Test 1 Flashcards
What is a virus?
A piece of bad news(Nucleic acid, DNA or RNA Genome) wrapped up in a protein
Why should we pay attention to viruses?
Because they can be zoonotic!
Defining Viruses
- Viruses are non‐living entities.
- Viruses contain nucleic acid genome (DNA or RNA) surrounded by a protein coat and, in some cases, other layers of material, such as a lipid envelope.
- Viruses do not possess standard cellular organelles such as mitochondria, chloroplasts, Golgi, and endoplasmic reticulum with associated ribosomes
- Viruses cannot make energy or proteins by themselves and have to rely on a host cell.
- All Viruses are obligate intracellular parasites. Outside the living cell, viruses are inert or dormant particles, whereas, inside the cell, the virus hijacks and utilizes the host cell machinery to produce its proteins and nucleic acid for the next generation of virus.
- Viruses do not have the genetic capability to multiply by division. The process of Virus reproduction resembles an assembly line in which various parts of the virus come together from different parts of the host cell to form new virus particles.
Can viruses grow on an agar plate?
No! They can’t grow on a non-living media
Do virusese have functional ribosomes?
No!
Virion
A complete virus particle that consists of an RNA or DNA core with a protein coat sometimes with external envelopes and that is the extracellular infective form of a virus.
CAN BE FOUND OUTSIDE OF THE HOST!
Virus
Virus is a broad general terminology used to describe any aspect of the infectious agent and includes: the infectious (Virion) or inactivated virus particle, or viral nucleic acid and protein in the infected cell.
Viroid
An infectious particle smaller than any of the known viruses, an agent of certain plant diseases. The particle consists only of an extremely small circular RNA (ribonucleic acid) molecule, lacking the protein coat of a virus.
CAUSES DISEASE IN PLANTS
Variolation
Inoculation was the method first used to immunize an individual against Small Pox (Variola) with material taken from a patient or a recently variolated individual in the hope that a mild but protective infection would result.
Louis Pasteur
injected dried, potassium hydroxide treated, infected rabbit brain material in two boys bitten by rabid dogs and both of them recovered.
Charles Chamberland
In 1884, the French microbiologist Charles Chamberland (1851–1931) invented a filter known today as the Chamberland filter. This filter had pores smaller than bacteria.
Martinus Beijerinck
called this filtered, infectious substance a “virus” and this discovery is considered to be the beginning of virology.
Oncogenic Viruses
viruses that cause cancer
Eradication of an infectious disease
involves complete elimination of the pathogen or
the disease‐causing agent from a defined geographic region.
Smallest Viruses
Porcine circovirus type 1 (17 nm diameter) Parvoviruses (18 nm diameter)
Largest Viruses:
Pandoravirus (400nm diameter)
Poxvirus (200 nm diameter & 300 nm in length)
What shape is this?
Filament-shaped
What shape is this?
Bullet-shaped
What shape is this?
Tadpole-shaped
What shape is this?
Rod-shaped
What shape is this?
Brick-shaped
What shape is this?
Spherical
Pleomorphism
the ability of some Virus to alter their shape or si
Most common shapes of virsues?
rod shaped or spherical
Electron Microscopy (EM)
Biological materials have little inherent contrast: need to be stained.
Requires negative staining with electron dense material such as Uranyl acetate, or Phosphotungstate.
Resolution range is usually 50-75 angstroms.
Detailed structural interpretation is not always possible, sometimes misleading.
This picture is from what kind of microscope?
Electron microscope
Cryo-Electron Microscopy (Cryo-EM)
Allows the observation of biological specimens in their native environment (not stained or fixed in any way) at cryogenic temperatures in EM (-180°C for liquid nitrogen stages, -269°C for He).
Resolution range is around 3.3-20 angstroms
NO STAINING!
What kind of microscope is this picture from?
Cryo-Electron Microscopy (Cryo-EM)
X- RAY crystallographic Method
4 Methods for to Determine the Morphology of Viruse
- x-ray crystallographic method
- Electron Microscopy
- Cryo-Electron Microscopy
- Nuclear Magnetic Resonance (NMR)
Why method took this picture?
X-ray Crystallographic Method
Describe a virus
A RNA/ DNA genome that in enclosed in a capsid, which is surrounded by an evelope which is a bubble of fat. There are glycoproteins in the evelope.
Capsid
the protein shell of a Virus that encases/envelopes the viral nucleic acid or genome
made up of capsomeres held together by non-covalent bonds
Most Viruses have one capsid, except Reoviruses which have a double layered capsid.
Nucleocapsid
Capsid + Virus Nucleic acid (DNA or RNA)/Genome
Capsomere
the basic subunit protein in the capsid of a virus.
What are the two most common types of capside symmetries?
Helical Symmetry and
Cubic/Icosahedral Symmetry
Helical Symmetry
- In the nucleocapsids with helical symmetry, the capsomeres and nucleic acid are wound together to form a helical or spiral tube.
- Because of the interactions between the nucleic acid and capsid protein, incomplete virions (empty helical particles) cannot form.
There are Naked Helical Virus and enveloped Helical Virus.
In all Animal Viruses, the Helical Nucleocapsid is enclosed within a Lipoprotein Envelope.
Naked Helical Nucleocapsids are common among Plant Viruses, eg. Tobacco Mosaic Virus
Cubic/Icosahedral Symmetry
An Icosahedron is a solid with 12 corners [vertices], 20 facets [Equilateral triangular faces], and 30 edges.
- Two types of capsomers are present in the icosahedral capsid. They are the pentagonal capsomers at the vertices (pentons) and the hexagonal capsomers making up the facets (hexons). There are always 12 pentons, but the number of hexons varies with the virus group.
- Seen in Spherical Viruses
We also have naked icosahedral and enveloped
Triangulation number
(T-number): Describes the relation between the number of pentagons and hexagons of the icosahedron. The larger the T-number the more hexagons are present relative to the pentagons.
Triangulation number is calculated using the formula:
Where h and k are the distances between the successive pentagons on the virus surface for each axis.
Simply remember, h and K are calculated based on the number of jumps from one pentagon to adjacent pentagon across the hexamer.
Triangulation Formula
T=h^2+ h x K+k^2
h= number of straight jumos
K= number of times that you change direction
Parvovirus
T=1, simplest icosahedron, the capsid consists of 60 copies of CP protein.
Reoviridae
The outer capsid has a T=13 icosahedral symmetry, the inner capsid a T=2 icosahedral symmetry.
Complex Symmetry:
Virions are composed of several parts, each with separate shapes and symmetries, such as Pox Viruses. Bacterial viruses (Bacteriophage) also contain complicated structures, i.e., Icosahedral heads and helical tails.
Functions of the Viral Capsid
Responsible for the structural symmetry of the Virus Particle
Encases and protects the viral nucleic acid from enzymes (nucleases), chemicals and physical conditions (pH and Temperature)
Receptor attachment proteins on Viral Capsid facilitates the attachment of the Virus to Specific Receptors on the Susceptible Host cells.
