"class" directed review Flashcards
1
Q
provide and overview on the 4 main structural components of a virus
A
• Viruses are non-living particles outside a host cell called virions.
• Virions house the genetic material and functions as a gene transfer system.
• Viruses are made up
of 2 key components:
- Capsid: A layer of protein covering the genetic material.
- Genetic material: Either a DNA or RNA, but never both.
Other components varies between viruses: - Envelop: May have an extra outer covering of the capsid called an envelop.
- Nucleocapsid: Made up of capsid + envelop.
Note: Viruses without capsid are referred to as naked
2
Q
Provide an overview on the functions on the capsid of a virus
A
Functions of the capsid
• A capsid protects the genetic component of a virus from inactivation.
• Another function is to recognize, attach and gain entry into host cells
for genetic replication.
• Capsid ensures that the virus is transported to the right host cell
where the genome can be transcribed and replicated.
• A capsid can alters its conformation and ensures the release of the
genetic material into host cells
3
Q
Provide an overview on the components that constitute a viral capsid, and their symmetry
A
• Capsids are made up of many molecules of the same or two or more
proteins.
• Molecules making up a capsid are asymmetrical but are arranged to
form symmetrical structures.
• Symmetrical capsids entails that the appearance looks unchanged if
rotated at several angles.
• Virus capsid are Polyhedrons (exist in many forms and shapes like rod,
spiral, spike, helical).
4
Q
what are the main types of capsid symmetry
A
- Helical shape
- Icosahedral shape
- Rod shaped
- Complex shape
5
Q
Provide an example of a virus with helical symmetry
A
• Helical shape: ssRNA spiral or coiled shaped viruses. E.g. Tobacco Mosaic virus (TMV), influenza viruses, measles, some bacteriophages.
6
Q
Provide an example of a virus with rod-shaped symmetry
A
• Rod-shaped viruses: Tobacco rattle virus (2 unequal RNAs).
7
Q
Provide an example of a virus with filamentous symmetry
A
• Filamentous: some bacteriophages have either ssDNA or dsDNA.
8
Q
Provide an example of a virus with icosahedral symmetry
A
• Icosahedral shape: Polyhedron shape. E.g. herpes simplex virus,
poliovirus, parvovirus.
9
Q
Provide an example of a virus with complex symmetry
A
• Complex shaped viruses: smallpox and bacteriophages.
10
Q
Provide an overview of helical symmetry
A
• Helical shaped capsids are found in many ssRNA viruses.
• The RNA is coiled in the shape of an helix and many copies of the
same protein are arranged around the coil
11
Q
provide an overview of icosahedral symmetry
A
• Viruses with icosahedral symmetry consist of a shell made from protein molecules and arranged in the form of a scaffold to form a polyhedron.
• The virus genome have
less contact with the
capsid proteins compared
to helical-shaped viruses.
A virus icosahedron has five-, three, and two-fold axis of rotational
symmetry
12
Q
What are virus capsomeres and provide an example
A
• Are the building blocks of a virus capsid arranged from discrete structures.
• Each capsomere is usually made up of many identical protein molecules.
• For example, the capsids
of the papiloviruses are
built from 72 identical
capsomeres.
• Note that the capsids of
some viruses can also be
constructed from more
than one type of capsomere;
E.g. Herpes viruses and
Adenoviruses
13
Q
Provide an overview on what a virion envelope is
A
(Membranes)
• Viruses may have an extra layer of protection of the capsid called an Envelop.
• Envelop is a lipid-protein structure which surrounds the capsid and the genetic material (nucleic acid) inside known as a Nucleocapsid.
• Although, the envelop may be positioned in the capsid for some viruses.
• Majority of the enveloped viruses are mostly spherical or nearly spherical
14
Q
Provie a few examples of viruses wiht envelopes
A
Many viruses infecting animals possess envelops with helical
configuration such a the influenza virus, while a good number have
icosahedral shape (E.g. Herpes viruses).
• Fewer enveloped viruses infect plants (potato yellow dwarf virus),
while infection is rear among prokaryotes [Pseudomonas phage
φ(Phi) 6]
15
Q
Provide an overview of complex viruses
A
- Have both the helical and icosahedral symmetry.
- Most have a icosahedral head and a helical tail attached at one of the vertices.
- The tail is attached to the head through a protein structure referred to as a Connector or Portal.
- The connector has a powerful motor that drives the virus DNA into the young virus head during assembly.
- The tail may be long (Phage λ) or short (Phage T7).
- Some specialized structures may be attached to the tail (fibres and/or base plate).
- The head houses the genome of the virus made up of dsDNA.
- Examples are the bacteriophages with complex architecture are T7 and Phage λ.
16
Q
Overview of viral classification and nomenclature
A
• Defined viral characteristics are required prior to placing them in
specific orders, family, subfamily and genus while species grouping is
based on a number of similar characteristics.
