide.
o proliferative disorders seen in some poxvirus
infections.
protein and NA synthesis
 proteins
 early
 VCP
 binds to C4b
 blocks the activation of classical complement
pathway.
 protein that binds to and neutralizes interferon
gamma.
 intermediate and late genes




DNA synthesis observed then
funtion
post-translational processing of viral proteins
structural proteins.
NA synthesis
 NA
 starts about 1-2 hrs PI
 makes - 10,000
copies/cell
 1/2 end up in virions
 self-priming
 may nick at one or both
ends
 from 3" end only - no
ozaki fragments
NA
synthesis
 NA
 formation of
high m.w.
concatemers
 cleaved and
repaired to
make virus
genomes.
Assembly
 some unknown contribution from the cell
 poxvirus gene expression and genome replication occur in
enucleated cells,
 maturation is blocked.
 Assembly not understood,
 probably involve interactions with the
cytoskeleton
 e.g. actin-binding proteins
 Inclusions are formed in the cytoplasmAssembly
 mature into virus particles.
 Actin 'comet tails' form
 shoot IEV through the cytoplasm
to the cell surface
o possibly into adjacent cells
o an alternative mechanism for
cell to cell spread?
 Highly processed and packaged genomic
DNA accumulates
 mature viral particles within 24 hours of
infection,
 minority of mature
enveloped virus fuse
with the host cell
plasma membrane
 released from the cell.
 responsible for spread
of the infection
throughout body.
 Most remains
associated with the
cell.
Release
AGENTS OF DISEASE
Smallpox
 variola virus (VV) and vaccinia are the best known.
 VV strains are divided into
 variola major (25-30% fatalities)
 variola minor
 same symptoms but less than 1% death ratei
 Incubation period is about 12 days
 Initial symptoms include
 high fever
 Fatigue
 head and back aches
 2-3 days later
 lesions appear
 progress from macules to papules,





and pustular vesicles.
small blisters that itch and are
extremely painful
begin developing on the bodies
extremities
spread to the rest of body..
Twelfth day, the blisters scab over
and leave permanent pitted scars.
Death usually results if the virus
reaches the brain, heart or lungs.
 There is no other reservoir for VV but humans
 VV causes only acute infections, from which the infected
person either:
 a) dies
 b) recovers with life-long immunity
 Vaccinia virus is an effective immunogen.
 first appeared in China and
the Far East at least 2000
years ago.
 The Pharaoh Ramses V died
of smallpox in 1157 B.C.
 skin lesions found on his
mummy
 Marcus Aurelius Antonius,
 Roman philosopher-emperor,
 another victim;
 During his reign
smallpox wiped out
2,000 people a day.
History
History
 reached Europe in 710 A.D.
 Americas by Hernando Cortez in 1520.
 3.5 million Aztecs
 more than half the population died of smallpox in two-years
 ravaged George Washington's troops at Valley Forge.
 Major contributor to defeat of Crow, Dakota, Sioux,
Blackfoot, Apache, Comanche and other American
Indian tribes
History
 plague proportions in the cities of 18th century Europe
 killed five reigning European monarchs during
the 18th century.
 most serious infectious disease in The West
 accounted for a substantial proportion of
deaths,
 especially among town dwellers.
 mortality rate varied regionally,
 10% in Europe and 90% in America
 Chinese healers used
variolation
 dried scabs from smallpox
victims,
 ground to a powder
 blown up the nose.
 worked better if use variola
minor
 widely practiced in the
middle east for many
centuries,
 Turkey
 fluid from smallpox vesicles
scratched into the
recipient's arm
Vaccination/
Variolization
Vaccination/
Variolization
 Lady Mary Wortley Montagu
 early 1700's
 noted beauty
 severe facial scarring and loss of her
eyelashes from smallpox
 wife of the British Ambassador to the
Ottoman Empire.
 had her 6 year old son variolated,
 smallpox epidemic broke out in Britian
 Lady Mary got children of the aristocracy
variolated,
 still disapproved by medical hierarchy
 Jenner
 underwent variolation in 1756 and






nearly died
noted milk maids, who developed
pustules on their hands
from milking cows
did not get smallpox.