Interaction with host cell membranes to form the envelope
Uncoating of the genome in host cell
Transport of the Viral Genome to the appropriate site. May dump the genome into the cytoplasm (most + ssRNA viruses), or serve as the core of replication (Reovirus and Retrovirus)
Facilitates Specific recognition and Packaging of the nucleic acid genome
Capsid contains Antigenic sites. Determines the Antigenicity of the Virus
envelope
which is usually a lipid bilayer with embedded proteins. The envelope facilitates virus entry into host cells and may also help the virus to adapt fast and evade the host immune system. Enveloped viruses can cause persistent infections.
How is the evelope accquired?
The envelope is acquired by budding of viral nucleocapsid through a cellular membrane, such as cytoplasmic membrane, golgi membrane or nucleus membrane.
Budding only occurs at the sites where the virus specific proteins have been inserted into host cell membrane.
When does budding occur?
occurs at the sites where the virus specific proteins have been inserted into host cell membrane.
What are the two types of viral proteins found in the evelope?
- Glycoprotein
- Matrix protein
Glycoproteins
The glycoproteins are anchored in the lipid bilayer by means of hydrophobic bonds.
These proteins have a large external domain and a small cytoplasmic domain. Theyare transmembrane proteins.
They are often the spikes seen on the virus surface.
What are two types of glycoproteins in Virus Envelope?
External glycoprotein anchored in the envelope by a single transmembrane domain, and a
short internal tail. These proteins are usually the major antigens of the virus and involved in functions such as hemagglutination, receptor binding, antigenicity, and membrane fusion. Example, Hemagglutinin (HA) and Neuraminidase (NP) in Influenza Virus.
Channel proteins, which are mostly hydrophobic proteins that form a protein lined channel through the envelope. This protein alters permeability of the membrane ( e.g. ion channel). Such proteins are important in modifying the internal environment of the virus.
Neuraminidase (NP)
Release progeny virus from host cell, Antigenic
Hemagglutinin (HA)
Binding, Fusion, Antigenic, Hemagglutination
Fusion protein
Glycoprotein; fuses lipid envelope of the virus, and then releases viral proteins into the cytoplasm
pH independent fusion proteins, such as in HIV and Measles
and
pH dependent (Acidic) fusion proteins, such as HA in influenza virus
Virus Envelope Matrix Protein
These proteins link the internal nucleocapsid to the lipid membrane envelope.
Play a crucial role in Virus Assembly
Allow stabilization of the lipid envelope
Interact with the RNP (ribonucleoprotein) complex as well as with the viral membrane.
Serves as the recognition site of nucleocapsid at the plasma membrane and mediates the encapsidation of the RNA-nucleoprotein cores into the membrane envelope.
May perform other regulatory functions during virus replication.
What are the ways that viral proteins can interact with viral envelope proteins?
- Directly
- Via a matrix protein
- via a muliprotein layer
Lipid Bi-Layer in Virus Envelope
Acquired from a cellular membrane of host cell, such as cytoplasmic membrane.
Maintained only in Aqueous or Moist Environments. The Lipid envelope is sensitive
to Desiccation, Heat, and alteration of pH.
Enveloped viruses can be inactivated by the dissolution of lipid membrane with lipid solvents such as:
Ether
Chloroform
Sodium deoxycholate, Detergents, etc.
Therefore, enveloped viruses are easy to sterilize than non-enveloped viruses and cannot survive for longer periods in environment.
Virus Nucleic Acids
Can be RNA(double or single stranded) or DNA (double or single stranded)
DNA can be linear or circular
RNA- + sense( can be translated into protein right away) or negative sense- noninfectious genome
RNA can be Non-Segmented Genome (Monopartite) or Segmented Genome (Multipartite)
Positive-sense viral RNA
is similar to mRNA and thus can be immediately translated by the host cell.
Negative-sense viral RNA
is complementary to mRNA and thus must be converted to positive-sense RNA by an RNA polymerase before translation.
Mutations are more common in DNA or RNA virsues?
RNA
Antigenic drift
Outcomes:
Most cases minor changes.
Virus may become resistant to Antiviral drugs.
May cause change in antigenicity. Detrimental effect on
silent mutations
no change in the nucelo tide, but a base pair is different
nonsense mutation
A STOP codon is created, which inhibits virus sythesis
Missense Mutation
Conservative- change in the nucleotide doesn’t change the amino acid
Non-Conservative- change in the nucleotide changes the amino acid
Recombination
Intramolecular recombination involves the exchange of nucleotide sequences between different, but usually closely related, viruses during replication.
Reassortment
Reassortment is the most important mechanism for high genetic diversity in Viruses with segmented genome
usually only occurs with segemented viruses
Size of Virus Genomes DNA Viruses:
For the double-stranded DNA viruses, the complexity ranges from the relatively simple circular super-coiled genome of the Polyomaviridae and Papillomaviridae (5– 8 kbp) to the linear Herpesvirinae (125–235 kbp)
The single-stranded DNA viral genomes are either linear (Parvoviridae) or circular (Circoviridae and Anellovirus), with sizes ranging from 2.8 to 5 kbp
Size of Virus Genomes RNA Viruses:
The size of animal RNA viral genomes ranges from less than 2 kb (Deltavirus) to more than 30 kb for the largest RNA viruses (Coronaviridae)
Chemical Composition of Viruses
Viral Proteins
Constitute up to 50-70% of the Virion.
The genomes of animal viruses encode from as few as one protein to more than 100.
Virion proteins fall into two general classes: modified proteins and unmodified proteins.
Important Viral Proteins Include:
Enzymes
Structural proteins
Viral Nonstructural Proteins Regulatory proteins
Inhibitors
Enzymes
Enzymes required for mRNA transcription Enzymes required for nucleic acid replication Reverse transcriptase, etc.
Lysins:
Hydrolytic enzymes produced by bacteriophages to cleave the host’s cell wall.
Retroviral integrase (IN)
Enzyme produced by a retrovirus (such as HIV) that enables its
genetic material to be integrated into the DNA of the infected cell
Reverse transcriptase (RT):
Enzyme used to generate complementary DNA (cDNA) from a RNA template.
Nucleic acid polymerases
Viral Genome Replication.
Neuraminidases:
Enzymes that cleave glycosidic bonds. Allows release of viruses from host cell.
Structural proteins
The proteins that form the Viral Capsid. Such as VP7 and VP4 proteins of Rotaviruses
Viral Nonstructural Proteins
Proteins encoded by a viral genome that are produced in the organisms they infect, but not packaged into the virus particles. Some of these proteins may play roles within the infected cell during virus replication or act in regulation of virus replication or virus assembly. For example, NSP1-NSP5 genes of Rotavirus.