• Thus, member of a virus species may vary in;
- Strain
- Serotypes: differences in virus antigen reaction to antibodies
- Genotype: differences in a virus gene sequence
17
Q
Virus classification based on taxonomy hierarchy
A
- Order (ending in virales)
- Family (ending in viridae)
- Subfamily (ending in virinae)
- Genus (ending in virus)
- Species (ending in virus)
- Subspecies (may end in a number)
- strain
18
Q
Modern virus taxonomy and nomenclature
A
aming of some virus taxonomic groups incorporated the old
name together with the taxonomic suffix. E.g. the generic and
family name for picornaviruses (small viruses) were derived by
placing the suffix (virales) and (viridae) respectively, in place of
the word virus
look at table 4 (purple) in the review slide set
19
Q
Genetic classification of viruses
A
• A virus is made up of genetic material which is either DNA or RNA.
• This differentiates viruses from cellular organisms whose genetic unit is a DNA.
• Viruses can be divided into four groups of based on the number of strand in its genome.
1. double-stranded DNA (dsDNA)
2. single-stranded DNA (ssDNA)
3. single-stranded RNA (ssRNA)
4. double-stranded RNA (dsRNA
20
Q
The genetic approach as suggested byDavid Baltimore
A
- DNA virus
- RNA virus
- Reverse-Transcribing Viruses
• seven classes of viruses exist based on: - type of genome
- the way in which the genome is transcribed and replicate
21
Q
What is Baltimore classification based on?
A
Viral genome replication
• Due to the unique mode of virus transcription among
dsDNA and ssRNA gene groups, the four groups of
viruses can be subdivided into seven.
• This discovery was made by David Baltimore and now
named after him.
• He developed the system for classifying viruses based
on genome type and was named Baltimore method
for virus classification.
• This classification places virus into classes I - VII
22
Q
What is the role of a capsid
A
• Capsid protects the nucleic acid when the virus is outside the host
cell.
• Helps bind to a suitable host cell surface receptors using spike
proteins on an envelop (if present) or transfer it’s genetic material
into host cell through endocytosis if it’s a naked virus (that is no
envelop).
• E.g. Poxvirus (complex virus) lacks a typical capsid and are covered by
a dense layer of lipoprotein
23
Q
What is the role of genetic material in viruses
A
• Carries gene necessary to invade host cells.
• Note that the number of genes varies for each virus (few to hundreds).
• Redirect or manipulate host cell machinery into producing viral cells.
• Viruses lack protein synthesizing machinery. However, they contain
parts needed to evade host cells and manipulate the cell replicating
machinery into producing more copies of the virus rather than normal
cellular proteins
24
Q
What are the basic steps of virus genome replication
A
- Attachment of virion to suitable host cell.
- Entry of virus into the cell
- Transcription of virus genes into mRNA (messenger RNA) molecules.
- Translation of virus mRNA into virus proteins.
- Genome replication
- Assembly of virus proteins and genomes into new virions.
- Exit of virions from host infected cells.
Note: not all viruses undergo all seven steps above, some viruses may need extra steps while some steps may occur concurrently in some viruses in which case steps 3-7 may occur at the same time
25
What are the important host compontents utalized by virions to attach to host cells
• Viruses that infect animals and bacteria must first attach themselves to host cell
surface barriers (cell wall for bacteria or cell membrane for animals)
• Cell membrane: Animal cells possess a cell membrane which the virus must
attach itself to via specific cell surface receptors in order to invade the cell.
• Cell wall: Likewise, bacteria cells possess a cell wall which the virus must attach
itself to via specific cell surface receptors in order to invade the cell.
• Note that virus attachment and entry in plants cell are mostly mediated by
vectors
26
Attachment of Animal viruses via cell receptors
* Host cell surface receptors are proteins which a virus can bind using its attachment structures prior to cell entry.
* Binding of cell surface receptors and virus attachment structures are specific like a ‘’Lock and key’’.
* Sometimes a virus may need to bind to a second host cell receptor known as a co-receptor prior to binding.
* Cell receptors and co-receptors function to mediate cell-to-cell contact and binding as well as receptors for chemokines and growth factors.
* Binding of cell surface receptors and virus attachment structures result in a conformational change in virus proteins which initiates the binding.
* Most host cell surface receptors used by viruses contain sugar molecules (glycoproteins) composed of folded domains similar to immunoglobulin.
27
Virus attachment sites
• Viruses possess multiple binding sites on their surfaces which is largely dependent on the absence (naked viruses) or presence of an envelop.
28
Virus attachment sites
naked viruses
* Naked viruses: The attachment sites for naked viruses are on the capsid which differ in topology and may be in the form of ridges (foot and mouth disease virus), or within depressions (poliovirus). Both belong to the picornavirus group of viruses.
* Also, attachment sites for some naked viruses could be on specialized structures such as spikes of rotaviruses, or on fibers and knobs of adenoviruses.
* Binding of picornaviruses to host cell receptors result in major structural changes in the virion.
29
Virus attachment sites
enveloped viruses
• Enveloped viruses: Host cell attachment structures for enveloped viruses are on glycoproteins present on the envelope.
30
Virus attachment sites
haemagglutinins
* Haemagglutinins: Some virions possess proteins which binds to immunoglobulin causing them to clump (haemagglutination). E.g. are measles virus and influenza virus.
