Virology

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Virology
Virology
Introduction
Structure , Genome and Classification of viruses
Replication of viruses
Diagnostic: Methods for Detection and Identification of viruses
Bacteriophages
Plant viruses and viral diseases in plants
Animal viruses
RNA silencing as an antiviral defence in host
Suppression of RNA silencing by virus
Control of viral diseases (chemotherapy, transgenic plants,…)
1
References
Internet:
ICTV (International Committee on Taxonomy of Viruses)
Virology Cours www.virology.net
Book:
Plant Virology (Matthews 1991)
Journal:
Journal of General Virology
Virology
Journal of Virology
Advance in virus research
2
History of Virology
Robert Koch (1843-1910)
In 1892, Dmitri Iwanowski, a Russian botanist,
presented a paper to the St. Petersburg
Academy of Science which showed that extracts
from diseased tobacco plants could transmit
disease to other plants after passage through
ceramic filters fine enough to retain the
smallest known bacteria. This is generally
recognised as the beginning of Virology.
Louis Pasteur (1822-1895)
Dmitri Iwanowski (1864-1920)
In 1898, Martinus Beijerinick confirmed and
extended Iwanowski's results on tobacco
mosaic virus & was the first to develop the
modern idea of the virus, which he referred
to as contagium vivum fluidum ('soluble
living germ').
Also in 1898, Freidrich Loeffler &
Paul Frosch showed that a similar
agent was responsible for footand-mouth disease in cattle. Thus
these new agents caused disease
in animals as well as plants.
Freidrich Loeffler (1852-1915)
Martinus Beijerinick (1851-1931)
Frederick Twort (in 1915) and Felix d'Herelle (in 1917) were the first to recognize viruses which infect bacteria.
3
Symptom of viruses
Typically, virus infections of plants might result in effects such as
growth retardation, Stunting, mosaic patterning on the leaves, yellowing, necrosis, wilting…
Hyperplasia: excessive cell division
Hypertrophy: excessive cell growth
Roguse
Ring spot
Plum Pox Virus
4
Introduction to Virology
Virus; Virion :
Morphology: Icosahedral, bacilli form, Rod shape, filamentous
Protein + Nucleic acid
Protein; polymers of CP (coat protein = subunit)
2130 copies of CP=Capsid of TMV
10 million TMV=1% of Tobacco cell
(do not grow or undergo division, lack of apparatus necessary for the generation of
metabolic energy or for protein and nucleic acid synthesis)
Obligate intracellular parasite (They are absolutely dependent on the host cell)
They cause alteration in host cell metabolites
Diseases: HIV, Influenza, Mosaic and stunting in plant
5
Genome of Viruses
DNA or RNA, single-stranded (ss) or double-stranded (ds), linear or circular
dsDNA
ssDNA
dsRNA
+ssRNA
-ssRNA
Virus genomes range in size from 3,500 nucleotides to approximately 280 kilobase pairs
6
Terminal Structures
P
5’
3’
OH
CAP
Poly A
VPg (viral protein linked genome
tRNA like
structure
5’phosphate
3’ OH
7
Positive-sense RNA genomes Purified (+)sense vRNA is directly infectious when applied
to susceptible host cells in the absence of any virus proteins
There is an untranslated region (UTR) at the 5' end of the genome which does not encode
any proteins & a shorter UTR at the 3' end. These regions are functionally important in
virus replication & are thus conserved in spite of the pressure to reduce genome size.
Terminal Structures: Both ends of (+)stranded eukaryotic virus genomes are often
modified, the 5' end by a small, covalently attached protein or a methylated nucleotide
'cap' structure & the 3' end by polyadenylation. These signals allow vRNA to be recognised
by host cells & to function as mRNA.