14th May 1796, used cowpox from
Sarah Nemes a dairy maid to
"vaccinate" 8 year old James
Phipps,
later challenged with variola virus
VV
showed that he was protected.
Vaccination/
Variolization
 For more than 100 years, the "vaccine strains" (many
origins) were propagated from arm-to-arm,
 Vaccination was almost universally adopted worldwide
around 1800
 for at least last 50 years, Vaccinia has been a distinct virus
from Cowpox
 origin?
 molecularly most similar to Buffalopox
 United States stopped vaccinating its military population in
1989
 civilians in the early 1980s
 Recently have started to vacccinate again
 current VACCINIA VACCINE
 Dryvax,®® the vaccinia (smallpox) vaccine
currently licensed in the United States, is a
lyophilized, live-virus preparation of infectious
vaccinia virus (Wyeth Laboratories, Inc.,
Marietta, Pennsylvania).
 Previously, the vaccine had been prepared from calf lymph
with a seed virus derived from the New York City Board of
Health (NYCBOH) strain of vaccinia virus and has a
minimum concentration of 108 pock-forming units
(PFU)/ml.
 Vaccine
administered by
using the
multiplepuncture
technique with
a bifurcated
needle.
 Side Effects and Less Severe
.Adverse Reactions
 Reaction at site
 swelling and tenderness of
regional lymph nodes,
 fever
 Approximately 70% of children
experience >1 days of temperatures
>100 F
 15%--20% of children
experience temperatures
>102
 Inadvertent inoculation at
. other sites -
autoinoculation
 face, eyelid, or other persons
(~ 6/10,000
 Erythematous or urticarial
rashes
 Moderate to Severe
Adverse Reactions.
 eczema vaccinatum,
 localized or systemic
dissemination of
vaccinia virus
 generalized vaccinia,
 vesicular rash
 ~3/10,000
vaccinations
 progressive vaccinia,
vaccinia necrosum
 severe, potentially fatal
illness
 progressive necrosis in
the area of vaccination
 postvaccinial
encephalitis
 15%--25% die,
 25% have permanent
neurological sequelae
ERRADICATION
 Less than 40 years ago, smallpox was
endemic in 31 countries.
 Yugoslavia as late as the early 1970s
 1960's over 2 million people/year die
 WHO in 1965 decided to to achieve
eradication
 last naturally occurring outbreak was in
Somalia on 26th October 1977.
 Endemic smallpox was declared eradicated
in 1980 by the (WHO).
ERRADICATION
 possible for 4 reasons:
 single stable serotype
 no other reservoir for variola virus but humans
 Infection spreads only from close contact with
infected persons
 Vaccinia virus is an effective immunogen
ERRADICATION
 variola virus causes only acute infections,
 infected person either:
 dies
 recovers with life-long immunity
 infectious from onset of eruptive exanthem,
 most commonly from days 3-6 after onset of fever.
 Thus only infectious after show signs and symptoms
 know who exposed so can isolate
 Strict quarantine with respiratory isolation
o minimum of 16-17 days
o incubation : 10 - 12 days.
After eradication
 What to do with existing stocks
 consolidated into t two collections
 1976, WHO urged 75 labs in several countries that retained
stocks of variola virus to destroy or transfer them to official
WHO repositories in U.S. and Soviet Union.
 South Africa was last to destroy its virus stocks in
December 1983.
 CDC keeps about 400 different strains,
 Moscow laboratory 200.
 Vector in Novizbersk, Russia
 extent of clandestine stockpiles in other parts of the
world unknown.
After eradication
 One year after the "last" human on earth died from
smallpox, a British photographer became infected and died of
smallpox which she contracted while taking pictures in a lab
at a medical school where some of the virus was stored
 January 1996, WHO's governing board recommended that all
stocks of smallpox be destroyed by 1999.