* Forces involved in virus-host cell binding are weak forces involving hydrogen bonding, van der Waals forces and ionic forces.
* Sugar molecules on host cell receptors and /or on the virion are involved in the binding forces
* Initial binding of viruses to host cells involves weak forces which are reversible, which become irreversible with binding of more receptor.
31
Provide an overview on entry of viruses into animal cells
Entry: a virus must gain entry into host cells either
through the cell surface or through the an endosome
membrane (tiny vesicle forming part of a plasma
membrane which break offs into the cytoplasm) in a
process called endocytosis.
• Endocytosis: A process which serve several roles for
cells including nutrients uptake and defense against
pathogens is hijacked by viruses to gain entry into
cells.
32
Provide an overview of endocytosis
Entry of viruses into animal cells (Endocytosis)
• Animal viruses gain entry into host cells through two main endocytic
mechanisms:
1. Clathrin-mediated endocytosis: Clathrin is a cell protein found around
the inner side of cell membrane. Clathrin forms a coat around viruses
resulting in pit and invagination of the cell membrane which is budded
off and shed allowing the virus entry into cell. Virus examples are the
adenoviruses and vesicular stomatitis virus.
2. Caveolin-mediated endocytosis: Similarly viruses such as simian virus
40 coat themselves with cell membrane protein (caveolin) allowing
them entry into cells through endocytosis
33
what are the two main mechanisms of viral entry into host cells
• Host cell entry may occur either by
1. Endocytosis or fusion of virus envelop containing glycoprotein with plasma membrane
2. Fusion of virus envelop with the cell endosome membrane.
Virus fusion proteins are hidden in the envelop and are released upon the binding of a virus to a
host cell receptor resulting in a series of conformational changes.
The changes fuse the virus envelop and cell membrane first from the outer layers in a process called
hemifusion, followed by fusion of the inner layers, completing the fusion process
34
Intracellular transport of cell-invading virus
• Microtubules: One of the cell transport systems exploited by viruses to arrive at
the nucleus.
• Most RNA viruses replicate in the cytoplasm.
• Exceptions are:
1. Retroviruses: They copy their RNA genome into DNA in the cytoplasm which
is then transported to the nucleus during cell division (mitosis) for
replication.
2. Influenza viruses: require the cell splicing machinery in the nucleus to get rid
of intervening sequences (introns).
35
Note that the lentiviruses, e.g. HIV can transport its DNA from the cytoplasm into
the intact nucleus with cell division in progress.
• Most DNA viruses can only replicate their genome in the nucleus and so utilize
the microtubule as a transport to the nucleus before entry through the nuclear
pores (parvoviruses, herpesviruses, retroviruses and adenoviruses).
• Exemptions are poxviruses and iridoviruses: replicate their DNA in the
cytoplasm.
36
provide an overview of uncoating
* Viruses shed their capsid on entry into a host cell releasing its genome.
* For animal viruses, uncoating can occur either in the cytoplasm, nuclear pore or in the nucleus.
* Virus gene replication may not occur immediately upon successful virus-cell entry.
* Host intracellular defenses such as lysosomal enzymes may neutralize infectivity before or after virus uncoating.
* In certain cases, invading viruses may initiate a latent infection rather than begin a complete replication cycle.
* Also, under favourable cell conditions (that is virus survives and in right cell), gene replication involving transcription can commence.
37
Provide an overview of host cell entry via bacteriophages
lysozymes to aid genome injection through the cell wall.
| • Mycoplasma: Are exemptions as they lack a cell wall.
38
Provide an overview on the different types of viral genome replication
Virus genome replication is the fifth step in replication cycle in which
an invading virus makes copies of its genes for progeny offspring.
• Most DNA viruses copy their genes directly to DNA
• Most RNA viruses copy their genes directly to RNA
• A few DNA virus replicate their genes through RNA intermediate
• A few RNA virus replicate their genome through DNA intermediate
39
Replication of viruses in eukaryotic cells
• Eukaryotic viruses upon cell entry either replicate
their genome in the cytoplasm or transported into the
nucleus. Destination is dependent on the type of
genome
```
The genome of most DNA viruses
are replicated in the nucleus; but
those of some dsDNA are
replicated in the cytoplasm.
Most RNA viruses replicate their
genome in the cytoplasm with
exemption of some minus-strand
RNA viruses.
The reverse transcriptase viruses
(retroviruses and pararetroviruses)
each replicates RNA to DNA in the
cytoplasm and DNA to RNA in the
nucleus
```
40
Check out figure 11 in the review slide set
provides an overview of the enzymes for genome replication in viruses
41
Provide an overview of replication of DNA viruses
• Class I (dsDNA) and class II (ssDNA) viruses replicate their genome via dsDNA
• ssDNA synthesizes its complimentary strand to become dsDNA.
• Each viral DNA has at least one specific sequence where replication is initiated
(replication origin).