+
5’
Ribosome
Proteins
8
Negative-sense RNA genomes are not infectious as purified RNA. This is because such
virus particles all contain a virus-specific polymerase. The first event when the virus
genome enters the cell is that the (-) sense genome is copied by the polymerase, forming
either (+)sense transcripts which are used directly as mRNA, or a double-stranded molecule
known either as the replicative intermediate (RI) or replicative form (RF), which serves as a
template for further rounds of mRNA synthesis. Therefore, since purified negative-sense
genomes cannot be directly translated & are not replicated in the absence of the virus
polymerase, these genomes are inherently non-infectious.
_
5’
RdRP
+
5’
Ribosome
Proteines
Ambisense Genome Organization:
Some RNA viruses are not strictly 'negative-sense' but ambisense, since they are part ()sense & part (+)sense:
Ribosome
Ribosome
9
Segmented & Multipartite Virus Genomes
Segmented virus genomes are those which are divided into two or more physically
separate molecules of nucleic acid, all of which are then packaged into a single virus
particle.
Multipartite genomes are those which are segmented and where each genome
segment is packaged into a separate virus particle. These discrete particles are
structurally similar and may contain the same component proteins, but often differ in
size depending on the length of the genome segment packaged.
Family:
Segments:
Geminivirus (group III) (single-stranded DNA)
Bipartite
Comovirus (single-stranded RNA)
Bipartite
Furovirus (single-stranded RNA)
Bipartite
Tobravirus (single-stranded RNA)
Bipartite
Partitiviridae (double-stranded RNA)
Bipartite
Bromoviridae (single-stranded RNA)
Tripartite
Hordeivirus (single-stranded RNA)
Tripartite
10
Open Reading Frames on viral genome
P0 (29KDa)
5’
RdRp
ORF0
ORF2
ORF3
ORF5
VPg
ORF1
ORF4
CP
RT
ORF3
ORF5
Subgenomique RNA 5’
ORF4
MP
Nomenclature: Polerovirus: PLRV (Potato Leaf roll Virus)
11
Gene Expression Strategies in viruses
Max proteins by Min genome
Cucumovirus +ssRNA
Geminiviridae (ssDNA),
bipartite Begomovirus
monocistronic mRNAs
RNA 1
120K
RNA 2
Polymerase
2a
RNA 3
MP
CP
NIa
Nib
1- Leaky Scan
CP
2- subgenomic RNA
BC1/MP
AC1/Rep
3- Segmented genome
2b
AC2
DNA A
4- Frame shift
5- Read through
DNA B
Potyvirus +ssRNA PVY
6- polyprotein
P1
HcPro
P3
6K1
C1
6K2
CP
VPg
PolyA
...
35K
P0 (29KDa)
5’ VPg
BV1
CMV
52K
50 71K (RNA binding/ATPase)
49K (Vpg/Pro)
58K
30K
RdRp
ORF0
ORF2
ORF1
ORF3
ORF5
ORF4
CP
RT
ORF3
ORF5
Subgenomique RNA 5’
ORF4
+ssRNA Polerovirus: PLRV
MP
12
Function of viral proteins
Structural proteins: coat protein (CP)
Capsid: protect the fragile nucleic acid genome from:
Physical damage: mechanical forces
Chemical damage: Chemical and UV irradiation (from sunlight) leading to chemical
modification.
Enzymatic damage: Nucleases derived from dead or leaky cells or deliberately secreted
by vertebrates as defence against infection.
Furthermore, the outer surface of the virus is responsible for recognition of the host
cell. Initially, this takes the form of binding of a specific virus-attachment protein to a
cellular receptor molecule. However, the capsid also has a role to play in initiating
infection by delivering the genome from its protective shell in a form in which it can
interact with the host cell.
13
Non Structural proteins:
Enzymes:
Proteinase
Polymerases: RdRP, DdRP, RT
Helicase
Replicase (polymerase + Helicase)
Other proteins:
Movement protein (MP)
Transmission protein
VPg
Protein recognizing host cell (glicoprotein)
Suppressor of RNA silencing
14
Replication of Viruses
1) َAttachment: specific binding of a viral attachment protein
Initiation phase:
•attachment
•penetration
•uncoating
(v.a.p.) to a cellular receptor
glycoproteins or glycolipids
Replication phase:
•DNA synthesis
•RNA synthesis
•protein synthesis
Release phase:
•assembly
•maturation
•exit from cell
The expression (or absence) of receptors on the surface of cells largely
determines the TROPISM of most viruses, i.e. the type of cell in which they
are able to replicate - important factor in pathogenesis.