After eradication
 May 21, 1999: World Health Assembly (WHA), the annual
meeting of the World Health Organization, passed a resolution to
retain the variola virus stocks stored at the CDC and Vector for
three more years for purposes of bio defense research. Target 21
December 2002
 May 18, 2002 Acting on fears of bioterrorism, the 191 World
Health Organization members on Saturday formally reversed a
long-standing order for the destruction of all smallpox virus
stocks and recommended they be retained for research into new
vaccines or treatment.
Arguments for destruction
 DNA sequences of representative strains already
determined.
 Smallpox vaccine derived from different virus than
variola virus, so live virus stocks not needed for
protection.
 Opportunity costs of using maximum-containment labs,
an extremely limited resource that could better be
devoted to study of current disease agents.
 Continued work with live variola virus would entail a
small but real risk of accidental release.
Arguments for destruction
 Lack of a realistic animal model for smallpox limits the
relevance of smallpox research for understanding the
human immune system.
 Normative benefits of virus destruction: applying moral
suasion on would-be proliferators and terrorists to desist
from using smallpox as a weapon.
 Opportunity for the U.S. to demonstrate moral
leadership.
 Decision to eradicate smallpox was a collective decision
of the world community
Arguments for retention
 Live virus needed to develop defensive drugs and vaccines.
 Other poxviruses are not identical to variola and hence not
ideal surrogates.
 Testing with live virus is needed for FDA licensure of new,
safer vaccine and antiviral drugs.
 Study of viral pathogenesis could provide useful scientific
knowledge, possibly new drugs.
Arguments for retention
 Potential therapeutic benefits of variola proteins in
modulating human immune responses.
 Rogue states are not influenced by ethical or legal
norms.
 destruction of the viruses in the two known repositories does
not guarantee the complete removal of the virus from the earth
(preserved corpses of smallpox cases, forgotten or hidden
stocks elsewhere);
 Effective medical defenses offer best deterrent against
terrorist use.
 Decision to destroy the virus is politically, and not
scientifically, motivated.
Bioterrorism
The Poor Man’s Weapon of Mass
Destruction
Past use
 Jeffrey Amherst and Smallpox Blankets
 commanding general of British forces in French & Indian
war
 In battle against Chief Pontiac's forces during the summer
of 1763:
 Captain Simeon Ecuyer had bought time by sending
smallpox-infected blankets and handkerchiefs to the Indians
surrounding the fort
 Colonel Henry Bouquet to General Amherst, dated 13 July
1763, suggests in a postscript the distribution of blankets
to "inocculate the Indians";
 Amherst to Bouquet, dated 16 July 1763, approves this plan
in a postscript and suggests as well as "to try Every other
method that can serve to Extirpate this Execrable Race.
Future use ?????
 Soviet Weaponization of Smallpox
 Kanatjan Alibekov (a.k.a. Ken Alibek), the first deputy director of
Biopreparat, defected to the U.S. in 1992 and revealed the Soviet
weaponization of smallpox.
 Initial development of smallpox as a weapon occurred at Center of
Virology in Zagorsk Mass production of variola virus in eggs reportedly
began in late 1940s.
 Viral suspension was stored in multi-ton quantities at Zagorsk.
 Planned delivery by intercontinental strategic bomber and ballistic
missiles against U.S. cities (and after 1968, Chinese cities as well).
 Possible military doctrine: eliminate the survivors of a nuclear
exchange.
Future Use ????
 Development of "improved" smallpox weapons at the Vector
laboratory in Koltsovo during the late 1980s.
 Development of large-scale production method
in bioreactors.
 Genetic engineering of chimeric strains,
including transfer of toxin genes and virulence
factors from other viruses.
 Experiments initially done in vaccinia virus,
with the intent of repeating them in variola.
Future Use ????
 Undeclared Stocks of Smallpox Virus ·
 Between 3 and 8 countries SUSPECTED
 Strongest evidence implicates Russia, North Korea, Iraq.
 Circumstantial evidence, no "smoking gun":
 Vaccination of troops in North Korea, Iraq.
 1993 Russian Federal Security Service report on North
Korea's biowarfare program mentioned work on
smallpox.
Future Use ????
 Outbreak of natural smallpox in the Middle East in 1970-72: clinical
specimens could have been preserved in freezers.