• Proteins involved in DNA replication binds to this site and they include:
1. A helicase (unwinds the double helix at that site)
2. A ssDNA binding protein (keeps the two strands apart)
3. A DNA polymerase
42
Provide an overview of replication of ds RNA viruses
• dsRNA replication is similar to dsDNA in that the double strand must
be unwound by helicase before replication can occur.
• Some dsRNA viruses replicate their genome by two mechanisms:
1. Semi-conservative mechanism: Some dsRNA viruses replicate their
genome similar to dsDNA whereby each double stranded progeny
molecule is made up of the parent strand and a daughter strand
(Pseudomonas phage phi 6).
2. Conserved mechanism: Some dsRNA viruses replicate their genome
in a process whereby the double stranded molecule of the infecting
virus genome is conserved
43
Provide and overview of replication in ss RNA viruses
• Viruses with ssRNA in class IV (+ssRNA) and class V (-SSRNA) replicate
their genome by synthesizing their complimentary strands.
• Synthesis of each RNA molecule requires the RNA-dependent RNA
polymerase at the 3’ end of the template.
• Plus-strand RNA viruses of eukaryotes replicate their RNA in
association with membranes derived from the cytoplasmic
membranous structures.
• Minus-strand RNA replicate by coating their RNA template with viral
protein and not host membrane.
44
Provide an overview of replication of rt viruses
• Some RNA viruses replicate their genes through DNA
intermediates.
• Some DNA viruses replicate their genome through RNA
intermediate.
• Both virus groups utilize the reverse transcriptase enzymes.
• Reverse transcription occurs within the viral structure in the
cytoplasm of host cell
45
list some viral culture techniques
1. Virus culture techniques:
• Virus cultivation or culture: techniques involving the use of specific characteristics of a virus for multiplication.
• Virus isolation: use for obtaining identical strains of a virus.
• Virus purification: E.g. centrifugation
46
list some virus id techniques
2. Virus identification techniques:
• Microscopy: the use of highly sensitive microscopes for the analysis of virus architecture for identification.
• Electrophoresis: separation of viral DNA and proteins by differential movement of charged particles through a membrane for identification.
• Gene analysis techniques: PCR, RT-PCR, Real-time PCR, Microarrays
47
Provide an overview of virus cultivation
• This refers to the propagation or growing of a virus for research
purposes.
2 major techniques:
1. Cell free culture (absence of living cells): Made from extraction of
internal components of different organisms for the cultivation of
viruses.
2. Cell culture: Majority of viruses requires supply of appropriate cells
to enable growth and multiplication
48
Provide and overview of virus-cell culture
• Cells for the culture of viruses are provided based on the host type
1. Phages: virus-infecting bacteria are supplied with bacteria cultures.
2. Plant viruses: may be supplied with specially grown plants or with
cultures of chloroplast.
3. Animal viruses: requires either:
• whole organism, such as transgenic mice,
• eggs containing chick embryos,
• insect larvae or
• cultured animal cells
49
Requirement for animal cell culture
cts cell line
• Continuous cell lines: This involves the propagation of viruses using
cells from animals or humans that have been immortalized either in
the body of an animal or in the laboratory.
• The cells can be sub-cultured for many years or indefinitely for
research purposes
50
Requirement for animal cell culture
media
2. Media: this provides nutrients for a virus to grow. Most media are
supplemented with animal serum which contains nutrients needed for the
growth of many cell lines.
Also, Media ensures the maintenance of optimum osmotic pressure and pH
51
Requirement for animal cell culture
growth vessel
3. Growth vessel: A virus can be cultivated in immortalized cells in glass or
plastic flask or plates where the cells bathed or suspended in the growth
medium.
Cells forming monolayer: cells growing in a single layer on a growth vessel.
Cells in suspension: Need to be stirred to keep them in suspension
52
Requirement for animal cell culture
antibiotic
4. Antibiotic: To prevent cell contamination with unwanted
| microorganisms (fungi and bacteria).
53
Requirement for animal cell culture
incubator
5. Incubator: Used to provide the optimum concentration of carbon
dioxide for the culture of viruses.
54
Requirement for animal cell culture
sterile cabinet
6. Sterile cabinet: provides a sterile work environment which help
prevent contamination of cells, self, others or the environment.
55
Provide an overview on virus isolation techniques
plaques
• Majority of viruses can be isolated due to their ability to form
discrete visible zones called plaques in layers of host cells.
• Plaques are formed as confluent areas on infected cell on the culture
plate or dish which shows signs of cell alteration or cell death.
• Plaques continues to spread as the virus infect more cells.
• Plagues could be formed as monolayers (single patches) if overlaid in
agarose gel or lawns by phages of bacterial growth (figure 5).
• Extraction and re-culturing of individual plagues result in a purified
plague
56
list a bunch of virus isolation techniques
Clone
isolate
strain
purified plaque
passaging
virus efficiency
lab strain
57
What is a clone
• Clone: If a plague is assumed to be from a single virus, it is referred to as a clone (genetically identical).
58
what is an isolate
• Isolate: Arises from a clone that in genetically identical.
59
what is a strain
• Strain: An isolate different from the parent isolate is regarded as a strain.