2) penetration : unlike attachment, viral penetration is an energy-dependent
process, i.e. the cell must be metabolically active for this to occur. three mechanisms
may be involved:
translocation of the entire virion across the cell membrane
endocytosis of the virus into intracellular vacuoles; eventually into the cytoplasm.
fusion of the viral envelope with the cell membrane. requires the presence of a viral
fusion protein in the virus envelope, e.g. influenza haemagglutinin; retrovirus envelope
glycoprotein.
3) uncoating : a general term for the events which occur after penetration, in which
the capsid is removed and the virus genome exposed, usually in the form of a
nucleoprotein complex.
Retrovirus cores are highly ordered nucleoprotein complexes which contain, in addition
to the diploid RNA genome, the reverse transcriptase enzyme responsible for
converting the viral RNA genome into the DNA PROVIRUS.
15
Replication of Viruses (+SSRNA)
Replication site: Viroplasme
16
17
MATURATION
The stage of the life-cycle at which the virus
becomes infectious. Usually involves
structural changes in the particle, often
resulting from specific cleavage of capsid
proteins to form the mature products, which
frequently leads to a conformational change
in the capsid, or the condensation of
nucleoproteins with the genome. For some
viruses, assembly and maturation are
inseparable, whereas for others, maturation
may occur after the virus particle has left the
cell.
18
For viruses which replicate in the cytoplasm )Picornaviruses( the genome is simply
released into the cell, but for viruses which replicate in the nucleus ) Herpesviruses( the
genome, often with associated nucleoproteins, must be transported through the
nuclear membrane. This is achieved by interactions of the nucleoproteins (or capsid)
with the cytoskeleton. At the nuclear pores, the capsid is stripped off, and the genome
passes into the nucleus.
+ssRNA
-ssRNA
dsRNA: (segmented genome: Reoviridae) by viral RdRP, formation of – sense which
serves as template for the synthesis of +strand
dsDNA:
In Nucleus: cellular DdRP II
In cytoplasm: viral DdRP
dsDNA with RNA intermediate: (Badnavirus, Calimovirus) RT
+ssDNA: (parvovirus) in nucleus, formation of – sence which serves as template for the
synthesis of +strand, rolling cyrcle
19
Viroids are small (200-400nt), circular RNA molecules with a rod-like secondary structure
which possess no capsid or envelope which are associated with certain plant diseases. Their
replication strategy like that of viruses. Replication does not depend on the presence of a
helper virus. No proteins are made. - they are obligate intracellular parasites. 20-25 different
viroids have been identified, e.g. potato spindle tuber viroid and coconut cadang cadang
viroid.
Virusoids (Satellite Viruses) are viroid-like molecules, somewhat larger than viroids
(e.g. approximately 1000nt) which are dependent on the presence of helper virus to
encapsidate, they are packaged into virus capsids as passengers.
Sattelite Nucleic Acids
are nucleic acids that do not code coat protein and are not
able to multiply therefore they depend on a helper virus for replication. This helper virus
also encapsidates them.
Prions
consist of a single type of protein molecule with no nucleic acid component.
Confusion arises from the fact that the prion protein & the gene which encodes it are also
found in normal 'uninfected' cells. These agents are associated with diseases such as
Creutzfeldt-Jakob disease in humans, scrapie in sheep & bovine spongiform encephalopathy
(BSE) in cattle.