These are never in a viron
Regulatory proteins
A broad category of viral proteins that play indirect roles in the biological processes and activities of viruses. Included here are proteins that either regulate the expression of viral genes or are involved in modifying host cell functions. Many of the proteins in this category serve multiple functions. For example, products of the retroviral NEF GENE.
imporant for immune response
Incomplete Virions
Virion without Nucleic Acid [Empty Capsid]
Defective Virions
A virus that cannot replicate because it lacks a full complement/ copy of viral genes. Defective viral particles result from mutations or errors in the production or assembly of virions.
Replication of Defective Virions
Replication of Defective Virions occurs only in mixed infection with a helper virus. Helper virus can supplement the genetic deficiency and make defective viruses replicate progeny virions when they simultaneously infect host cell with defective viruses.
DIP (defective interfering particle)
When the defective viruses can not replicate, but can interfere other congeneric mature virion entering the cells, we call them defective interfering particles (DIP).
Pseudovirion
Contains non-viral genome within the viral capsid, such as host nucleic acid instead of viral nucleic acid. Look like ordinary Viral particles under electron microscope, but do not replicate.
Synthetic Viral particles (Pseudovirions) are being used to inject foreign nucleic acid into a cell, such as delivery of DNA Vaccines.
viruses have the host genome, not the viral genome
Pseudotypes
When related viruses infect the same cell, the genome of one virus may be
enclosed in the heterologous capsid of the second virus
Mechanisms of Survival of Viruses
- stability
- population size
- Host range
- Persistent Infections
- Avoid Exposure to the outside enviornment
The physical stability of a virus
affects its survival in the environment:
Example: Viruses that are transmitted by the respiratory route have low environmental stability, whereas those transmitted by the fecal–oral route have a higher stability.
Population Size is crucial
A virus may disappear from a population if supply of susceptible hosts is exhausted. This depends on size of population, immunity and pattern of virus shedding.
Host Range
Many viruses can infect more than one host. Example, Foot and Mouth Disease Virus and Rotavirus.
Persistent Infections
Virus remains in host for long periods
Avoid exposure to external environment
Vertical Transmission, Venereal Transmission, Vector Transmission
Deterimnants of Disease
Viruses depend on 3 factors:
- host factor
- Enviornment
- Pathogen
All of these factors contribute to the developement of a disease.
Determinants of Viral Disease- Environmental Factors:
Vegetation, Deforestation, Natural Calamities
Overcrowding, poor ventilation, housing
Stress factors, diet
Level of hygiene
Vector & Reservoir Populations
Virus Factors:
Evolution/Selection Pressure
Mechanisms of Genetic Diversity Transmission Efficiency
Portal of entry of Virus in Host
Tropism to Host Organs
Dose of Infection
Immuno evasion
Host factors:
Host Species
Host Immunity
Expression of Critical Receptors
Host Physiological factors (Nutrition
status, Age, Hormonal Factors, Stage
of Cell Differentiation)
Interferons, Other Inhibitors
Fever
With globalization, the study of Viral diseases has become more ________
With globalization, the study of Viral diseases has become more complex. The transmission dynamics of zoonotic pathogens are now influenced by several anthropogenic factors, such as human travel, trade of animals and other resources, deforestation, development and land use, and human demographics and behavior.
Herd Immunity
is a form of immunity that occurs when the vaccination of a significant portion of a population (or herd) provides a measure of protection for individuals who have not developed immunity.
Incubation period
refers to the interval between infection and the onset of clinical signs. In many diseases there is a period during which animals are infectious before they become sick.
Prodromal period
The first signs and feelings of illness after incubation period.
The period of early symptoms of a disease occurring after the incubation period and
just before the appearance of the characteristic symptoms of the disease.
Acute period
When the disease is at its height. Severe clinical signs.
Decline period
Period when clinical signs begin to subside
Convalescent period
The body gradually returns to its pre-diseased state, and health is restored.
Course of a Typical Disease
4 Risk Groups of Viruses
Risk Group 1: no to low individual to community risk
i.e. AAV
Risk Group 2: moderate individual risk; low community risk
i.e. herpes, foot and mouth disease, adenoviruses
Risk Group 3: high individual risk, low community risk
i.e. HIV, Hep. B, yellow fever
Risk Group 4: high individual risk and high community risk
i.e. Ebola, smallpox
Treatment is not known/ hard to get for risk 4 pathogens
four biosafety levels (BSL-1to BSL-4)
based on the risk group(s) of pathogens it handles.
Ross has a BSL- 2 lab
BSL-4 Laboratory
BSL-4 is the maximum containment laboratory. BSL-4 labs handle dangerous and exotic pathogens belonging to the highest risk group, i.e. risk group 4 (e.g. Ebola virus).
Lab workers should wear a one-piece, positively air-pressurized, HEPA-filtered, supplied- air suit.
Negative air-pressure must be maintained in the laboratory room.
Incoming and outgoing air should be HEPA-filtered
(HEPA: High Efficiency Particulate Air)
Sterilization through double door autoclaving system
Suit decontamination shower after leaving the containment laboratory area.
Biohazard
Biological substances that pose a threat to the health of living organisms, primarily that of humans
Biosafety
Laboratory biosafety describes the containment principles, technologies and practices that are implemented to prevent the unintentional exposure to pathogens and toxins, or their accidental release.
Aerosol
Very small droplets of fluid that can spread via air. Viruses can spread in lab through aerosol route.
Biosafety Cabinets (BSC)
An enclosed, ventilated laboratory workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens requiring a defined biosafety level.
Biosecurity
Laboratory biosecurity describes the protection, control and accountability for valuable biological materials (VBM, see definition below) within laboratories, in order to prevent their unauthorized access, loss, theft, misuse, diversion or intentional release.
Successful detection of viruses from a sample depends upon:
Collection of the sample from the right site
At the right time
And from the most appropriate animal
Proper Transport and Storage of Sample Performing the correct diagnostic test Proper interpretation of results
Diagnostic Laboratory also requires data on:
Epidemiological data
Case histories
Clinical signs
The Timing of Sample Collection is Important: For virus isolation
specimens should be collected as soon after onset of symptoms as possible, because maximal amounts (titers) of virus are usually present at the onset of signs . The chance of viral recovery is best during the first three days after onset and is greatly reduced beyond 5 days with many viruses.
The Timing of Sample Collection is Important: For serological tests
For serological tests, two blood specimens are generally collected – one during the acute phase of the illness and the second sample during the convalescence period (varies upon type of virus, 10-14 days after 1st sample or even more).
Can have a positive test with one titer due to exposure! This is why you MUST take 2!
The Timing of Sample Collection is Important:molecular diagnostics
As a general rule, specimens collected for molecular diagnostics, such as PCR, should be obtained during the early part of the illness.
The site from which the specimen is collected will be influenced
The site from which the specimen is collected will be influenced by the clinical signs and knowledge of the pathogenesis of the suspected virus.
The Transport and Storage of Sample is Important:
All specimens should be delivered to the laboratory
All specimens should be delivered to the laboratory as soon after collection as possible, since a loss of infectivity occurs over time.
