department of microbiology, islamic azad university, falavarjan branch advanced virology
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IN THE NAME OF GOD. Department of Microbiology, Islamic Azad University, Falavarjan Branch Advanced Virology dsDNA Viruses By: Keivan Beheshti Maal. Large Enveloped dsDNA Viruses. POXVIRUSES. Genus Orthopoxvirus. buffalopox virus {buffalo, cattle, human} - PowerPoint PPT PresentationTRANSCRIPT
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Department of Microbiology, Islamic Azad University,
Falavarjan Branch
Advanced Virology
dsDNA Viruses
By:
Keivan Beheshti Maal
IN THE NAME OF GOD
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POXVIRUSES
Large Enveloped dsDNA Viruses
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Genus Orthopoxvirus buffalopox virus {buffalo, cattle, human} camelpox virus {camel} (CMLV) cowpox virus {rodents, felines, bovines, human} ectromelia virus {mousepox} 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}
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Poxvirus
Variola (smallpox) virus is most closely related to camelpox
Both are believed to have evolved from a common ancestor
probably a rodent poxvirus
5,000-10,000 years BC
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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
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Virus Structure Among largest, most
complex animal viruses
brick‑shaped membrane bound viruses
Non-obvious helical or icosahedral symmetry
360 x 270 x 250 nm
Size of chlamydia under light microscope
complex internal structure
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Virus Structure "core"
biconcave = dumb bell shaped
tightly compressed nucleoprotein
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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).
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Virus Structure Core DNA
most essential genes located in the central part of the genome (highly consereved)
e.g assembly and replication
non‑essential genes are located at the ends (much more diverse)
e.g genes encode unique biological determinants:- Host range- Virulance factors- Evasion from immune system
General size: 130‑300kbp Vaccinia
- 190,000 nucleotide base pairs- completely sequenced
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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)
- in virions flanked by 2 "lateral bodies"
function unknown
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Virus Structure
surface of virus covered with filamentous protein
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
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Proteomics of Poxvirus
Proteomics: The protein composition of virion
Aims: 1) important prerequisite for functional
studies
2) Important prerequisite for studying pathogenicity responsible proteins
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Proteomics of Poxvirus
Poxvirus gene classes:
Early genes: 1) early and 2) immediate early (most of the core
enzymes) Intermediate genes
Late genes: 1) virion structural proteins 2) morphogenesis factors for assembly
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Proteomics of Poxvirus Infectious Forms:1) Intracellular Mature Virions (IMV)
2) Intracellular Enveloped Virus (IEV)
3) Cell-Associated Extracellular Enveloped Virus (EEV)
4) Extracellular Enveloped Virus (EEV) Recent Classification: IMV = MVs (Mature Virions) IEV = WVs (Wrapped
Virions) EEV = Evs
(Extacellular Virions)
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Proteomics of Poxvirus Early Studies for Protein Analysis in VACV MVs
SDS-PAGE
Combination of SDS-PAGE and N-terminal a.a sequencing
Identification of 12 unique virus encoded proteins (Takahashi et
al., 1994)
Two dimensional gel electrophoresis and a.a sequencing or immunoprecipitation
Identification of 12 unique major membrane and core
proteins (Jensen et al., 1996)
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Proteomics of Poxvirus 10 years later methods:
1) Gel-free liquid chromatography Identification of 75 viral proteins and their relative
abundance (Chung et al., 2006)
-enzymes-transcription factors-membrane proteins-core proteins- host interacting proteins
A4: most abundant protein in MV particles (core protein complexes with core protein p4a/4a; morphogenesis)
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Proteomics of Poxvirus
2) High Performance liquid Chromatography or SDS-PAGE in combination with electro-mass spectrometry
(Yoder et al., 2006)
Identificaton of 63 VACV virion proteins Confirmed the presence of most previously identified
proteins Some previously identified proteins were not
identified ( A2.5, A6, A9, A18, A21, A22, A25, A26, A28, A31, A45, C6, D7, D13, C5.5, G9, H2, H6, I2 and L5)
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Proteomics of Poxvirus
3) Gradient centrifugation with sucrose and cesium chloride (Resch et al., 2007)
Identification of 80 proteins including 69 previously identified and 11 novel protein
15 previously reported proteins were not identified Determination of 10 most abundant MV proteins:[major proteins: F17, A3, A4, A10, A17 that perform 80% of MV protein mass)]
All these studies highlight: 1)important role that different proteomics technologies
have played in detection of proteins 2) importance of defining virion purity
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Replication
Replication in cytoplasm of host cell.other DNA viruses in nucleus- Use host enzymes for DNA synthesis
Replication without any 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
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Replication attachment - vaccinia
host cell receptors for epidermal growth factor (EGF)
VGF for vaccinia growth factor.
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Replication 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
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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
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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
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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[proliferative disorders in some poxvirus
infections]
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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 post‑translational processing of viral proteins structural proteins
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Nucleic Acid synthesis starts about 1-2
hrs
makes - 10,000 copies/cell
self‑priming
may nick at one or both ends
from 3" end only - no Okazaki fragments
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Nucleic Acid synthesis
formation of high m.w. concatemers
cleaved and repaired to make virus genomes.
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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
cytoskeletone.g. actin-binding proteins
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Assembly
Inclusions formed in cytoplasm mature into virus particles Actin 'comet tails' form
- shoot IEV through the cytoplasm to the cell surface- possibly into adjacent cells- an alternative mechanism for cell to cell spread?
Highly processed and packaged genomic DNA accumulates
mature viral particles within 24 hours of infection
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Release 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
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VACV Life Cycle
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AGENTS OF DISEASEIN
POXVIRUSES
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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 rate
Incubation period is about 12 days Initial symptoms include
high fever Fatigue head and back aches
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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.
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Poxvirus Infection
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.
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History 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.
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Vaccination/ Variolizatio
n
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
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POX Vaccinaion For more than 100 years, the "vaccine strains"
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 molecularly most similar to Buffalopox
United States stopped vaccinating its military in 1989 civilians in the early 1980s Recently have started to vacccinate again
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Pox Vaccination current VACCINIA VACCINE
Dryvax: the vaccinia (smallpox) vaccine currently licensed in the United States
a lyophilized, live-virus preparation of infectious vaccinia virus
(Wyeth Laboratories, Inc., Marietta, Pennsylvania).
Previously: 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 (PFU)/ml.
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. inoculation at other sites autoinoculation face, eyelid, or other
persons (~ 6/10,000)
Erythematous or urticarial rashes
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. 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
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Moderate to Severe Adverse Reactions eczema vaccinatum
localized or systemic dissemination of vaccinia virus
generalized vaccinia vesicular rash ~3/10,000
vaccinations
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vaccinia necrosum severe, potentially fatal
illness progressive necrosis in
the area of vaccination
postvaccinial encephalitis 15%-25% die 25% have permanent
neurological disorders
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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 achieve eradication
last naturally occurring outbreak was in Somalia on 26th October 1977
Endemic smallpox was declared eradicated in 1980 by the (WHO).
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ERRADICATION possible for 4 reasons:
single stable serotype
no other reservoir for variola virus than humans Infection spreads only from close
contact with infected persons
Vaccinia virus is an effective immunogen
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ERRADICATION variola virus causes only acute infections
infected person either: dies recovers with life‑long immunity
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- minimum of 16‑17 days- incubation : 10 ‑ 12 days
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After eradication What to do with existing stocks consolidated into 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 1983
CDC keeps about 400 different strains Moscow laboratory 200 strains in Novizbersk,
Russia
extent of stockpiles in other parts of the world unknown.