60
what is a purified plaque
* Purified Plaque: Are genetically pure virus strain derived from the re-culturing of a plaque derived from monolayers of the 2 or more virions.
* Passaging: A term used for each virus sub-culture process.
61
what is virus efficiency
• Virus efficiency: Viruses replicate more efficiently after repeated subculture.
62
what is a lab strain
• Laboratory strain: Occurs when an isolated virus strain has undergone numerous replication cycles in the lab and is now quite different genetically from the wild type of virus
63
Provide an overview of centrifugation
• Centrifugation employs rotational gravitational force to separate particles in a
solution by density.
• The process separates virus strain from host cell debris and other
contaminants to obtain a pure concentrate for various experimental purposes.
64
What are the two main types of centrifugation in virology
explain them
1. Differential centrifugation: involves alternating cycles of low and high
speed resulting in partial purification of the virus.
Results in partial purification of a virus
2. Density gradient centrifugation: involves the centrifugation of viral particles
in a solution of increasing concentration which separates each particle by
density. This results in a more purified form of the virus.
• Results in a more purified form of the virus using a solution of
increasing concentration and density.
• Sucrose is commonly used as a solute due to its high solubility.
• Two types of density gradient centrifugation are:
i. Rate zonal Density gradient centrifugation
ii. Equilibrium Density gradient centrifugation
65
light microscopy
1. Light microscopy: Most are not efficient in virus identification due to their low resolution
power compared to the minute size of a virus
66
confocal microscopy
2. Confocal microscope: Is a more sensitive type of light microscope used in virus investigation to
study cytopathic effects.
Generally, it employs the use of a laser to scan the virus particle, producing excellent images of thick
specimens and fluorescing specimens.
It can be used to investigate live viral cells or transfer of proteins from a virus or host cell which are
tagged with appropriate fluorescence labels. E.g. green fluorescence protein from a jelly fish protein.
Images of specimens arising from confocal microscopy can be represented in 3-D
67
electron microscopy
3. Electron microscopy: Highly sensitive and commonly used in virus investigation to obtain fine details of the
suspected organism.
Most procedure requires staining of suspected virus particles or ultrathin section of a virus infected cell in form
of a micrograph.
Two methods are used to reveal fine details of the virus or ultra-thin section of a virus-infected cell
68
em: negative staining
i. Negative staining: generate contrast using heavy metal-containing compounds. E.g. potassium
phosphotungstate and ammonium molybdate.
In an electron micrograph of virus, the stains appears as dark areas around the virions, allowing the overall
visualization of virion shape and size.
Examples are scanning electron microscope and transmission electron microscope
69
em: cryo cooling
ii. Cooled to ultra-low temperature or both
Cryo-electron microscopy: wet samples are frozen to below -1600C, freezing the water in the glass-like material.
Images are recorded while the specimen is frozen and processed by a computer. Thereafter, data from multiple
images of a specimen are constructed into a three-dimensional images of the virus particle
70
xray crystallography
• It reveals very fine details of the three dimensional structures of a
virion including DNA, proteins and DNA-protein complexes.
• It involves producing crystals of the virus particle which are placed in
a beam of X-ray, where they are diffracted based on repeating
arrangements of the molecules/atoms in the crystal.
• Analysis of diffraction patterns of molecules or atoms will help
determine their position in the cell/tissue
71
provide and overview of electrophoretic techniques
* Electrophoretic techniques: Mixtures of DNA or proteins can be separated in gel composed of agarose or polyacrylamide.
* Each DNA or protein in a mixture is visualized as a band (horizontal line) in the gel matrix after staining in appropriate reagent.
* Each DNA molecule or protein in a complex mixture is allowed to migrate through a gel matrix according to its molecular weight or size and charge which are monitored my reference markers.
* There 3 major form of electrophoresis
72
What are the three major forms of electrophoresis
1. Agarose gel electrophoresis: Used for detecting the size of a virus specific DNA using already-known DNA templates as markers.
2. One-dimensional Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE): Used for estimating molecular weight of a virus protein using known weights of proteins as reference.
3. Two-dimensional SDS-PAGE: Proteins are separated in two dimensions based on their isoelectric point and molecular weight
73
sds page
One-dimensional SDS-PAGE
showing separation of virus
proteins based on individual
molecular weights:
74
2d sds page
```
First, the proteins in the complex
mixtures are first separated
based on isoelectric charge
(horizontal arrow)
Second, the proteins are
separated based on individual
molecular weights (vertical
arrow)
```
75
what are the main categorizes of the detection of viral components for diagnosis
• The techniques can be categorized into four:
i. Detection of virions
ii. Detection of virus infectivity
iii. Detection of virus antigens
iv. Detection of virus nucleic acid
76
1. Detection of virions via microscopy
• Its involves negative staining of virions for examination using confocal or electron microscopy.
• Negative staining: generate contrast using heavy metal-containing compounds. E.g. potassium phosphotungstate and ammonium molybdate.
• In an electron micrograph of virus, the stains appears as dark areas around the virions, allowing the overall visualization of virion shape and size.
• Examples are scanning electron microscope and transmission electron microscope.