The Transport and Storage of Sample is Important
Because of the lability of many viruses,
Because of the lability of many viruses, specimens intended for virus isolation must always be kept cold and moist
If immediate delivery is not possible,
If immediate delivery is not possible, refrigerate specimens (2 °C to 8 °C), or place them on wet ice or a cold pack.
Avoid
Avoid freezing. But, if samples must be frozen, freeze them rapidly at –20 °C or –70 °C.
Specimens for histopathologic examination should never be
Specimens for histopathologic examination should never be frozen. They should be fixed in 10% buffered formalin or fixatives.
Viral transport medium (VTM)
Stabilize the infectivity of specimens, especially swabs. VTM prevents specimen from drying, helps maintain viral viability and retards the growth of microbial contaminants.
The VTMs consist of a buffered salt solution to which has been added protein (e.g., gelatin, albumin, or fetal bovine serum) to protect the virus against inactivation and antimicrobials to prevent the multiplication of bacteria and fungi.
We don’t want the swabs to get dry.
The three potential hazards associated with transportation of pathogens are
(1) breakage of containers resulting in spillage,
(2) Resulting in exposure to possible infection, and
(3) A delay in package delivery to the diagnostic laboratory, which may have serious
implications during an outbreak situation.
To prevent spillage, it is recommended to follow the
To prevent spillage, it is recommended to follow the basic triple packaging system while transporting infectious materials.
Processing of Samples: Tissue Homogenization
Finely minced and homogenized in a glass or mechanical homogenizer. Homogenates are diluted in 1:10 and centrifuged at 10,000 g for 15 minutes
Tools:
Mortar and pestle or Ten Broeck Tissue Grinder
For Feces Samples:
Use a vortex mixer:
Phosphate buffer saline or water is added to liquefy the fecal, if pasty or solid. Then vortexed and centrifuged, supernatant collected.
For Swab Samples:
Twirl the swab in VTM and vortex the VTM in the vial.
Diagnosis/Detection of Viruses/Viral Diseases
- Clinical Signs
- necropsy
- Histopathology
Classical Swine Fever- Necropsy Sign
Turkey egg-shaped Kidney
Clinical signs for Blue tongue in Sheep
Cyanosis of tongue, appears purplish, blue
Histopathological Sign of Rabies
Negri body or inclusion bodies in an infected neuron
Detection of Viruses by Cultivation/Isolation
Cultivation/ isolation of viruses in cells/tissue culture
and
inovulation in eggs
Cytopathic effect or cytopathogenic effect (abbreviated CPE)
refers to damage or morphological changes to host cells during virus invasion.
Diagnosis/Detection of Viruses/Viral Diseases Electron Microscopy
Can be used to demonstrate viruses in samples and detect viruses that cannot be grown in in-vitro
Negative-stain electron microscopy
The virus sample is mixed with a solution of heavy metal salt that is highly opaque to
electrons (sodium phosphotungstate or uranyl acetate).
The mixture is then spread on a thin layer on a carbon-coated copper grid and dried.
How Negative stain electron microscopy works:
Following bombardment with an electron beam, the stain absorbs electrons in much
higher amounts than the sample.
On the other hand, the parts of the viral particles that are not penetrated by the stain
appear as electron-lucent (low affinity, less electron density) areas on an opaque (high
affinity, electron-dense) background.
To detect virus particles by negative-stain electron microscopy, the fluid matrix must contain
approximately 106 -107 virions per ml.
TEM
The method used in TEM is based on
transmitted electrons.
TEM seeks to see what is inside or
beyond the surface.
SEM
The method used in SEM is based on
scattered electrons.
SEM focuses on the sample’s surface
and its composition.
Advantages of SEM vs TEM
Assay
Qualitative or quantitative measurement of a target entity/analyte, such as a
drug or biomolecule.
Gold Standard Test
A diagnostic test that is considered to be the most accurate and best available under a particular condition or set of conditions.
Negative predictive value (NPV)
The probability that a negative test result accurately indicates the absence of infection.
Positive predictive value (PPV):
The probability of a positive result accurately indicating the presence of infection.
Sensitivity
The probability (percentage) that cases with the infection (determined by the result of the reference or ‘gold standard’ test) will have a positive result using the test under evaluation.
Specificity
The probability (percentage) that cases without the infection (determined by the result of the reference or ‘gold standard’ test) will have a negative result using the test under evaluation.
Serum
The clear yellowish fluid obtained upon separating whole blood into its solid and liquid components after it has been allowed to clot.
The clot is removed by centrifugation and the resulting supernatant, designated serum, is carefully removed using a Pasteur pipette.
How to collect Serum
Red-top Vacutainer Tube
Plasma
is produced when whole blood is collected in tubes that are treated with an anticoagulant. The blood does not clot in the plasma tube. The cells are then removed by centrifugation. The supernatant, designated plasma is carefully removed from the cell pellet using a Pasteur pipette.
Plasma – clotting factors
Plasma – clotting factors = Serum
How to collect Plasma
Lavender-top EDTA Vacutainer Tube
Enzyme-Linked ImmunoSorbent Assay (ELISA):
Typical ELISA:
Antigen coated in a well
Add antibody tagged with an Enzyme
Antigen binds to enzyme-tagged antibody
Wash the excess unbound antibodies
Add substrate
Enzyme tagged to antibody which is bound to antigen will change color
of substrate. Intensity of color indicates more positive reaction.
Direct ELISA:
Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration. The specificity of the primary antibody is very important.
Indirect ELISA:
Primary antibodies are not labeled, but detected instead with enzyme- conjugated secondary antibodies that recognize the primary antibodies.
Sandwich ELISA:
The antigen to be measured is bound between a layer of capture antibodies and a layer of detection antibodies. The two antibodies must be very critically chosen to prevent cross-reactivity or competition of binding sites.
Competitive ELISA
The antigen of interest from the sample and purified immobilized antigen compete for binding to the capture antibody. A decrease in signal when compared to assay wells with purified antigen alone indicates the presence of antigens in the sample.
Unlabeled antibody is incubated in the presence of its antigen (sample).
These bound antibody/antigen complexes are then added to an antigen-coated
well.
- The plate is washed, so unbound antibody is removed. (The more antigen in the sample, the more Ag-Ab complexes are formed and so there are less unbound antibodies available to bind to the antigen in the well, hence “competition”.)
- The secondary antibody, specific to the primary antibody, is added. This second antibody is coupled to the enzyme.
- A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent
signal.
- The reaction is stopped to prevent eventual saturation of the signal.
- Weaker signal indicates presence of antigens in sample.
Fluorescence Antibody Test (FAT)
The antibodies are labelled with a fluorescent dye (most commonly used is fluorescein isothiocyanate [FITC] or rhodamine). Visible fluorescence appears following antigen- antibody reaction.
