1

Department of Microbiology, Islamic Azad University,

Falavarjan Branch

Advanced Virology

dsDNA Viruses

By:

Keivan Beheshti Maal

IN THE NAME OF GOD

POXVIRUSES

Large Enveloped dsDNA Viruses

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}

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

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 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

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).

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

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

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

Proteomics of Poxvirus

Proteomics: The protein composition of virion

Aims: 1) important prerequisite for functional

studies

2) Important prerequisite for studying pathogenicity responsible proteins

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

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)

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)

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)

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)

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

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

Replication attachment - vaccinia

host cell receptors for epidermal growth factor (EGF)

VGF for vaccinia growth factor.

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

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

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[proliferative disorders 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 post‑translational processing of viral proteins structural proteins

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

Nucleic Acid synthesis

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

cytoskeletone.g. actin-binding proteins

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

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

VACV Life Cycle

AGENTS OF DISEASEIN

POXVIRUSES

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

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.

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.

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.

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

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

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.

. inoculation at other sites autoinoculation face, eyelid, or other

persons (~ 6/10,000)

Erythematous or urticarial rashes

. 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

Moderate to Severe Adverse Reactions eczema vaccinatum

localized or systemic dissemination of vaccinia virus

generalized vaccinia vesicular rash ~3/10,000

vaccinations

vaccinia necrosum severe, potentially fatal

illness progressive necrosis in

the area of vaccination

postvaccinial encephalitis 15%-25% die 25% have permanent

neurological disorders

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).

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

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

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.