20
Detection of Viruses
1- Physical methods: Electron microscopy, X-ray crystallography
2- Chemical methods:
overall composition of viruses & the nature of the nucleic acid and the construction of the particle
3- Molecular Methods:
For Protein: Serological methods ELISA and Western blot
For Nucleic acid: PCR and Hybridization (Northern blot and southern blot)
21
Serology; Based on utilization of antibody
Western blot
Variable part (Bind antigene)
antigene specific
Light chain: Fab: antigene-bindidng fragment
ELISA
Heavy chain: FC: Crystalisable fragment
Constant part
Animal specific
Antibody (IgG): Polyclonal and Monoclonal
X
Specific polyclonal
antibody for virus X
Y Y
YY Y
serum
Polyclonal:
Different antibodies products
of different B cells against
different epitopes
B cells
Epitopes:
MKTFLIFVLLAMAMKIATAARELNPSNKELQSPQQSFSYQQQPFPQQPY
PQQPYPSQQPYPSQQPFPTPQQQFPEQSQQPFTQPQQPTPIQPQQPFPQ
QPQQPQQPFPQPQQPFPWQPQQPFPQTQQSFPLQPQQ
22
Monoclonal Antibody Production
Antigen is injected into a mouse to
stimulate production of antibodies.
Cancer cell, often a
B cell derived from
a tumor
Antibody
Lymphocytes from
spleen ‫طحال‬
Screen for hybrid cell that
produces specific antibodies
and clone to produce
unlimited quantities of
monoclonal antibody.
Selection on HAT medium
(hypoxanithine)
Myeloma cells
Fuse lymphocytes with
myeloma cells to produce
antibody producing hybrid
cells.
23
Serology: 1. precipitation test
and Agar Diffusion
Precipitation lines
2. ELISA
(Enzyme-Linked ImmunoSorbant Assay)
More sensitive
Rapid
Commercial kits
24
Y
3
YY
Health
YY
YY
YY
4
YY
2
YY
Infected
Indirect
Direct
Y
1
Ab-alkalin phosphatase substrate (phosphate)
Y
Y
DAS
TAS
Double Ab Sandwich
Triple Ab Sandwich
Different types of ELISA
Y
Specific Ab Virus
Immuno Blot
(DIBA)
25
3. Western Blot
Protein Extraction
Gel electrophoresis (PAGE)
Transfer on membrane
Western blot (revelation by antibody)
Enzyme: Peroxidase
Substrate: Luminol + H2O2
H I
H I
H I
Y
Gel Acrylamid
membrane
Immuno Blot
Autoradiogarphy
(film)
26
Detection of Nucleic acid:
1. PCR
Polymerase Chain Reaction: Amplification of a DNA segment
Denaturation: Denature Template DNA
94ºC
Annealing: Anneal Primers
(Forward & Reverse Oligonucleotides)
40-60ºC
Extention: Extend new strand of DNA
(DNA polymerase, DNTPs)
72ºC
27
PCR
Electrophoresis
H I
Gel Agarose
(Ethidium
Bromide)
30-40
Cycles
94ºC 10sec
50ºC 30sec
72ºC 45sec
28
RNA viruses
DNA viruses
PCR
RT
RNA
cDNA
PCR
Reverse Transcription:
Template RNA
Reverse primer
dNTPs
Reverse Transcriptase
37-50 ºC 1h30
29
2. Hybridization (Nucleic acid):
Northern Blot (RNA)
Southern blot (DNA)
RNA or DNA Extraction
Gel electrophoresis (Agarose) denaturizing (Temprature and Formamid)
Transfer on membrane
Prehybridization
Hybridization with probe (RNA or DNA)
5’
5’
Template linear DNA with a promotor (T3 or T7)
Template DNA
RNA polymerase (T3 or T7)
Primer
Mix rNTPs –UTP
PNK (poly nucleotide Kinase)
UTP (32P) radioactive
ATP (32P) radioactive
37ºC 1h30
37ºC 1h30
30
Northern or Southern Blot
H I
H I
H I
Prehybridization
probe
hybridization
Gel Agarose
Autoradiogarphy
(film)
membrane
H
Infected
31
Localization of virus in tissue
Preparation of tissue (section)
ELISA with fluorescent antibody
Observation by microscope
32

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