Direct FAT
Labelled antibodies are added onto the sample (Antigen). Visible fluorescence
appears at the binding sites of the specific antibodies (antigen-antibody binding).
Indirect FAT (IFAT)
IFAT employs a secondary antibody labeled with a fluorescent marker
that recognizes the primary antibody bound to antigen.
Immunohistochemistry
The antibody is tagged with a enzyme, generally horseradish peroxidase. The enzyme reacts with a substrate to produce a colored product that can be visualized in the infected cells with a standard light microscope.
Direct assay
Enzyme tagged with primary antibody that binds to antigen. Upon
successful antigen-antibody binding, tagged enzyme catalyzes substrate to produce
color product.
Indirect assay:
Enzyme tagged to a secondary antibody that is specific against
primary antibody.
Immunochromatography (lateral flow devices)
A form of POC (Point-of-Care) test that is simple to perform, easy to carry, and does not require specialized equipment.
i.e. pregnacy tests
Point of Care (POC)
Diagnostic testing performed at or near the patient’s site of care.
How lateral flow devices work:
Lateral Flow Assay Archeicture
One of the antibodies is immobilized on the chromatographic paper, and the other is labeled with colloidal gold and infiltrated into sample pad.
When the liquid sample is dropped on the sample pad, the antigen in the sample forms an immunocomplex with the antibody labeled with colloidal gold.
Its complex moves along with the liquid sample in the nitrocellulose membrane, and makes a contact with the antibody immobilized on the membrane, followed by forming an immuno- complex with the immobilized antibody, resulting in generating a colored red purple line.
Appearance of this red purple line on the membrane indicates the presence of antigen of interest in the sample.
The excess unbound colloidal gold labeled antibodies migrate further forward and bind to antibodies in membrane against these antibodies, also yielding test control band.
Agglutination
Agglutination is a method using the property of specific antibodies to bind many antigens (antigens on pathogen, or antigen coated particles- latex beads) into single clumps thereby forming large complexes, which are easily precipitated. The precipitation can be macroscopically or microscopically visible.
Same Disease, Different Names
Disease caused by same virus, but known by different names in different parts of the world. Example, Hog Cholera in North America is known as Classical Swine Fever in rest of the world. Both are same disease caused by members of genus Pestivirus.
However, the Disease linked Nomenclature/Naming of Viruses may cause Confusion
Different Viral Diseases, but known by the same name.
Example, Hand-Foot and Mouth Disease in Humans is different from Foot and Mouth Disease of Livestock. The former being caused by members of genus Enterovirus, whilst later being caused by members of genus Aphthovirus.
The Baltimore Classification system
Baltimore classification (first defined in 1971) is a classification system based on Viral Genome.
Using this system, viruses are classified into one of the seven groups depending on a combination of their nucleic acid (DNA or RNA), strandness (single-stranded or double- stranded), Sense, and method of replication.
Another Baltimore Classification System Graphic
The International Committee on Taxonomy of Viruses (ICTV) Classification
The International Committee on Taxonomy of Viruses (ICTV) Classification System is followed by Virologists Worldwide
The ICTV is the only body charged by the International Union of Microbiological Societies with the task of developing, refining, and maintaining a universal virus taxonomy.
Established in 1966.
Considers the following for Classification of Viruses: Nature of Virus Genome and Virus Genetic Diversity Virus Replication Strategies
Virus Morphology
An example of ICTV Classification System Example: Herpes Simplex Virus
Order: Herpesvirales
Family: Herpesviridae
Subfamily: Alphaherpesvirinae
Genus: Simplexvirus
Species: Herpes simplex virus 1 (HSV-1) Herpes simplex virus 2 (HSV-2)
Inclusion Bodies in Host Cell during Viral Infection
An abnormal structure in a cell nucleus or cytoplasm, such as aggregates of proteins, having characteristic staining properties and associated especially with certain viral infections
Found in Nucleus or Cytoplasm, or for some viruses in both
Positive Sense Genome
can be translated into a protein
negative sense genome
Can’t be translated into the genome
Is it easy to cultivate viruses on an agar plate?
Viruses do not have the genetic capability to multiply by division. Outside the host cell, they are inert/dormant particles.
Viruses need a Living Host Cell to Replicate and Generate Next Progeny of Viruses. Inside the host cell, the virus hijacks and utilizes the host cell machinery to produce its proteins and nucleic acid for the next generation of virus.
How can you cultivate viruses?
- Cell/Tissue Culture
- Inoculation in Embryonated Egg
- Laboratory Animals
Organ Culture
Parts of an organ or a whole organ are cultured in-vitro. New Technique. Technical difficulties
CELL CULTURE
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.
Cell culture involves the growth of dispersed cells in-vitro, either as cells in suspension, or as a monolayer on a solid surface such as a inner surface of polystyrene culture flask.
Suspension cultures
Cells which do not require attachment for growth or do not attach to the surface of the culture vessels. Can be propagated in suspension.
Monolayer cultures
When the bottom of the culture vessel is covered with a continuous layer of cells, usually one cell in thickness, they are referred to as monolayer cultures.
Primary Cell Culture
This is the maintenance of growth of cells dissociated directly from the parental tissue (such as kidney or liver) of human or animal origin.
Cells have the same chromosomes and same number of chromosomes as the original tissue. They are morphologically similar to cells of parent tissue
Monolayer
Advantages and Disadvantages of Primary Cell Culture
Advantages:
Best culture systems for isolation and propagation of viruses.
Heterogeneous – many cell types
Closest to animal (Similar to Animal Tissue cells)
Used in producing Viral Vaccines
Disadvantages::
Difficult to obtain
Relatively short lifespan in culture [Capable of only 5-20 subcultures] Very susceptible to contamination
May not fully act like parent tissue due to complexity of culture media
Secondary Culture/Transfer Culture
When a primary culture is sub-cultured, it becomes known as secondary culture or cell line.
Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture vessel.
This is periodically required to provide fresh nutrients and growing space for continuously growing cell lines.
You grow the cells on two plates.
Cell Line
After the first subculture, the primary culture becomes a cell line
Finite/Diploid Cell Lines
The cell lines which have a limited life span and go through a limited number of cell generations.
Homogenous population of a single cell type – fewer cell types
Limited life span: May be sub-cultured up to 100 times before the cells die
Derived mainly from embryos; or from secondary cell cultures
Cell retains original morphology and diploid chromosome number
These cell lines exhibit the property of contact inhibition, density limitation and anchorage dependence
The growth rate is slow and doubling time is around 24-96 hours
Technically, less hassle to use
Continuous Cell Lines
Acquire the ability to divide indefinitely.
Continuous cell lines/Immortal Cell Lines/Heteroploid Cell Lines –
Cell cultures of a single cell type - Most homogeneous
Derived directly from cancer cells; or induced transformation of a primary or diploid cell strain to divide indefinitely
Genetically weird – furthest from animal. Abnormal morphology & chromosome number
Absence of contact inhibition and anchorage dependence
The growth rate is rapid and doubling time can be 12-24 hours
Hassle free to use
FDA prohibits their use in Vaccine Production
HeLa Cells- Example
Fibroblastic
(or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate.