 Iraqi BW research on camelpox virus, a close relative of variola that
could have served as a surrogate for studies of production and
weaponization.
 No indications of large-scale production of variola virus in suspect
countries.
 No apparent preparations for use as a weapon.
Smallpox Significance as a
Potential Bioterrorist Agent
 High aerosol infectivity;
 stability of virus in aerosols
 Infectious dose is thought to be low
 as low as a few virions)
 Increasing susceptibility of the population
Smallpox Significance as a Potential Bioterrorist Agent
 High mortality rate in the non-immune




 variola major = 20-40%
 variola minor = 1%
Potential for significant ongoing transmission due to secondary
spread
Ease of large-scale virus production
Existence of clandestine smallpox virus stockpiles
Worldwide supplies of smallpox vaccine are limited
Smallpox Significance as a Potential Bioterrorist
Agent
 prime candidate for genetic engineering
 engineer it so that the current vaccines are no longer effective
 add virulence factors to the smallpox genome (e.g. botox gene) that
would make it virtually 100% fatal.
 extremely hardy; surviving on fomites for days or weeks.
 no known treatment to abate the course of the disease other than
routine medical care.
Smallpox
 spread from one person to another by saliva droplets
 30% of susceptible contacts became infected
 face-to-face contact with the ill person.
 most infectious during the first week of illness
 there is no proven treatment for smallpox
 patients with smallpox can benefit from supportive therapy
(intravenous fluids,medicine to control fever or pain, etc.) and
antibiotics for any secondary bacterial infections that occur.
HUMAN INFECTION
 Cowpox –
 acquired by humans by milking cows
 ulcerative lesions ("milkers nodules") on the hands
 protects against smallpox
 rodents are the main reservoir of
 spreads secondarily to cows and domestic cats.
 Molluscum
contagiosum
 minor infectious
warty papule of the
skin with a central
umbilication,
 transferred by direct
contact
 sometimes as a
veneral disease.
HUMAN
INFECTION
 Monkey pox
 acquired from monkeys or wild
squirrels
 occasionally spreads from
human to human in
unvaccinated communities
 Antigenically cross-reacts with
other poxviruses
 Sick monkeys have not been
identified
 apparently healthy animals have
antibodies.
HUMAN
INFECTION
Distribution
of Human
Monkeypox
(1970 1982)
Sierra
Leone
Ivory
coast
Nigeria
Liberia
Tropical rain forest
Location of human
Monkeypox cases
Zaire
HUMAN INFECTION
 Pseudocowpox
 occurs worldwide
 disease primarily of cattle.
 In humans it causes non-ulcerating "milker's nodes".
 ORF
 worldwide occupational disease associated with handling sheep and
goats afflicted with "scabby mouth".
 In humans it manifests as a single painless, papulo-vesicular lesion on
the hand, forearm or face.
Non human infections
 Ectromelia – mouse
pox
 Fowl pox
EXPRESSION VECTORS:
 homologous
recombination in
infected cells
 introduces foreign
DNA coupled to
VV promoter
(e.g. tk) into virus
genome.
 Use to make
vaccines by
introducing gene
for key antigen(s)
Vaccinia Expression Vector
Plasmid
DNA
Recombinant plasmid
cloning vector
Foreign
gene
Vaccinia
DNA
Introduced by transfection
Nucleus
Gene from
vector
Gene from
rescuing
virus
Recomb.
of vaccinia
sequences
Replication
Maturation
Infection of cell
Infectious
vaccinia virus
“rescuing virus”
Release of
recombinant
vaccinia virus
containing foreign
gene
Foreign
gene
Vaccination w/
live virus capable
of producing
foreign
immunizing antigen
EXPRESSION VECTORS:
 Problems for human vaccines:
 % of the human population already vaccinated  lifelong protection may result in poor response to
recombinant vaccines
 Dangerous in immunocompromised hosts.
 solution use Avipoxvirus vectors, e.g. fowlpox or
canarypox
 'suicide vectors' - undergo abortive replication in mammalian
cells:
 can express high levels of foreign proteins
 no danger of pathogenesis
 no natural immunity in humans