• The technique can be applied in detecting rotaviruses in faecal sample from patient suffering gastroenteritis.
• Disadvantage of the technique are; high cost of equipment, limited sensitivity
requiring a minimum detectable concentration of 106 virions
77
Detection of infectivity using cell cultures
virus infectivity
• Virus infectivity: refers to the ability of a virus to replicate itself in a
host.
78
Detection of infectivity using cell cultures
virus inoculation
• Virus inoculation: Suspected viruses in infected host cells can be
detected by inoculation of suspected specimen in a culture of cells, or
an host organism.
79
Detection of infectivity using cell cultures
microscopy
• Microscopy: After incubation in desired temperature, cultures are
observed under the light microscope for virus-induced pathology
(cytopathic effect).
80
3. Detection of virus antigens
• Virus proteins/antigens can be detected using various
immunological techniques involving antigen-antibody
interactions.
• It can be done using specific antisera or monoclonal antibodies
raised against the virus protein.
• Direct testing: Probing of virus antigen with anti-virus antibody
could be direct if antibody was probed on the antigen directly.
• Indirect testing: A secondary antibody (anti-IgG) was used
after reacting the virus antigen and anti-virus antibody to
detect the immunoreaction.
81
Detection of virus nucleic acid
hybridization
1. hybridization: Employs sequence specific-DNA probes carrying appropriate
labels for the detection of specific viral DNA or messenger RNAs (mRNAs).
• It may be done on the surface of a membrane after Southern blotting (DNA
probing) or northern blotting (RNA probing).
• In situ hybridization: Thin sections of a virus-infected tissue are probed for the
presence of virus specific nucleic acid.
• DNA microarrays: Made up of a substrate containing hundreds to millions of
tiny spots of specific DNA probes where specific DNA or RNA molecules in a
sample can be detected by hybridization with specific spots
82
Detection of virus nucleic acid
pcr
2. Polymerase chain reaction (PCR): Enables the multiplication of DNA fragment into millions of copy. Important for detecting small amount of viral DNA in a sample.
PCR technique requires specific oligonucleotide primers specific to viral sequences. Agarose gel electrophoresis can be used to separate the amplified DNA, which is detected by probing in specific labels.
83
Detection of virus nucleic acid
rt pcr
. Reverse Transcriptase (RT) PCR: The RNA of viruses can be copied to DNA using Reverse Transcriptases and amplified using Reverse Transcriptase PCR.
84
Detection of virus nucleic acid
4. Real-time PCR: Is a quantitative technique for detecting the
number of copies of a specific nucleic acid in a sample. Increase
in DNA is monitored using fluorescing labels whose glow
increases as number of DNA is multiplied.
85
what is an infectivity assay
* Measures the titer or concentration of infective virus in a specimen or preparation.
* Test virus samples are inoculated into suitable hosts: bacteria culture, plant or animal and responses are observed for infective virus
86
what are the two categories of infectivity assays
explain them
1. Quantitative assays: Host response can be assigned values. E.g. number of
plagues on a culture plate.
• A plague assay estimates the concentration of an infective virus in a culture dish and results are calculated as plague-forming unit per milliliter solution (PFU/ml).
2. Quantal Assay: It detects if a host has responded to the presence of a virus
or not depending on the desired variable.
• In cell culture, it determines the virus dose that can infect 50% of inoculated tissue culture (TCID50 : Tissue culture Infective dose)
• In animal assay, it determines either the amount of virus that can infect 50% of test animal (ID50) or cause the death of 50% of test animals (LD50).
87
provide and overview of genome sequencing
* Genome sequencing: developed by Fred Sanger and colleague at Cambridge, UK.
* It is used to determine the sequence of bases in a DNA molecule usually after PCR.
* Derived DNA sequences are search against the genome database (BLAST) to find a match which in turn provide information on the identity, role, function or characteristics of the gene.
88
what is gene manipulation
• Gene manipulation: Enable the manipulation of virus nuclei acid for
research and health purposes.
89
what are restriction endonucleases
• It involves the isolation of specific fragments of a genome using
restriction endonucleases, the cloning of the fragments in bacterial
plasmids and the introduction of specific-site mutations into the virus
genome.
90
what is gene recombination
• Gene recombination: The natural processes of recombination and re-
assortment that produce new combinations of virus genes can be
harnessed to produce new viral genotype in the laboratory.
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Investigating gene function and expression
The technique assesses the role of a virus gene by blocking its expression using specific enzymes
resulting in a mutant gene. Hence, the mutant gene is unable to replicate or perform the same role as
the wild type and becomes a conditional lethal mutant
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Techniques in studying gene function
gene mutation
1. Gene mutation: Alteration of the normal conditions (temperature, chemical) for a gene (DNA) to
generate a mutant whose functions are compared with the wild type.
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Techniques in studying gene function
reverse genetics
2. Reverse genetics: The gene of an RNA virus can be Reverse Transcribed into DNA where mutations
are induced before transcription back to mRNA.
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Techniques in studying gene function
rnai
3. RNA interference (RNAi) or RNA silencing: Short sequences of specific dsRNA molecules can be
used to inhibit the expression of virus genes and thereafter investigate the function of the gene.
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Techniques in studying gene function
microarray
4. Microarray technology: Allows researchers to monitor the expression of hundreds and thousands
of genes.
Virus RNA are detected by copying RNA from infected cells into DNA using a reverse transcriptase,
amplify the DNA via PCR, label with fluorescing dye and added to the microarray. The probes that
bind DNA from the sample are detected by scanning with a laser at a wave length that excites the
fluorescent dye
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Provide and overview on viroids
• Viriods are very tiny particles with covalently closed, single stranded circular RNA.
• The RNA of viriods are not protected by a protein coat (capsid).
• The name viriods was coined from their similarity with viruses;
• Viriods = Vir (Virus) + iod (like)
• Thus, viriods are regarded as subviruses.
• Viriod genome do not encode proteins, but are capable of independent replication in
hosts leading to disease conditions.
• Viriods are so far known to infect mainly plants, but one species have been found to
causeHepatitis D in humans
• The length of the viriod RNA varies between 246 - 434 nucleotides
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Viriods are classed into two families
1. Pospiviriodae
| 2. Avsunviriodae
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pospiviriodae
Pospiviriodae: The members of this group are named after the potato spindle tuber viriod.
Members have rod-like shape, a small single stranded region and a central conserved region.
They replicate in the nucleus, where their RNA is replicated by plant RNA polymerase II.
The ends of the synthesized RNA are ligated to form a circular RNA
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how are viroids trasmitted
1. Cell to cell transmission of viriod progeny occur through;
I) Plasmodesmata (channel between two adjacent cells through the cell wall).
II) Phloem: Progeny viriods can be transported through the phloem to infect new cells.
From these cells, viriods penetrate the pollen, ovule and seed. Young plant arising from an infected seed becomes infected with the viriod.
New infection can occur through:
2. Viriod contaminated seeds, cutting and tubers
3. Viriod contaminated equipment and implements
4. Insect vectors (aphids)
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Symptoms (Signs) of viriod infected plants
* The most common symptoms of viriod infected plants are:
* stunted growth,
* deformation of leaves and fruits,
* stem necrosis,
* death of plant (final resort)
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Provide and overview of virophages
• Virophages are viruses capable of infecting other larger viruses in a
host for the acquisition of some its needed replication machinery
from as well as parasitizes the helper virus.
• Thus, virophages are obligate parasites
• Similar to satellite viruses, but virophages mostly are parasitic to the
helper virus (large virus) resulting in death in most cases
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differentiate viriophages and satellite viruses
Early findings on virophages indicates that they belong to the satellites viruses.
• However, differences in their molecular, structural and behavioral characteristics enable the
International Committe on Taxonomy of Viruses (ICTV)to place them into separate families.
1. By definition: Virophages are not subviral agents.
According to ICTV, Virophages are viruses with functional genome which encodes structural proteins
and those required for DNA replication. Virophages possess the full complement of a virus in having
a capsid, nucleic acid which can self-assemble into a nucleocapsid and uses the cell ribosome-
encoding machinery for replication. Also, viriophages have been regarded as a small-bonifide viruses
infecting other viruses
Conversely, satellite viruses are subviral agents without genes that can code for functions needed
for replication and are dependent on coinfection of the host by a second viruses (helper viruses).
* So far, viriophages are known to be DNA viruses while satellite viruses are known to possess genomes that are either DNA or RNA.
* Virophages suppresses the replication capacity of its viral host by its parasitic mode of life thereby enhancing replication of host cells and limiting pathology, while satellite viruses may not hinder the replication of its helper virus.
* Replication in viriophages occurs almost entirely in the giant virus by hijacking the replication machinery of the virus host to replicate its own genome. Conversely, replication in satellite viruses involves the utilization of host cell machinery to initiate expression and replication of its genome in the nucleus. thereafter, transport to the cell cytoplasm to hijack the gene replication machinery of the helper virus to replicate its progeny.
* thus, the initial classification of viriophages into dsDNA satellite viruses is now placed in a divergent family of dsDNA satellite viruses infecting protists that are not subviral agents
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virophage classification
• Virophages have been classified into the virus family- Lavidaviridae.
• The lavidaviridae family is a divergent family from the dsDNA satellite
viruses infecting protists that are not subviral agents.
• Two genera have been carved out from the family:
• Genus: Sputnikvirus
• Genus Mavirus
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giant viruses
Giant viruses
• Giant viruses are the most complex of all viruses (genetically and structurally) whose genome was isolated in 2003.
• They replicate their genome inside the host cell cytoplasm and encode a large number of genes similar to some bacteria.
• The first identified giant virus was the Acanthamoeba polyphaga Mimivirus (APMV).
• AMPV has a capsid size of 500 nm, with a dense fibrile layer of about 140 nm in length.
• The genome of AMPV is dsDNA, and quite unique in having a huge size of 1.2 Mbp (megabase pairs), encoding about 979 putative proteins.
• These unique characteristics led to the creation of the virus family mimiviridae in 2005.
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Viriophages as parasites to the megaviruses
* Giant viruses were found to be hosts to viriophages by a team of researchers in 2008.
* Viriophages are the first true parasites of viruses, thought share similarity with the satellite viruses.
* They hijack the replication machinery of the prey (giant viruses) for the replication of its own genome
* Further, the parasitic activity of virophages induces partial inhibition of the giant viruses in the host cell resulting in the synthesis of defective particles which are harmless to the host.
* Molecular sequencing have shown evolutionary relationship between virophages, giant viruses and host cells as sequences of the former have been demonstrated in genome of the giant viruses and host cells.
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Phage diversity
• A variety of phages have been identified and grouped into families
based on morphological and genetic characteristics.
• Genetic characteristics may be either DNA or RNA, single or double
stranded, circular or linear, and usually present as a single copy.
• Morphology diversity in phages differs from simple, icosahedral and
filamentous phages to more complex phages with a tail and an
icosahedral head.
• Most phages are have tails.
• Phages are more common in environments where their bacteria host are
more abundant.
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whare are virulent phages
Virulent phages convert the cell replication machinery of
their bacteria host for viral gene replication resulting in
cell lysis, especially in obligately lytic phages, and release
of progeny virions. Most members undergo the phage
lytic lifecycle as opposed to the lysogenic lifecycle.
In exceptional cases, the filamentous ssDNA phage M13
releases progeny virions continously from cells without
lysis and is commonly known as a ‘’chronically infecting’’
phage
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what are temperate phages
2. Temperate phages exhibit alternating replication cycles involving:
i. Productive (lytic) infection
ii. Reductive (lysogenic) infection: occurs when the phage lies latent or dormant in the bacterial host usually due to unfavourable conditions.
The phage at the latent stage is known as a lysogen
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the lysogeny cycle
* In lysogeny, the phage genome is repressed for lytic functions and most time integrates into the chromosome of the bacteria.
* An example is the phage lambda (λ) capable of existing extrachromosomally.
* In phage P1, the prophage replicates together with the host and remain dormant until the lytic cycle is initiated usually under conditions that result in the disruption of host DNA.
* Inactivation of the repressor gene follows the damage of host DNA, initiating a lytic cycle.
* Superinfection immunity: prophages in bacterial host (resident phages) can prevent superinfection by the same or other phages by repressing the incoming phage genome resulting in protection of the host
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Genomic groups of phages
```
1. RNA phages:
• ssRNA phages
• dsRNA phages
2. DNA phages:
• ssDNA phages
• icosahedral ssDNA phages
• Filamentous ssDNA phages
• dsDNA phages
• Phage T4
• Phage T7
• Phage lambda
```
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vertebrate innate defence barriers post-infection
* Innate immune cells encountered by viruses are:
* Complement
* Interferons
* Natural killer (NK) cells
* APOBEC3 proteins
* Tetherin
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why is Vertebrate adaptive immunity against viruses important
* Is an essential outcome due to virus infection in a vertebrate host, whereby virus-specific immune response is developed.
* This is due to the presence of virus-specific antigen for which the host immune system identify and bind to specific receptors on the virus antigen known as epitopes resulting in a plathoria of events known adaptive immune response
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what are the two main types of lymphocytes
explain what they are
• The key cellular players involved in an adaptive immune response are known as the
lymphocytes.
• two classes of lymphcytes exist:
1. B lymphocytes (B cells): Are synthesized in the bursa of fabricus in birds and in the bone
marrows for mammals.
2. T lymphocytes (T cells): Are produced in the thymus.
Individual lyphocyte has receptors on the surface, specific for each epitopes on a virus
antigen.
Lymphocyes without contact with virus-specific epitopes are known as naive lymphocytes.
This group differ in their surface molecules and circulation patterns from the epitope-
specific lymphocytes
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provide a structural overview of ab
* Antibodies are made up of a group of glycoproteins referred to as immunoglobulin
* An antibody is made up of:
* two heavy polypeptide chains,
* two light polypeptide chains
* two antigen binding sites and
* fragment crystalization (Fc) region
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list some Virus-induced cancers
* Papillomavirus-linked cancers
* Polyomavirus-linked cancers
* Epstein-Barr virus-linked cancers
* Kaposi’s sarcoma
* Adult T cell leukemia
* Hepatocellular carcinoma
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What is a vaccine?
* A vaccine is any material injected or inoculated into a vertebrate, is capable of stimulating an immune response which protects against infection with specific pathogen.
* Immune response may involve induction of both B cells (which mature into antibody producing cells) or T cells (involved in cell-mediated immunity)
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Mechanism of vaccines
• The aim of the majority of vaccines is to stimulate the immune cells
(B and T cells) of the recipient to generate long-lasting immunity
against specific viral infection.
• Strong immunogenic material is required as a vaccine candidate to
generate a strong immune response against a specific or group of
virus.
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list the Types of vaccines
* Live attenuated virus vaccine
* Inactivated virus vaccine
* Virion subunit vaccines
* Live recombinant virus vaccines
* Virus-like particles
* Synthetic peptide vaccines
* DNA / RNA vaccines