Epithelial-like
cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches.
contact inhibition
is a growth mechanism which functions to keep cells growing into a layer one cell thick (a monolayer). If a cell has plenty of free space, it replicates rapidly and moves freely.
. Epithelial-like
cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches.
Lymphoblast-like
cells are spherical in shape and usually grown in suspension without attaching to a surface.
float, detached
Culture Medium
Provides all the necessary nutrients (amino acids, inorganic salts, Vitamins and Glucose) required for growth of Cells
i.e. Eagle’s Basal Medium, Leibovitz L-15 Medium
Serum in Culture Media
Vital source of adhesion factors, attachment and spreading factors, low molecular weight nutrients, and hormones and growth factors.
Carrier proteins for lipoid substances and trace elements into the cell
Regulate cell membrane permeability
The most widely used animal serum supplement is fetal bovine serum, FBS.
For the growth medium, you add _____ serum.
5-10 % serum
For maintenanace medium, you add _____
0-2 % serum
How do you tell the pH of the tissue culture?
Phenol red pH Indicator
red- acidic
orange- alkaline
Why would the pH change?
CONTAIMINATION! it will turn orange
Carbon dioxide (CO2) Level
The growth medium controls the pH of the culture and buffers the cells in
culture against changes in the pH
Usually, this buffering is achieved by including an organic (e.g., HEPES) or CO2-bicarbonate based buffer.
Changes in the atmospheric CO2 can alter the pH of the medium.
Therefore, it is necessary to use exogenous CO2 when using media buffered
with a CO2-bicarbonate based buffer
4 – 10% CO2 is common for most cell culture experiments.
Antimicrobial Agents
To prevent contamination with bacteria, mycoplasma, yeast, molds, etc.
Temperature of the Cell Culture
Cell line and Temperature
Human and Mammals- 36-37
Insect Cells- 27
Avian Cell lines- 38.5
Cold- blooded animals- 15-26
Proteases
Trypsin and Collagenase
Dissociation/ Detach Cells
transfering cell into a new medium plate
Treat primary cell culture with Trypsin and EDTA, which releases the cells.
Dissociation of Cells from Culture Vessels with Enzyme-free Cell Dissociation Buffers
Incubating cells with too high a trypsin concentration for too long a time period will damage cell membranes and kill the cells. Therefore, researchers now often use enzyme-free dissociation buffers to maintain integrity of cells.
Containers to grow Viruses
- Polystyrene Flasks
- Polystyrene Dishes
- Microwell Plates
- Roller Bottles
- Leighton Tube
How do you know if your cells are growing?
You can check using an inverted Tissue Culture Microscope
Cytopathic effect or cytopathogenic effect
(abbreviated CPE) refers to damage or morphological changes to host cells during virus invasion.
The Shell Vial Technique
Shell Vial: Small borosilicate glass vial with a coverslip
grow the cells on a coverslip
Co-Cultivated Cells
- Single monolayer consisting of multiple [different] cell types
- Ideal for the isolation of multiple viruses • Detection of viral antigens
Detected using a pool of fluorescein-labeled monoclonal antibodies
R- Mix
is a mixed monolayer of mink lung cells (strain Mv1Lu) and human Adenocarcinoma cells (strain A549). In combination, these cells support the detection of many viruses, in particular those of the respiratory group that includes Influenza A and B, RSV, Adenovirus, and Parainfluenza viruses 1,2, and 3.
Co-cultured cells
What do you need to grow viruses in eggs?
Specific-Pathogen-Free
(SPF) Eggs
Egg Candler
Betadine
Sterile Swabs
Drill
Micro Drill Bits
Do you want to use fertile eggs?
YES
Routes of Egg Inoculation
Evidence of Virus Growth in the eggs
- Death of the embryo
- Paralysis [sluggish movement]
- Stunted growth
- Urate deposits in the mesonephros • Hemorrhage and congestion
- Hemagglutins in embryonic fluids
- Extracellular membrane lesions
Pocks on Chorioallantoic Membrane (CAM)- Pocks formed by vaccinia virus on CAM
Rate-Zonal Centrifugation
-Type of ultracentrifucation
Sample is layered as a narrow zone on the top of a density gradient, such as Sucrose of different densities. Under centrifugal force, particles move at different rates depending on their mass.
particles that are light will settle down slower and vise versa
Sucrose gradient centrifugation
Isopycnic Centrifugation
Buoyant Density: If the object has exactly the same density as the fluid, then its buoyancy equals its weight. It will remain submerged in the fluid, but it will neither sink nor float.
Isopycnic Point: The point where the buoyant density of as particle equals that of the surrounding Density Gradient Medium.
Suitable Density Gradient Medium:
Sucrose
In isopycnic separation, also called buoyant or equilibrium separation, particles are separated solely on the basis of their buoyant density. By this method, the particles will never sediment to the bottom of the tube, no matter how long the centrifugation time. Starting with a uniform mixture of sample and density gradient under centrifugal force, particles move until their density is the same as the surrounding medium (Isopycnic Point).
Cesium chloride
Hemagglutination assay
adding viruses, causes the RBC to clump
If there is no hemmagglutination, then the RBC settle down at the bottom of the tube and form a button
Plaque
Monolayer Plaque Assay
Plaque: A circular zone of necrotic cells surrounded by viable cells in a monolayer.
The assay is elegantly simple and is the most accurate of the quantitative biological assays.
Only viruses that cause visible cell damage of cultured cells can be quantitated using this assay. It is a functional measurement, and has no relation to actual number of viruses.
Unit: Plaque-forming Units/ml (PFU)
Measures the number of virus particles capable of forming plaques per unit volume. For example, if a solution has a PFU of 1000 pfu/ml, then every ml of solution contains enough virus to form 1000 plaques.
The Principle of Plaque assay
Each plaque respresents cel lysis initiated by one viral particle(agar restricts movement so that virus can infect only contiguous cells)
Transformation Assay
• Quantitative determination of titers of oncogenic viruses
Oncogenic viruses transform cells in culture
- Transformed cells lose contact in- hibition and become heaped upon one another
- Unit: Focus-forming units/ml
Transformed cells
Loss of contact inhibition
Instead of a mono-layer, you get clumps of cells!
Calculation of TCID50 Using the Reed and Muench Method
Adding this proportional factor to the dilution next above 50% 10-2.3
A virus suspension with dilution 10-2.3 represents 1 TCID50/0.1ml, i.e. at such dilution 50% of cultures inoculated will become infected. Titer is 102.3 TCID50/0.1ml
One-Step Virus Growth Curve
- Let us infect monolayer cell cultures with a virus
- Perform sequential sampling of virus at different time intervals
• Examine the Virus titer by measuring:
PFU/ml of intracellular virions and extracellular virions
- You have a monolayer, which you add the viral suspension to.
- Allow ~1 hr for virus to adsorb in cells. Culture cells
- Collect aliquots of Cells and Cell-Culture fluid at different time intervals for measuring titer of intracellular and extracellular virions
Burst size: Number of Virions released. Height of curve = Burst size
Steps of Viral Replication
- Attachment- very specific lock and key reaction
- Penetration
- Uncoating- open it’s protein capsid
- Synthesis of Viral Components (Nucleic acid and Protein)
- Assembly and Maturation
- Release in large numbers
Co-Receptor
In some cases, binding to a cellular receptor is not sufficient for infection. An additional cell surface molecule, or co-receptor, is required for entry.
Endocytosis
{Endo (within) cytosis (cell) } is a process in which a substance gains entry into a cell without passing through the cell membrane. The process involves invagination and pinching off of small regions of the cell membrane, resulting in the nonspecific internalization of molecules .
Membrane Fusion/Surface Fusion only happens in ______________
Only Enveloped Viruses
Viral glycoproteins are retained on the cell surface, and since these are antigenic, the cell can become a target of the immune system of the host.
Pore-mediated penetration of viral genome into host cell
(Non-enveloped Viruses)
Some non-enveloped viruses inject their genome into the host cytoplasm through creation of a pore in the host membrane.
Antibody Mediated Attachment and Penetration
i.e.
Feline Infectious Peritonitis (FIP)Virus
- where a defective antibody attachs to the surface of the virus and it allows the virus to enter the host cell
At same time, Antibodies against spike protein of FIP virus cannot clear the virus from host. Rather these antibodies bind to FIP virus spike proteins and facilitate entry of viruses into host cells through the antibody (IgG)-Fcγ receptor. These enhances the entry of viruses into host cell.
- Virus Uncoating
Release of Viral Genome in Host Cell.
Virion can no longer be detected; known as the “eclipse period”
Uncoating
Some animal viruses begin to uncoat after binding to the external receptor, but are not completely uncoated until inside the cell (Poliovirus).
Uncoating in some animal viruses requires a complex series of steps involving both host and viral gene products (Poxvirus).
In some other viruses, such as retroviruses and reoviruses, the first stages of the viral replication cycle (transcription, replication) actually occur inside the capsid.
Loss of infectivity of Virions.
Flow of Genetic Information in a Eukaryotic Cell
Replication of a Double-Stranded DNA Viral Genome and production of Viral mRNA
Replication of a Single-Stranded DNA Viral Genome and Production of Viral mRNA
Replication of a Double-Stranded RNA Viral Genome and Production of Viral mRNA
Replication of a Single-Stranded Minus RNA Viral Genome and Production of Viral mRNA
Replication of a Single-Stranded Plus RNA Viral Genome and Production of Viral mRNA
viral genome serves as both erves both, as genomic & mRNA
Replication of a Single-Stranded Plus RNA Viral Genome and Production of Viral mRNA by way of Reverse Transcriptase
Replication of Double-Stranded DNA Viral Genome and Production of Viral mRNA by way of Reverse Transcriptase
Polycistronic mRNA
- can either translate into polyprotein and then proteases turn it into functional proteins
- or an endonuclease turns into monocistronic mRNAs, which are then translated into functional proteins
i. e. HIV: protease inhibitors
Budding Through the Plasma Membrane (Enveloped Viruses)
Exocytosis
Flaviviruses, Arteriviruses, Coronaviruses and Bunyaviruses mature by budding through the membranes of Golgi apparatus or Endoplasmic Reticulum (ER). Vesicles containing the virus then migrate to the plasma membrane and are released by exocytosis.
Part on the right- exocytosis
Replication of Retroviruses
The Iceberg Concept of Infection and Disease
Course of a Typical Acute Infection
Virus Spread in Host
A. Local Spread on Epithelial Surfaces
- Replicate in epithelial cells at the site of entry
- Local spread by infecting contiguous cells
- Produce localized infections
- Shedding
- May or may not proceed to adjacent subepithelial tissues or beyond
Neural Spread of Viruses to CNS through Olfactory Routes
can enter from the blood or from the nose
“Bulls Eye”
Rash of Lyme Disease
Viral-bacterial synergism
Infection of the Respiratory Tract
E. Viral Infection of the Hemopoietic System
Damage to Endothelium
Hemorrhages
Petechiae hemorrhage (Pin-Point/Small spots )
Ecchymoses hemorrhage( on bottom right) (larger areas of hemorrhage, ill-defined margins)
Disseminated intravascular coagulation [DIC]
Complication arising from Viral Infection of Blood Vessels.
Widespread activation of the clotting cascade that results in the formation of blood
clots in the small blood vessels throughout the body.
Some of these clots can clog the vessels and cut off blood supply to organs such as
the liver, brain, or kidneys.
Lack of blood flow can damage the organ and it may stop working properly, often
resulting in Ischemia and Necrosis.
Over time, the clotting proteins in your blood are consumed or “used up.” When this
happens, severe bleeding can occur from various sites.
F. Transplacental Infection of the Embryo and Fetus
Congenital hydranencephaly, calf showing recumbence, depression, and a slightly dome-shaped skull (BVDV
infection)
Porencephaly (Bovine Viral Diarrhea Virus, BVDV)
Transplacental Transmission of Bovine Viral Diarrhea Virus (BVDV) and Outcomes
Non-pregnant animal Usually Mild infection, Scours, Milk drop, reduced WBCs
Vasculitis mediated by Immune-Complex Formation
Spike proteins on Feline Infectious Peritonitis Virus (FIPV) bind to antibodies. These antibodies fail to neutralize the virus. Instead there is antigen-antibody immunocomplex formation which activates the complement cascade and the result is vasculitis (inflammation in the blood vessels) and edema.
Feline Infectious Peritonitis
Feline Infectious Peritonitis (Immune Complex-Mediated Vasculitis)
Distended abdomen
Latent Infection (Persistent Infection)
Latent infections, in which infectious virus is not demonstrable except when reactivation occurs. Reactivation is often stimulated by immunosuppression and/or by the action of a cytokine or hormone.
Viral latency may be maintained by:
Restricted expression of genes that prevents the expression of proteins which,
otherwise, would have killed the infected cells.
Virus genome maintained indefinitely in cell by integration of viral nucleic acid
into host cell DNA, or by carriage of viral nucleic acid in episomal form.
Example: Infectious Bovine Rhinotracheitis
Chronic Infection (Persistent Infection)
Acute infection followed by chronic infection in which the virus is continuously shed from or is present in infected tissue.
Established if host immunity is unable to clear virus from acute infection. Not all chronic infection may start as acute infection.
Example: Foot and Mouth Disease in Cattle
Slow Infection (Persistent Infection
Prolonged incubation period, lasting months or years.
Quantities of infectious virus gradually increase during a very long preclinical
phase
Slow progressive lethal disease. Example: Prions
Cytopathic effects (CPE)
Cell lysis
Cell rounding
Cell detachment
Vacuoles in cytoplasm
Inclusion bodies
Syncytium formation
Antigenic changes in cell membrane Swelling and Clumping
Cell fusion (Syncytium or polykaryon formation)
Involves the fusion of the plasma membranes of four or more cells to produce an enlarged cell with four or more nuclei. Prone to premature Cell Death.
Result from the fusion of an infected cell with neighboring infected or uninfected cells.
Enveloped viruses specifically direct the insertion of their surface glycoproteins, including fusion proteins, into host‐cell membranes as part of their budding process, often leading to membrane fusion and syncytium formation.
Syncytia formation may be the only detectable CPE of some paramyxoviruses.
Inclusion Bodies in Host Cell during Viral Infection
An abnormal structure in a cell nucleus or cytoplasm or both, such as aggregates of proteins, having characteristic staining properties and associated with certain viral infections.
Help to identify certain viral infection.
Inclusion bodies can be:
Accumulation of viral components, such as Negri bodies consist of ribonuclear proteins
produced by the rabies virus.
Result from degenerative changes in cell, such as Owl’s eye inclusion bodies seen in
herpesvirus infection
Crystalline aggregates of virions, such as in adenovirus infection.
Apoptotic Pathways
Activation of host‐cell caspase enzymes mediate death of the cell. Once activated, caspases are responsible for degradation of the cell’s own DNA and proteins.
Antibody-Dependent Cell Mediated Cytotoxicity
N/K cells
The Cell Cycle
The story of Oncogenic Papillomaviruses
Papillomaviruses produce papillomas (warts) on the skin and mucous membranes of most animal species.
These benign neoplasms are hyperplastic epithelial outgrowths that generally regress spontaneously.
Occasionally, however, they may progress to malignancy, which in part is a property of specific virus strains. Example, cervical carcinoma in women.
In benign warts, the papillomavirus DNA is episomal, meaning it is not integrated into the host-cell DNA and persists as an autonomously replicating episome.
In papillomavirus-induced malignant cancers the viral DNA is integrated into that of the host. Thus, integration probably is necessary for malignant transformation.
Integration disrupts one of the early genes, E2, which is a viral repressor.
However, the viral oncogenes (e.g., E6 and E7) remain intact and cause the malignant
transformation.
block p53
Oncogenic RNA Viruses
All RNA tumor viruses belong to the family Retroviridae.
Acutely Transforming Retrovirus
vs slow transforming
Acutely Transforming Retrovirus
Inserted proto-oncogene becomes oncogene/v-onc gene in virus. >Mutation
>Loss of introns/cell elements that control the expression of this gene
C-onc/proto-oncogene is inserted in virus genome by recombination between proviral DNA and Host DNA
Acute transforming retroviruses are directly oncogenic by carrying an additional viral oncogene, v-onc.
The retroviral v-onc originates from a host c-onc gene/proto-oncogene which is inserted into the virus genome usually by recombination events between provirus DNA and host DNA, i.e. v-onc genes are c-onc genes separated from host genome and inserted in virus genome.
Remember: c-onc genes/proto-oncogenes are host genes that encode important cell signaling products that regulate normal cell proliferation.
v-onc genes are separated from the cellular machinery that normally controls gene expression (lacks introns), so they have power of unregulated expression.
Moreover, the c-onc/proto-oncogene (inserted in virus genome) also mutates accentuating its transformation activity.
v-onc genes are under the control of the viral LTRs(long term repeats) , which are strong promoter- Allows the gene to express more and more, which makes the condition worse
Tumor Antigens
New antigens appear on the surface of tumor cells that may provoke an immune response. These are abnormal proteins that arise from mutations and are broadly classified into.
Products of Mutated Oncogenes and Tumor Suppressor Genes (Tumor-Specific antigens)
Products of Other Mutated Genes (Tumor-associated antigens) Tumor antigens are usually classified into 5 types ( in picture)
The immune system recognizes these abnormal antigens as foreign and tries to destroy the tumor cells.
However, these abnormal molecules often are not appropriately presented to cells of immune system, such as cytotoxic T cells.
Activating and inhibitory receptors of natural killer (NK) cells.
A. Healthy cells express self class I MHC
molecules, which are recognized by inhibitory receptors, thus ensuring that NK cells do not attack normal cells. Note that healthy cells may express ligands for activating receptors (not shown) or may not express such ligands (as shown), but they do not activate NK cells because they engage the inhibitory receptors.
B. In virus-infected cells, class I MHC expression is reduced so that the inhibitory receptors are not engaged, and ligands for activating receptors are expressed. The result is that NK cells are activated and the infected cells are killed.
Type I Inferons
Type-I: There are many type I interferons. IFN- :
Leukocyte interferon
Produced in large quantities by plasmacytoid dendritic cells
Produced in smaller amounts by macrophages, monocytes, and lymphocytes). Not host specific
IFN- :
Fibroblast interferon. Secreted by virus-infected fibroblast. Generally Host species-specific.
The two major type-I interferons (IFN- and IFN-) are produced by virus-infected cells within a few hours of viral invasion. The IFN binds to specific receptors of IFN-responding host cells, triggering the intracellular synthesis of antiviral proteins.
Dual Function of Interferon and in Antiviral Response
Autocrine: The infected cell secretes the IFN that acts on same cell, triggering apoptosis. Paracrine: The infected cell secretes IFN which acts on adjacent cells.
Type –II Interferon
Type-II: Only one type of interferon, IFN-gamma( looks like y). Mostly Immunoregulatory. Produced by antigen-stimulated T cells and Natural Killer (NK) cells Labile at pH 2
Demonstrates host specificity
Gene Silencing
Cells utilize small, interfering, RNA molecules (RNAi) to silence genes as a means of regulating normal developmental and physiological processes, and potentially to interfere with virus replication.
Production of RNAi initiates formation of the RNA-silencing complex that includes an endonuclease that degrades those mRNAs with a sequence that is complementary to that of the RNAi.
Cells can utilize this mechanism to disrupt virus replication through the production of RNAi that are complementary to specific viral genes.
siRNA
cell mediated immunity
Incidence
The number of new cases that occur in a population over a specified period of time.
The incidence rate, or attack rate, is a measure of the occurrence of infection or disease in a population over time—for example, a month or a year, and is especially useful for describing acute diseases of short duration.
Prevalence
The number of occurrences of disease [old and new cases], infection, or related attributes [antibodies] in a population, at a particular point of time.
It is difficult to measure the incidence of chronic diseases. For such diseases it is customary to determine the prevalence—that is, the ratio, at a particular point in time, of the number of cases currently present in the population divided by the number of animals in the population.
Agar gel immunodiffusion test
Example: Detection of antibodies against avian influenza (matrix antibodies), equine infectious anaemia (Coggins test) and enzootic bovine leukosis.
Complement fixation test:
Immunoblotting:
