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
Yolk Sac Inoculation-
5-7 days
Allantoic Cavity Inoculation
7-13 Days
Amniotic Cavity Inoculation
7-13 year old eggs
Chorioallantoic Membrane Inoculation (CAM)
9-12 days old
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
Laboratory Animal Inoculation
Intracerebral or Intraperitoneal Mouse Inoculation
Ultracentrifuge
Ultracentrifuges have played a vital role in virology because they provide sufficient gravitational force (0,000 RPM and 200,000 x g to 150,000 RPM and 1,000,000 x g) to efficiently sediment even the smallest viruses.
very high centrifugucation
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
Virus Purification with Membrane Chromatography
Chromatographic membranes are now utilized in purification of viruses and virus like particles and for impurity removal. The macroporous structure of the membrane allows large viruses to enter it and to bind to the inner pore surface easily.
Binding of the viruses on ion exchange membranes depends on charge distribution on the virus. Adsorption and subsequent elution of viruses can be achieved by anion or cation exchange membranes.
Virus quantification
counts the number of viruses in a specific volume to determine the virus concentration.
The Virus Titer
is lowest concentration of virus that still infects cells. Also defined as the number of infectious units per ml of the sample.
There are basically two types of viral quantification tests
Biological: Depend on a virus particle initiating a successful replication cycle. Plaque assays
Pock assays
Various endpoint titration methods
Physical: Do not depend on any biological activity of the virus particle. Electron microscopic particle counts
Hemagglutination
Immunological assays, such as ELISA
Quantitative PCR assays Flow Cytometry
- Direct Particle Counts by Transmission Electron Microscopy (TEM)
The most direct method to determine the concentration of virus particles in a sample.
Not routinely carried out as:
Expensive
Required Trained Staff
Cannot assess biological activity of the preparation
Compared with a known concentration of a standard particle Ideal for viruses with unique geometrical shapes.
Chances of error high.
Virus Counter 2100:
The technology behind the Virus Counter 2100 is based on a specialized version of flow cytometry developed specifically for use with nanometer scale particles.
When using a Virus Counter, each sample is stained with two different fluorescent dyes, one specific for nucleic acid, and the other specific for protein, and analyzed as they flow through a laser beam.
This dual channel system measures intact virions through detection of co-localized protein and nucleic acids.
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
“HA titer” of the virus suspension.
The inverse of the greatest dilution that completely agglutinates the red blood cells is defined as the “HA titer” of the virus suspension.
High Performance Liquid Chromatography (HPLC):
The concentrations of specific viral antigens may be quantified through UV (Ultra Violet) analysis of fractions generated during HPLC..
Single Radial Immunodiffusion (SRID):
Radial diffusion of purified viral antigens (standards) and viral particles through agarose gel seeded with polyclonal antisera against a viral antigen
Quantitative Polymerase Chain Reaction (qPCR)
Utilizes polymerase chain reaction chemistry to amplify viral DNA or RNA to produce high enough concentrations for detection and quantification by fluorescence. Fluorescent probes are used in a conventional PCR system.
helps to guessestimate viral concentrations
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)
Monolayer Plaque Assay- stained with
Crystal Violet
Plaques- clear circles
Viable cells- bluish purple
Determination of Titer (Plaque-Forming Units/ml)
Average plaque count x reciprocal of the dilution selected
• Titer: PFU/ml
Pock Assay
• Pock
Necrotic area on chorioallantoic membrane [CAM] of embryonated egg
- Titration of herpesviruses and pox- viruses
- Unit: Pock-forming units/ml
CAM inoculation
pock forming units correspond to the titer of the virus
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!
Quantal Assay
Measures the presence or absence of infection
Used for certain viruses that do not form plaques or for determining the virulence of a virus in animals or eggs
• Endpoint
Virus dilution that affects 50% of the test subjects
- Tissue Culture Infectivity Dose50 • Lethal Dose50
- Embryo Lethal Dose50
- Paralytic Dose50
Lethal Dose50
50% of the lab animals die
TCID50
TCID50 is the tissue culture infectious dose which will infect 50% of the cell monolayers challenged with the defined inoculum.
Multiplicity of infection (moi)
Multiplicity of infection (moi) is the average number of virus particles infecting each cell.
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
Replication of Viruses
Viruses do not have the genetic capability to multiply by division.
Virus hijacks and utilizes the host cell machinery to produce its proteins and nucleic
acid for the next generation of virus.
The process of Virus replication in the host cell resembles an assembly line.
Permissive Cell
A cell in which a virus is able to replicate, i.e. the cell machinery supports replication of the virus.
Non-Permissive Cell
Cells in which a factor or factors necessary to viral reproduction is not present or one detrimental to viral reproduction is present. eg. Absence of appropriate receptors.
MOI (Multiplicity of Infection):
Refers to the number of virions that are added per cell during infection
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
Adsorption
During this period, virus attaches to and enters cells, and the
titer of free virus in the medium may actually decline.
Eclipse period
Time interval between uncoating [“disappearance” of viruses] and appearance, intracellularly, of first infectious progeny virions.
No infectious virus can be detected during this time. Usually ranges from 2-12 hours for most virus families.
Latent period
The time before new infectious virus appears in the medium, i.e. time from uncoating to just prior to the release of the first extracellular virions.
During this phase no extracellular virions are detected.
Burst size
Number of infectious virions released per average cell.
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
Attachment
Receptor: In cell biology, a structure on the surface of a cell (or inside a cell) that selectively receives and binds a specific substance, and mediates its entry or action into the cell. Receptors may be proteins, carbohydrates, glycoproteins, lipids, lipoproteins, or even complexes.
1. Attachment to Host Cell Surface
- Mediated by interactions between the Virus and Complimentary Receptor on Host Cell
surface. Cell that lack the appropriate receptor escape being infected by viruses. - 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.
- Some Viruses may use more than one host cell receptor, such as HIV.
- Host cells certainly do not maintain receptors specifically for viruses, these cellular receptors carry out normal functions for cell, such as uptake of proteins. Viruses have evolved to use this receptors.
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 .
What are the ways that the virus can penetrate the host cell?
- Endocytosis
- Surface Fusion
- Pore- Mediated Penetration
- Antibody- Mediated Pentration
Receptor-Mediated Endocytosis
Used by:
- Naked viruses
- Most enveloped viruses
Clathrin-mediated endocytosis
Clathrin-mediated endocytosis
Look in powerpoint- Virus Replication
In Most Non-Enveloped Viruses
In Most Non-Enveloped Viruses: Lysis occurs when a viral capsid induces rupture of endosomal membrane, releasing Viral Capsid or genome.
local permeabilization
In Some Non-Enveloped Viruses: Some non-enveloped viruses induce local permeabilization of host endosomal membrane to allow virus capsid penetration into the cytoplasm.
Other types of Receptor-Mediated Endocytosis
Caveolin-mediated endocytosis of virus by host. (specialized lipid rafts)
Clathrin- and caveolin-independent endocytosis of virus by host.
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.
Fusion Protein
Facilitates Membrane Fusion
- present on the lipid evelope of the virus
Can be pH independent such as in HIV and Measles
or
pH dependent (Acidic) fusion proteins, such as HA in influenza virus- Low pH in endosome promotes fusion of envelope with endosomal membrane; lysis of nucleocapsid by lysosomal proteases, and release of viral genome
The F (Fusion) protein catalyzes membrane fusion at the cell surface at neutral pH. The viral nucleocapsid is then released into the cytoplasm
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
Processing of Primary RNA Transcript (Pre-mRNA)
Viral mRNAs are translated by cellular protein synthetic apparatus.
The Viral mRNA must conform to the requirements of host cell translation system, i.e. the host cell can recognize the mRNA and translate same.
A series of modifications occur, known as Processing of Primary RNA Transcript/Pre- RNA
After processing, mRNAs are translated in the cytoplasm.
Viral mRNAs produced in the nucleus must also be exported to the cytoplasm.
To be reconized by the host ribosome, the virus must first have a cap and a tail
Processing of Primary RNA Transcript (Pre-mRNA) Steps
- Capping
- Addition of 3’ poly-adenylated tails
- Splicing
Capping
- Capping:
Addition of 7-methylguanosine to the 5 ́ end of RNA. Stability of mRNA
Binding of mRNA to Ribosomes
Mark mRNA as “Self”
How Caps are Synthesized:
Can be Synthesized by host cell enzymes. –e.g. Retroviruses, Adenoviruses
Can be Synthesized by viral enzymes. –e.g. Poxviruses, Reoviruses
Cap snatching: virus steal caps from host
Cap
mRNAs. –e.g. Influenza
- Addition of 3’ poly-adenylated tails
PolyA tails interact with PolyA-binding protein: important for translation.
Viral mRNAs can be polyadenylated by host or viral enzymes
3’-Polyadenylation: ESSENTIAL FOR TRANSLATION
A stretch of adenylate residues are added to the 3’ end. The poly-A tail contains ~ 250 A residues in mammals. The major signal for the 3’ cleavage is the sequence AAUAAA. Cleavage occurs at 10-35 nucleotides downstream from the specific signal sequence. A second signal is located about 50 nucleotides downstream from the cleavage site.
- Splicing
- Splicing
RNA splicing is a process that removes introns and joins exons in a primary transcript.
An exon
An exon is the portion of a gene that codes for amino acids
An intron
An intron is a portion of a gene that does not code for amino acids.
- Splicing
A. Constitutive Splicing: Every intron is spliced out; Every exon is spliced in.
B. Alternative Splicing: All introns spliced out; Only selected exons spliced in. Result: mRNAs having different coding information derived from a single gene.
Types of Viral mRNA
• Monocistronic
mRNA that encodes one polypeptide •
Polycistronic
mRNA that encodes several polypeptides
UTR
open reading frame
codes for the protein
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
Important Viral Proteins Include
Enzymes
Structural proteins
Viral Nonstructural Proteins Regulatory proteins
Inhibitors
Assembly and Maturation of Virus
Assembly of Virus Genome and Proteins into New Virions follow a Specific Order. All components, including nucleic acids and proteins, are packaged to form mature
Virions.
May take place in Nucleus
Cytoplasm
Plasma/Cell Membrane (Most Enveloped Viruses)
Release of Progeny Virions
• Naked virions
Lysis of host cell
• Enveloped virions
Budding
Lysis
Bursting of cell
popcorn coming out of a popcorn bag
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
Cell-to-Cell Spread of Viruses
- Extracellular Spread
Same Replication cycle repeated in new host cell and so on…..
Released Viruses in Extracellular Milieu (environment). Travel to new cell.—
- Intercelluar spread
- nuclear spread
Intercellular Spread:
Cell-to-Cell Spread of Viruses
- Intercellular Spread:
Spread from cell-to-cell without contact with extracellular milieu. Results in Rapid Virus Dissemination, evasion of Immune system, and Persistent Infections.
Viruses can either utilize existing cell-cell contacts or exploit basic cell adhesion biology to deliberately establish contact between infected and uninfected target cells for the purpose of efficient spreading.
Inter-Cellular Transmission of Viruses:
Cell–cell plasma-membrane fusion followed by movement of infectious viral material (shown as viral cores) into the uninfected target cell. Herpesviruses, paramyxoviruses and retroviruses
Passage of virions across a tight junction. Herpesviruses
Movement of virions across a neural synapse. Rabies Virus
Viral induction of actin- or tubulin-containing structures. Poxviruses
Viral subversion of actin-containing structures, form Filopodial bridges. Retroviruses
Membrane nanotube subversion. HIV-1
Virological synapses. Retroviruses
Seen in HIV, Herpesvirus, Measles, etc.
Nuclear Spread of Virus Genome
The viral genome is integrated into the host cell genome and passed down to next progeny or generation of host cells, eg., Retrovirus
Pathogenicity
refers to the ability of a Virus to cause Disease in Host (i.e., harm the host).
Pathogen
The Virus which causes Disease is called a Pathogen
Pathogenesis
Pathogenesis is the manner/mechanism of development of a Disease.
Virulence
The term Virulence is used as a quantitative or relative measure of the degree of pathogenicity of the infecting virus.
Avirulent
Not Virulent (Not harmful to the host).
Virulence is not an absolute property of a Virus, but depends
Virulence is not an absolute property of a Virus, but depends on many variables
Virus variables:
Virus strain (Genetic Makeup of the Virus/Strain Variation)
Portal of entry of Virus in Host
Tropism to Host Organs
Dose of Infection
Immuno evasion
host variables:
Host Species
Host Immunity
Expression of Critical Receptors
Host Physiological factors
(Nutrition status, Age, Hormonal Factors, Stage of Cell Differentiation)
Interferons, Other Inhibitors
Fever
Lethal dose 50 (LD50)
The dose of the virus required to cause death in 50% of animals
Infectious dose 50 (ID50):
The dose of virus that will infect 50% of an experimental group
of hosts/animals.
ID50 : LD50 ratio:
Ratio of the dose of a particular strain of virus that causes infection in 50%
of individuals [infectious dose 50 (ID50)] to the dose that kills 50% of individuals (LD50).
The lower the ID50 and LD50,
The lower the ID50 and LD50, the more viru- lent the organism
Ways of Assessing Degree of Virulence
Degree of Severity of illness (Clinical signs)
Incubation Period
Degree of Severity, location, and distribution of gross, histologic, and/or
ultrastructural lesions in affected animals.
Severity of illness
Virulence can be measured as the proportion of infections that are fatal (case: fatality rate) or by the survival time until death. In this example, various strains of myxoma virus, a poxvirus of rabbits, were compared after subcutaneous inoculation NC: not calculable.
Degree & Severity of Pathological lesions
Degree & Severity of Pathological lesions can be used to measure virulence.
The Sequential Steps in Viral Infection (Pathogenesis)
- Entry of Viruses & Primary Replication
- Spread, Tropism, & Infection of Target Organs
- Virus-Cell Interactions, Secondary replication
- Tissue & Organ Injury
- Shedding
Routes of Entry of Viruses into Host- 1. The Skin
You have to have a breach in the skin.
Defenses
Dense outer layer of Keratin
Low pH
Presence of Fatty Acids
Bacterial flora
Dryness
Components of Innate and Adaptive Immunity
(migratory dendritic cells: Langerhans cells)
Routes of Entry of Viruses into Host
Transcutaneous Injection
- Bite of Arthropods
- Bite of Infected Animal
- Contaminated Objects
Routes of Entry of Viruses into Host
- Mucous membrane
Conjunctiva
Oropharynx
Genitourinary Tract
Rectum
Defenses
IgA (Antibodies)
Virucidal proteins
The Iceberg Concept of Infection and Disease
Routes of Entry of Viruses into Host
- Gastrointestinal Tract
Defenses:
Mucous membrane of oral cavity & esophagus
Acidity of the stomach
Alkalinity of Intestine
Layer of mucus covering the gut.
Lipolytic activity of Bile.
Proteolytic activity of
Pancreatic enzymes
Defensins (host defense
peptides) IgA
Scavenging Macrophages
Routes of Entry of Viruses into Host
- Respiratory Tract
Defenses:
Mucociliary blanket
Alveolar macrophages
NALT (nasal associated lymphoid tissue)
BALT (bronchus-associated lymphoid tissue) Temperature Gradient
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
Virus Spread in Host
B. Subepithelial Invasion and Lymphatic Spread
After replication at site of entry, viruses can remain localized or spread to other tissues.
After traversing the epithelium, Viruses may reach the subepithelial layers, which may be facilitated by:
(1) inflammatory response to virus infection and/or destruction of epithelium, or
(2) by transport pathways like transcytosis.
Viruses should overcome local host defense.
Directional shedding of viruses from the infected epithelium is critical to subepithelial spread. Apical release facilitates virus dispersal, whilst basolateral release provides access to underlying tissues, facilitating systemic spread.
In subepithelial tissues, viruses get access to lymphatics, phagocytes and tissue fluids.
argeted migration and replication of virus within phagocytic leukocytes, specifically dendritic cells & macrophages, and, or lymphocytes.
Some viruses may pass straight through lymph nodes to enter blood stream.
Some viruses are processed and epitopes presented to Helper T-Cells: Immune response.
Disseminated Infection
Infection spreads beyond the primary site of infection.
Systemic Infection
If a number of organs or tissues are infected.
C. Spread via the Bloodstream:- called
Viremia: The presence of a virus in the blood. Virus may be free in blood or in a cell, such as lymphocytes.
Primary vs secondary viremia
Primary Viremia: Initial entry of virus into the blood after infection
Secondary Viremia: Virus has replicated in major organs & once more entered the circulation
Passive Viremia:
Passive Viremia: Direct inoculation of virus in host, such as contaminated syringe or bite of arthropods. No initial replication elsewhere in host before.
Active Viremia
Active Viremia : Viremia following initial virus replication in host. Release of virions from the initial site of replication, such as lymphatics or epithelium of intestine, to the blood stream.
What Happens to Viruses in Bloodstream
- Free in Plasma (Parvovirus) Short duration Viremia
- Cell-Associated Viruses
Tend to cause Prolonged Viremia. Multiply in Macrophages or Lymphocytes.
Interaction between Virus and Macrophage
Varies
- Macrophages may fail to phagocytose host cells containing virions. Prolonged Viremia
- Virions may be phagocytosed & transferred passively to adjacent cells, where replication begins
- Tissue invasion via carriage of virus inside monocytes/macrophages that emigrate through the walls of small blood vessels (Trojan Horse)
- Virions may be phagocytosed and destroyed
- Virions may be phagocytosed by macrophages & then may replicate in them
*
Trojan Horse
(Trafficking Monocytes Transport Viruses)
Wooden horse= macrophage
city= tissue cell
virus enters wooden horse and then the macrophage then goes to the tissue and the viruses come out of the wooden horse
Clearance of Virus from the Bloodstream
• Mononuclear phagocytes in the spleen, liver, bone marrow
- Antibody clearance
- Complement-mediated clearance
Virus Spread in Host
D. Spread via Nerves
Examples:
Herpes simplex virus exhibits low neuroinvasiveness of the central nervous system, but high neurovirulence. It always enters the peripheral nervous system, but rarely enters the central nervous system. When it does, the consequences are almost always severe, if not fatal.
Mumps virus exhibits neuroinvasiveness, but low neurovirulence. Most infections lead to invasion of the central nervous system, but neurological disease is mild.
Rabies virus exhibits high neuroinvasiveness and high neurovirulence. It readily infects the peripheral nervous system and spreads to the central nervous system with 100% lethality unless antiviral therapy is administered shortly after infection.
Neurotropic virus:
Neurotropic virus: Viruses that can infect neural cells.
D. Spread via Nerves
Infection may occur by neural or hematogenous spread.
Neuroinvasive virus:
Neuroinvasive virus: Viruses that enter the central nervous system (spinal cord and brain) after infection of a peripheral site.
Neurovirulent virus:
Neurovirulent virus: Viruses that cause disease of nervous tissue, manifested by neurological symptoms and often death.
Neural Spread of Viruses
- Transport of virus within axons
- Transport of virus in perineural lymphatics
- Transport of virus in the endoneu- ral space
- Transport of viruses via infected Schwann cells
Centripetal movement of virus: Towards the CNS/Brain
Centrifugal movement: From CNS, within peripheral nerves, to other locations in body.
Retrograde Spread:
Retrograde Spread: Travel opposite direction of nerve impulse flow. Invades axon terminals and then spread to Dendrite or cell body, and then cross synapse to reach next axon terminal.
Anterograde Spread:
Anterograde Spread: Travel in direction of nerve impulse flow. Virus invades dendrites or cell bodies and then spread to axon terminals, and then cross synaptic contacts to invade dendrite of next neuron.
Neural Spread of Viruses to CNS through Olfactory Routes
can enter from the blood or from the nose
Centripetal movement of virus:
Towards the CNS/Brain
Centrifugal movement:
From CNS, within peripheral nerves, to other locations in body.
RABIES
Spread of Viruses to CNS through Blood-Brain Barrier
Methods:
Increasing permeability of endothelial cells through the secretion of TNF (Tumor Necrosis factor).
Breakdown of endothelial cell junctions through Matrix- metalloproteinase (MMP).
Trojan horse (Trafficked by Monocytes)
Important Features of Localized and Systemic Acute Viral Infections
Local Systemic
Site of pathology portal of entry distant sites
Incubation short long
period (IP)
Viremia NO YES
Duration variable lifelong
of immunity
Secretory IgA very important not important
Virus Shedding
Shedding of infectious virions is crucial to the maintenance of infection in
populations.
Critical to virus transmission: The amount of virus shed in an excretion or secretion is important in relation to disease transmission.
Some viruses are shed from a variety of sites and several sites.
Acute infection: usually intensive shedding over short time period.
Persistent infections: can be shed at lower titers for months to years.
can be shed in as many ways as you can get it!
Tropism
The specificity/Affinity of a virus for a particular host tissue
An enteric virus replicates in gut, but not in lungs.
A respiratory virus replicates in lungs, but not in nerves.
Pantropic Viruses
Can Replicate in more than one host Organ/Tissue
Viral Tropism
Determinants of Viral Tropism
Receptors on Host Cell
Viral attachment proteins
Viral enhancers: Gene activators that increase the efficiency of transcription of
viral or cellular genes, facilitating Virus Replication.
Cellular protease requirement
Temperature of replication
Acid lability and protease digestion
Transcriptional control of tropism
Anatomic barriers
Host organ response to infection
Rash
- A general term applied to any temporary eruption on the skin
- A rash usually is a shade of red Varies with the disease in question
“Bulls Eye”
Rash of Lyme Disease
Virus Injury to Organs/Tissues
A. Skin
Can be localized (Papilloma) or disseminated (lumpy skin disease).
Vesicles- rupture with ulcers- FMD in Pig, Vesicles on coronary band
Ulcers in FMD
Nodules in Lumpy Skin Disease, Cattle
Warts caused by Papillomavirus
Warts are benign (not cancerous) skin growths that appear when a virus infects the top layer of the skin.
Papules(solid, crusty elevated), Papular stomatitis in cattle
Erythema in Hog cholera
Erythema, Reddening of skin, consequence of systemic viral infections (endothelial injury in blood vessels throughout body, including those of the subcutaneous tissues)
Virus Injury to Organs/Tissues
B. Gastrointestinal tract
Rotavirus, Norovirus are INGESTED
Parvovirus, Pestivirus Hematogenous spread, systemic infection
- Destruction of enterocytes due to Viral replication, Hypersecretion
- Gastrointestinal disease, Malabsorption, Diarrhea
- Pronounced de4qhydration, Acidosis, Hemoconcentration
C. Respiratory tract
Tropism for different parts of the respiratory tract with considerable overlap.
Loss of ciliary activity
Loss of integrity of the lining
mucus layer
Multifocal destruction of
epithelium
Inflammation
Example: Avian Infectious Bronchitis
Exudation
Influx of inflammatory cells
Obstruction of air passages
Hypoxia & Respiratory distress
Secondary Bacterial Infection
Viral-bacterial synergism
Infection of the Respiratory Tract
Virus Injury to Organs/Tissues
D. Central Nervous System
Lytic infections of neurons by togaviruses, flaviviruses, herpesviruses, or other viruses leads to encephalitis or encephalomyelitis characterized by neuronal necrosis, phagocytosis of neurons (neuronophagia), and perivascular infiltrations of inflammatory cells (perivascular cuffing).
Examples:
Neuronal vacuolation in Prion disease
Progressive Demyelination- Canine Distemper
Damage to Endothelium
Petechial and ecchymotic hemor- rhages
Disseminated intravascular coagu- lation [DIC]
Edema
Infarction
Ischemic necrosis
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
Teratogenesis
• This is the abnormal development or arrests in development of the embryo or fetus
May result in death or malformations during the antenatal period
• Susceptibility to teratogens
Varies with the species and stage of develop- ment, decreasing with fetal age
i.e. cerebellar hypoplasia, Arthrogryposis
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
Virus-induced Immunopathology
Defined as Tissue injury mediated by host immune response to Virus infection. It is the price paid by the host to clear a viral infection.
Depends on the delicate balance between the protective and destructive effects of the host immune response to viruses.
Immunopathology is often the cause of damage with viruses that are relatively non- cytolytic and persistent, i.e. infected cells are not immediately destroyed and immune response becomes chronic.
If the immune response clears the infection by destroying a small number of virus- infected cells, the host survives with minimal symptoms and no permanent damage.
On the other hand, if a large number of cells are infected before immune induction, the same immune mediated destruction can cause severe or fatal pathological consequences.
Immunopathology
Tissue damage mediated by hypersen- sitivity reactions
Autoimmune diseases, eg, moon blind- ness in a horse
Inflammation-mediated tissue damage, eg, fibrosis
Immunodeficiency disorders
Some Mechanisms of Virus-induced Immunopathology (The role of T cells)
T cells, for example, can directly destroy virus-infected cells or release cytokines, such as tumor necrosis factor (TNF), that damage cells.
There may be Cytotoxic cell mediated lysis/killing of infected host cells.
With some non-cytopathic virus infections, such as HCV and HBV, destruction of
infected cells by CD8+ effector T cells is the main cause of damage to the liver.
There may be release of cytokines from T cells (CD4+ and CD8+) and other cells that cause inflammation and tissue damage that becomes chronic against persistent virus infections.
CD4+ cells elaborate far more cytokines than CD8+ cells and also activate other non-specific effector cells, such as neutrophils.
Cytokines
Cytokines are a broad and loose category of small proteins that are important in cell signaling. They act as mediators and regulators of immune processes, but also cause inflammation.
These include:
Monokines, cytokines produced by mononuclear phagocytic cells
Lymphokines, cytokines produced by activated lymphocytes, especially Th cells Interleukins, cytokines that act as mediators between leukocytes
Some Mechanisms of Virus-induced Immunopathology (The role of Innate Immunity)
Invading viruses and their replicative intermediates can be recognized by several innate immune receptors expressed either at the host cell surface or within cells, such as Toll-like receptors (TLRs).
Persistent activation of these receptors of innate host cells by viruses causes production of pro-inflammatory cytokines and interferons, as well as signals that recruit and activate cells involved in inflammation.
Injury can also be mediated by free radicals, such as Nitric oxide and Superoxide. Normally, they inhibit viral replication. But when produced in abundance, cause cell damage.
Some Mechanisms of Virus-induced Immunopathology (Toxicity from antibody responses)
Antibody responses to viruses may also contribute to tissue damage.
This occurs when antibody binds to an infected cell, activates complement and causes
an inflammatory reaction.
Alternatively, antibody mediated inflammatory reactions involve toxicity following: Engagement of IgG with Fc receptors on inflammatory cells, which causes
inflammatory mediator release.
Following deposition of viral antigen–antibody complexes in capillary beds,
leading to activation of the complement cascade.
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
Some Mechanisms of Virus-induced Immunopathology:
Infection and damage to mononuclear phagocytes
Infection and damage to mononuclear phagocytes can protect an invading virus from phagocytic removal. Moreover, these infected cells may exhibit aberrant behavior, resulting in a cascade of events that are detrimental to the host.
Infectious Bursal disease
Infectious Bursal disease: Virus replication causes atrophy of the bursa and a severe deficiency of B lymphocytes, resulting in immuno- suppression. As a result, infected birds become susceptible to other pathogens.
Inapparent Infections
- Clinical signs and symptoms are not evident • Too few cells may be infected
- Stimulate host immune response
- Possible source of virus spread
Acute Infection
(Short-term Infection)
Short clinical course.
Rapid clearance from host immune response.
Persistent Infection
Persistent infections, per se, in which infectious virus is demonstrable continuously, whether or not there is ongoing disease. Disease may develop late, often with an immunopathologic or neoplastic basis, or in other instances, there may be no manifestation of clinical signs.
The pathogen is not cleared efficiently by the adaptive immune response
Persistent Infections are Important:
Recrudescent episodes of disease in the individual host.
Immunopathology
Survival of virus in host and continuous shedding, source of spread of Virus.
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
Cytocidal
Cell Death
Lysis
Apoptosis
Non‐Cytocidal
Persistent Infection
Cell Transformation
Tumor cells
Cytopathic effect or cytopathogenic effect
Cytopathic effect or cytopathogenic effect (abbreviated CPE) refers to damage or morphological changes to host cells during virus invasion.
Primary effect induced by viral replication and viral proteins toxic to host
cells.
Secondary effects of metabolic needs of the virus.
- Complete destruction of cells- Most severe form of CPE. All cells in the monolayer rapidly shrink, become dense (pyknosis), and detach from the glass within 72 hours Eg. Enteroviruses
- Subtotal destruction of Cells- Consists of detachment (death) of some, but not all of the cells in the monolayer.Eg. Togaviruses, some picornaviruses and some paramyxoviruses
- Focal destruction of cells- Produce localized areas (foci) of infection. Eg. Herpesviruses and poxviruses.
Pyknosis:
a degenerative condition of a cell nucleus marked by clumping of the chromosomes, hyperchromatism, and shrinking of the nucleus
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.
Acidophilic Staining
recognizes/Affinity for acid dyes, such as eosin. Appear Pinkish upon staining.
Basophilic Staining
recognizes/affinity for basic dyes, such as hematoxylin. Appear Purplish Blue upon staining.
General mechanisms of virus‐induced cell injury and death
Inhibition of Host‐Cell Nucleic Acid Synthesis:
Large DNA viruses, such as poxvirus, produce enzymes that may degrade cellular DNA.
General mechanisms of virus‐induced cell injury and death
Inhibition of Host‐Cell RNA Transcription (mRNA production and processing)
Indirect consequence of viral effects on host‐cell protein synthesis that decrease the
availability of transcription factors required for RNA polymerase activity.
Certain viruses encode specific transcription factors to regulate the expression of their
own genes, and these factors sometimes modulate the expression of cellular genes as
well, such as herpesviruses.
Some viruses, such as influenza and herpesvirus, encode proteins that inhibit
polyadenylation and splicing of host cell primary mRNA transcripts required to form mature mRNA.
General mechanisms of virus‐induced cell injury and death
Inhibition of Host‐Cell Protein Synthesis :
Inhibition of Host‐Cell Protein Synthesis :
Production of viral enzymes that degrade cellular mRNAs
Production of factors that bind to ribosomes and inhibit cellular mRNA translation
Alteration of the intracellular ionic environment favoring the translation of viral mRNAs
over cellular mRNAs
Production of large excess of viral mRNA that outcompetes cellular mRNA for host
ribosomes
Some viruses cause lysosomes to release their hydrolytic enzymes, which then destroy the host cell
Fusion Proton
proton on the outside of a virus that helps it fuse to the cell membrane
General mechanisms of virus‐induced cell injury and death
Interference with Cellular Membrane Function:
Interference with Cellular Membrane Function:
Promotion of Cell Fusion (Syncytium Formation)
Affect ion exchange and membrane potential
Induce the synthesis of new intracellular membranes or the rearrangement of
previously existing ones
Damage to the cytoskeleton resulting in changes in cell shapes. The cytoskeleton is
responsible for the structural integrity of the cell, for the transport of organelles through the cell, and for certain cell motility activities.
Apoptosis
Apoptosis is the process of programmed cell death, which is essentially a mechanism of cell suicide that the host activates as a last resort to eliminate viral factories before progeny virus production is complete.
Different from Lysis where viral replication is complete, host cell is destroyed and new virions released.
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.
The Intrinsic (Mitochondrial) Pathway
The mitochondrial pathway is activated as a result of increased permeability of mitochondrial membranes subsequent to cell injury, such as that associated with a viral infection.
apoptotic pathway
The Extrinsic (Death Receptor) Pathway
The Extrinsic (Death Receptor) Pathway: The extrinsic pathway is activated by engagement of specific cell‐membrane receptors, which are members of the TNF receptor family (TNF, Fas, and others). Thus binding of the cytokine TNF to its cellular receptor can trigger apoptosis.
Similarly, cytotoxic T lymphocytes that recognize virus‐infected cells in an antigen‐specific manner can bind the Fas receptor, activate the death domain, and trigger the executioner caspase pathway that then eliminates the cell before it becomes a functional virus factory.
perforin and granzyme
Apoptotic Pathways
Cytotoxic T lymphocytes and natural killer cells can also initiate apoptosis of virus‐ infected target cell, utilizing preformed mediators such as perforin and granzyme that directly activate caspases in the target cell.
Antibody-Dependent Cell Mediated Cytotoxicity
N/K cells
Non‐Cytocidal Changes in Virus Infected Cells
Non‐cytocidal viruses usually do not cause immediate death of cells in which they replicate.
They often cause persistent infection during which infected cells produce and release virions, but overall cellular metabolism is little affected.
In many instances, infected cells even continue to grow and divide.
Can produce important pathophysiologic changes in their hosts by affecting crucial functions that are associated neither with the integrity of cells nor their basic housekeeping functions.
Damage to the specialized functions of differentiated cells may still affect complex regulatory, homeostatic, and metabolic functions, including those of the central nervous system, endocrine glands, and immune system.
With few exceptions (e.g., some retroviruses), these slow progressive changes ultimately may lead to cell death.
However, in the host animal, cell replacement occurs so rapidly in most organs and tissues that the slow fallout of cells as a result of persistent infection may have no effect on overall function.
But, terminally differentiated cells such as neurons (Nerve Cells), once destroyed, are not replaced, and persistently infected differentiated cells may lose their capacity to carry out specialized functions.
This type of interaction can occur in cells infected with several kinds of RNA viruses, notably pestiviruses, arenaviruses, retroviruses, and some paramyxoviruses.
Cell transformation
Cell transformation is the changing of a normal cell into a cancer cell.
Neoplasia
Neoplasia is a descriptive term that denotes an abnormal tissue overgrowth that may be either localized or disseminated. It is the process that leads to the formation of neoplasms (syn. carcinogenesis).
Oncology
Oncology is the study of neoplasia and neoplasms
benign neoplasm
A benign neoplasm is a growth produced by abnormal cell proliferation that remains
localized and does not invade adjacent tissue
malignant neoplasm
A malignant neoplasm (syn. cancer) is locally invasive and may also be spread to other parts of the body (metastasis)
Oncogenic Viruses
Oncogenic Viruses: Viruses that cause or give rise to tumors.
Neoplasms (tumors)
Neoplasms (tumors) arise as a consequence of the dysregulated growth of cells derived from a single, genetically altered progenitor cell.
Metastasis
Metastasis is the spread of cancer cells from the part of the body where it started (the primary site) to other parts of the body. When cancer cells break away from a tumor, they can travel to other areas of the body through the bloodstream or the lymph system, and develop secondary tumors in other parts of body.
Proto-oncogenes
Encode proteins that function in normal cellular growth and differentiation
Encode growth factor proteins
Encode growth factor receptors
Encode transcription factors
Encode intracellular signaling proteins Encode Signal Transducers
Proto-Oncogenes
Proto‐oncogenes are often involved in growth signaling and anti‐apoptotic pathways.
Encode growth factor proteins
Encode growth factor receptors
Encode transcription factors
Encode intracellular signaling proteins Encode Signal Transducers
Normally mitosis (cell division) is a carefully regulated event, requiring the activation of one protein to activate another, in what is known as a signal transduction cascade.
tumor supressor genes
Plays a role in keeping cell division in check. Encodes proteins that regulates and inhibits uncontrolled growth
The Cell Cycle
Oncogenes
Mutated forms of proto-oncogenes or aberrantly expressed proto-oncogenes.
Encode proteins that somewhat resemble products of proto-oncogenes, but differ
WHY?
These onco-proteins function in an unregulated manner, i.e. do not respond to regulatory signals.
Synthesis of aberrant growth factors, growth factor receptors, etc. that trigger uncontrolled proliferation of cells.
Defective differentiation of cells.
Failure to undergo apoptosis.
Under normal circumstances membrane-bound receptors require the binding of their ligand to be in an activated state. In contrast, receptors encoded by oncogenes do not require the regulatory step of ligand binding to be active.
Tumor Suppressor Genes
Encode proteins that inhibit cell proliferation (by holding cell cycle at G1 phase).
Functions:
Inhibits the expression of genes that are essential for the continuing of the cell cycle.
Connecting the cell cycle to DNA damage, i.e. if cell has damaged DNA it will not
divide.
Repair of damaged DNA
If repair effort fails, apoptosis of cells
Adhesion proteins known as metastasis suppressors that prevent spread of cancer
cells, or metastasis.
Rb: Retinoblastoma protein
One of the first tumor‐suppressor genes to be discovered was the Rb gene.
The Rb protein alternates between a phosphorylated state and Un‐phosphorylated
state. Rb is phosphorylated by enzyme Cyclin Dependent Kinases (CDKs).
The E2F transcription factor is necessary for the expression of a number of cell‐cycle‐ specific genes.
The role of Un‐phosphorylated Rb is to bind to the transcription factor E2F, preventing its activity, and not allowing cell division to proceed from G1 to S phase.
On the other hand, Phosphorylated Rb cannot bind E2F, this releases E2F from its inhibition and allows the cell cycle to progress.
A loss of normal Rb function leads to the loss of control over the cell cycle
Another tumor suppressor protein P16 blocks CDK. So CDK cannot phosphorylate Rb. Rb is free to bind and inhibit action of E2F
p53 Protein
P53 activates the DNA repair system and stops the cell cycle at the G1 checkpoint (before DNA replication).
if not then apopotisis
Tumor Viruses/Oncogenic Viruses
Viruses that cause cancer.
Oncogenic viruses generally have a DNA genome, or generate a DNA provirus after infection (Retrovirus)
Cells transformed by oncogenic viruses usually have viral DNA integrated into host genome. These integrated DNA may be a part or all of the viral genome.
However, the genome of few viruses may remain episomal, replicating in each step with host cell chromosome, i.e. the genome never integrates into host genome, but has an autonomous replicating system. Found in B cells infected with Epstein-Barr virus and in cells infected with Papillomavirus.
Oncogenic DNA Viruses
Although retroviruses are the most important oncogenic viruses in animals, certain DNA viruses are also significant, including papillomaviruses, polyomaviruses, herpesviruses
DNA oncogenes are an essential part of the viral genome, encoding proteins which alter patterns of gene expression and regulation of cell growth (induce transformation).
In every case the relevant genes encode early proteins having a dual role in virus replication and cell transformation.
Oncogenes of DNA viruses have no homologue or direct ancestors (c-onc genes/ protooncogenes) among cellular genes of the host.
DNA tumor viruses interact with cells in one of two ways:
(1) productive infection in Permissive cell, in which the virus completes its replication
cycle, resulting in cell lysis, or
(2) non-productive infection in Nonpermissive cell, in which the virus transforms the
cell without completing its replication cycle.
During such non-productive infection, the viral genome or a truncated version of it is
integrated into the cellular DNA; alternatively, the complete genome persists as an autonomously replicating plasmid (episome).
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
In acute transforming virus, it steals a proto-onco gene via
Gene recombination
V-onc +
after the proto oncogenes has been put into the host’s DNA and it starts mutating under no regulatory contro
Slow/Chronic Transforming Retrovirus:
Virus gene do not contain v-onc gene. The integration of retroviral genes into host chromosomal DNA can occur at promoter or enhancer sites that drive the increase in proto- oncogene/c-onc gene expression, leading to malignant transformation of the cell.
Promoter
DNA sequence at which DNA-dep RNA poly- merase binds to initiate transcription
Enhancer
A transcriptional regulatory sequence located some distance from the promoter; it increases the rate of initiation of transcription
Characteristics of Transformed (Neoplastic) Cells
- Cells are more spindle-shaped
- Loss of Contact Inhibition
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.
FOCMA antigen
Feline oncornavirus membrane- associated antigen
Innate Immunity
Innate immune defenses exhibit neither antigen specificity nor memory.
They provide a critical line of first defense against viral infections because :
They are constantly present
They are operational immediately after viral infection
Are the only immune defense available for the first few days after viral infection
A. Primary Physical and Chemical Defenses:
Viruses must first breach the barriers at their portal of entry before they can cause infection in their respective hosts.
These barriers often form the first line of defense.
Primary Physical and Chemical Defenses at Portal of Virus Entry
- The Skin
- Mucous membrane
- Gastrointestinal Tract
- Respiratory Tract
Defensins
(host defense peptides) with antiviral activity.
Modulate the host immune response.
Mucociliary blanket
The respiratory tract from the nasal passages to the distal airways in the lungs is protected by the “mucociliary blanket,” which consists of a layer of mucus produced by goblet cells that is kept in continuous flow by the coordinated beating of cilia on the luminal surface of the epithelial cells that line the nasal mucosa and airways. Larger particles (larger than in diameter) are trapped on the mucociliary blanket and then swallowed or coughed out.
Temperature Gradient
There is a temperature gradient between the nasal passages (33°C) and the alveoli (37°C) that plays an important role in the localization of infection. Thus, rhinoviruses, which infect the nasopharynx and cause the common cold, replicate well at 33°C but grow poorly at 37°C, while influenza virus, which infects the lower respiratory tract, shows the inverse temperature preference.
Natural Killer (NK) Cells
NK cells are large lymphocytes that are defined by their cytoplasmic granules.
They provide early and non-specific resistance against viral infections.
The cytoplasmic granules of NK cells contain:
Perforin which can produce pores in plasma membranes Granzymes – proteins that can initiate apoptosis.
When NK cells release their cytoplasmic granules, they can kill adjacent cells.
Natural killer cells are not antigen specific; rather, their activation requires differential engagement of cell-surface receptors in combination with stimulation by proinflammatory cytokines
NK cells mediate death of virus infected cells via apoptosis, and therefore, can eliminate infected cells before they release progeny virions.
Natural killer cells synthesize and release a variety of cytokines, including type II IFN and several interleukins that stimulate their own proliferation and cytolytic activity.
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.
Cellular Pattern Recognition Receptors
Cells at portals of virus entry possess surface receptors (pattern recognition receptors (PRR)) that recognize specific pathogen-associated molecular patterns (PAMPs), which are macromolecules present in viruses and other microbes, but not on host cells.
One class of PRRs are the toll-like receptors (TLRs)
These pattern recognition receptors (PPR) are expressed on and in a variety of different cells, including macrophages, dendritic cells, neutrophils, natural killer cells, endothelial cells, and mucosal epithelial cells.
Cells of the innate immune system respond in several different ways when their PRRs are ligated (attached) by a PAMP.
expression of IFNs and inflammatory cytokines such as tissue necrosis factor and interleukins-1 and -12 (IL-1, IL-12).
activation of phagocytic cells and endothelial cells with increased production of inflammatory mediators and cell-surface expression of adhesion molecules
Macrophages may initiate chemotaxis, bringing neutrophils to a site of inflammation.
However, persistent activation of these receptors by viruses can cause continuous production of inflammatory mediators (pro-inflammatory cytokines and interferons) and chemotaxis, resulting in immunopathology (damaging effects to host).
TLR
Toll-like Receptors
Interferons
Interferons (IFN) are a group of cytokines (complex glycoproteins) that are secreted by somatic cells in response to viral infections and to other stimuli.
They possess potent antiviral, immunomodulating and anti cancer properties.
Interferons show no virus specificity.
RNA viruses are stronger inducers of interferon than DNA viruses.
Being glycoproteins, interferons are orally inactive, and should be administered to the patient by parenteral route (Injection).
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.
Roles of type-I IFNs:
Inhibit Virus replication in Host Cells
Activate Natural Killer (NK) Cells to kill infected cells.
Increase expression of MHC-I molecules and antigen presentation.
Type-I IFNS also stimulate differentiation of monocytes into dendritic cells. Maturation of dendritic cells.
Stimulates memory T cell proliferation.
How type-I IFNs inhibit virus replication:
Activate a latent ribonuclease called RNAse L. RNAase L degrades viral RNA.
Induces synthesis of Mx proteins that bind and trap viral nucleocapsid and inhibit virus assembly.
Induce synthesis of Protein Kinase R (PKR) which prevents initiation of translation of viral RNA.
Protein Kinase R
Induce synthesis of Protein Kinase R (PKR) which prevents initiation of translation of viral RNA.
How type-I IFNs inhibit virus replication:
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
Type –III Interferon
Type-III: At least three type 3 interferons have been identified, IFN-1, IFN-2 and IFN- 3.
Recently discovered.
Expressed in response to viral infections and activation of Toll-like-Receptors. Primarily functions as immunoregulator.
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
Adaptive Immunity
Adaptive immunity includes humoral and cellular components.
Humoral immunity is mediated principally by antibodies released from B lymphocytes
Cellular immunity is mediated by T lymphocytes
Adaptive immunity is antigen specific, so that these responses take time (several days at least) to develop, and this type of immunity is mediated by lymphocytes that possess surface receptors that are specific to each pathogen
Adaptive immunity stimulates long-term memory after infection
Internal viral antigens usually elicit protective Cell Mediated Immune (CMI) response
Surface antigens elicit protective humoral and CMI responses
Viral infections usually do not provoke granulocytosis. Leukocytosis observed is commonly due to lymphocytosis and other mononuclear cells.
Granulocytosis
presence in peripheral blood of an increased number of granulocytes, i.e. Neutrophils, eosinophils, basophils, mast cells.
viral infections do not provoke this
Antibody-Mediated Immunity (Humoral Immunity)
Virus capsid and envelope proteins are antigenic.
Antibodies may be directed against viral proteins on free virions (capsid or envelope),
or against viral proteins expressed on surface of infected cells.
Virus Neutralization:
Neutralizing antibodies prevent virus attachment and entry into host cells. They bind to the viral capsid or host envelope.
Opsonization
Coating of virions with antibodies. Antibody coated virion is recognized and phagocytosed by macrophages, and sometimes by neutrophils.
Clumping of Viruses
Clumping of Viruses (Immunocomplex formation) : Reduces the number of viral particles available for cell invasion
Activation of Complement System
Opsonization - enhancing phagocytosis of antigens
Chemotaxis - attracting macrophages and neutrophils
Lysis - rupturing membranes of foreign cells/Pathogens
Agglutination- clustering and binding of pathogens together (sticking)
Antibody-dependent cell-mediated cytotoxicity
Antibodies bind to viral antigens expressed on surface of host cells. These host cells are destroyed by phagocytosis (macrophages and NK cells), or by complement mediated cytolysis
cell mediated immunity
Cytotoxic T Lymphocytes (CTL)
Portions of immunogenic viral proteins produced in the cytosol of the infected cell are transported to the endoplasmic reticulum, where they associate with class I MHC molecules. This complex is directed to the cell surface, where the viral peptides can be recognized by antigen-specific cytotoxic T lymphocytes.
Cytotoxic T-lymphocytes (CTLs) are then able to recognize peptide/MHC-I complexes by means of their T-cell receptors (TCRs) and CD8 molecules and kill the cells to which they bind.
Binding of the CTL to the infected cell triggers the CTL to release pore-forming proteins called perforins, proteolytic enzymes called granzymes, and chemokines. Granzymes pass through the pores and activate the caspase enzymes that lead to apoptosis of the infected cell.
Antigenic Plasticity
Antigenic Plasticity: Rapid changes in the structure of the viral antigen. May be the result of mutation, reassortment or recombination.
Due to change in antigen structure, the virus may become resistant to immunity generated by previous infection.
Antigenic Multiplicity:
Antigenic Multiplicity: Antigenic variants with little or no cross-reactivity. Example, there are more than 100 serotypes of rhinoviruses which are
antigenically different from each other. So immunity against one serotype may not work against the other variants.
Negative Cytokine Regulation
Negative Cytokine Regulation:
Blocking interferon receptor signal.
Virokines: Some viruses synthesize proteins which are homologs of
cytokines/interferons. Epstein-Barr virus synthesizes vIL-10, a homolog of IL-10,
and suppresses cytokine production by TH1 CD4+ cells.
Viroceptors: Some viruses encode proteins that are homologous to the receptors
for cytokines. The secreted cytokine receptor homologs bind to cytokines and serve as competitive antagonist. For example, Poxviruses.
Down-regulation of MHC class I pathway:
Down-regulation of MHC class I pathway: Interfere with expression of MHC class-I molecules and inhibit antigen presentation.
Inhibition of complement activation:
Vaccinia virus codes for a protein called VCP [Vaccinia Viral Protein], which binds
to C4b, inhibiting classical complement pathway.
A glycoprotein component of Herpes Simplex virus bind to C3b, inhibiting both
classical and alterative complement pathways.
Evasion of Neutralizing Antibodies
Evasion of Neutralizing Antibodies:
Some viruses produce large amounts of soluble proteins that “soak up” antibody.
Variation in the viral antigens
Production of antibodies that are incapable of neutralization. Example: Caprine
Arthritis-Encephalitis of goats.
Slow production of antibodies compared to fast infection of cells by virus.
Latency:
Latency:
Although the viral genome is in host cell, transcription of viral genes that encode
viral antigenic proteins (that are presented on surface of infected host cell with
MHC-molecules) are blocked.
Integration of viral genome in host cell genome, example retroviruses.
Cell-to-Cell spread of viruses:
Cell-to-Cell spread of viruses: Not exposed to host immune mediators (refer to lecture on virus replication, slides 74-75).
Inhibition of Apoptosis:
Inhibition of Apoptosis: Poxvirus and Herpesvirus produce caspase inhibitors that protect the infected cells against death and allow virus replication to be completed.
Virus Epidemiology
The study of the determinants, frequency, dynamics, and distribution of viral diseases in populations.
Porcine Epidemic Diarrhea Virus (PEDV)
Porcine Epidemic Diarrhea Virus (PEDV) causes severe Diarrhea and Vomiting in Pigs. First identified in the United States in May 2013. By the end of January of 2014, PEDV spread quickly across many US states, causing severe economic losses.
Why study epidemiology of Viral Diseases?
The risk of infection and/or disease in an animal or animal population is determined by: Characteristics of the virus (e.g., genetic variation from evolution)
The host and host population (e.g., passive, innate, and acquired resistance)
Behavioral, environmental, and ecological factors that affect virus transmission from
one host to another
Epidemiology
Epidemiology is an interdisciplinary field of science that takes into consideration all the above factors to provide data on the pattern, spread, occurrence and impact of a viral disease in a population, and also on the potential of occurrence of a viral disease in a population.
The role of Epidemiology in Viral Diseases:
To identify and/or clarify the role of viruses in the etiology of diseases
Understanding the interaction of viruses with environmental determinants of
disease
In determining factors affecting host susceptibility
In unraveling modes of viral disease transmission
Studying the impacts of viral disease on health, economy and society
Studying role of infectious pathogens in the pathogenesis of chronic non-
communicable diseases, such as oncogenic viruses in cancer
In large-scale testing of vaccines and drugs, i.e. clinical trials.
super-spreaders
This diagram detailing SARS transmission in Singapore shows the important role of “super-spreaders” in transmitting the disease. Five people caused more than half of the 205 cases there.
originial SARS comes from
Bats to civet to humans
Case Fatality Rate
The number [%] of deaths among the clinically ill animals
Mortality rate
The number [%] of animals in a population that die from a particular disease over a specified period of time.
Morbidity Rate
The morbidity rate is the percentage of animals in a population that develop clinical signs attributable to a particular virus over a defined period of time (commonly the duration of an outbreak).
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.
Sporadic Viral diseases
Viral diseases occurring occasionally, singly, or in scattered instances, and in a irregular and haphazard manner.
Enzootic Viral diseases (Endemic in humans)
The constant presence of a Viral disease within a given geographic area or population group.
Epizootic Viral diseases (Epidemic in humans)
The occurrence of more cases of Viral diseases than expected in a given area or among a specific group of people/animals over a particular period of time. Refers to peaks in disease incidence that exceed the endemic/ enzootic baseline or expected incidence of disease.
Panzootic viral disease(pandemic in humans)
a virus epidemic occuring over a very wide area( several continents or countries) and usually affecting a large proportion of the population
Carrier
Animals that have contracted an infectious viral disease, but display no clinical symptoms.
Shed virus, transmitting disease to others.
Shedding of virus may be continuous or intermittent.
Incubatory [acute] carriers
Animals that shed virus during the incubation period
of the disease.
Convalescent [chronic] carriers
Animals that shed virus during recovery from
disease.
Inapparent carriers
Carrier state may exist in an animal with an infection that is
inapparent throughout its course.
Transmission of African Horse Sickness caused by an Orbivirus
- Zebras, Inapparent Carrier
- Culicoides Vector
- Healthy Horse bitten by Culicoides and become infected.
*
Contagious disease
A disease that is spread from one person or organism to another by direct or indirect contact.
Period of contagiousness
refers to the time during which an infected animal sheds virus.
Exotic Disease
A disease not known to occur in a particular country or geographical area.
Reservoir
The habitat in which an infectious agent normally lives, grows and multiplies; reservoirs include human reservoirs, animals reservoirs, and environmental reservoirs. May be animate or inanimate.
Surveillance
The systematic collection, analysis, interpretation, and dissemination of health data on an ongoing basis, to gain knowledge of the pattern of disease occurrence and potential in a community, in order to control and prevent disease in the community.
Seroepidemiology
Methods to study Epidemiology of Viruses
Seroepidemiology simply denotes the use of serological data as the basis of
epidemiological investigation, as determined by diagnostic serological techniques.
Determine the prevalence or incidence of particular infections
Evaluate eradication and immunization programs
Assess the impact, dynamics, and geographic distribution of new, emerging, and re-
emerging viruses.
Correlation of serologic data with clinical observations makes it possible to determine the
ratio of clinical to subclinical infections
Study of duration of viral infection
Molecular Epidemiology of Viruses
The use of molecular biological data as the basis of
epidemiological investigation of Viral Diseases.
Outcome of Molecular Epidemiology
In addition to general prevalence, crucial information on genetic diversity and origin of viruses. Important for identifying novel viruses, viral load in host, designing or judging efficacy of vaccines, developing diagnostics, and for conclusive evidence for transmission of viruses from animals to humans (Zoonosis) or vice versa
Horizontal [Lateral] Transmission
Defined as the spread of an infectious agent from one person/animal or group to another person/animal or group.
Most viruses are transmitted by the horizontal route.
Contact, Vector, Airborne, Fomites, Vehicle borne, Iatrogenic
Contact Transmission
Spread of the infectious agent by direct or indirect contact
Direct-Contact Transmission
Direct-contact transmission involves actual physical contact between an infected animal and a susceptible animal (e.g., licking, rubbing, biting). This category also includes sexual contact , as seen in HIV or Herpesvirus infection.
Droplet Transmission
Contact Transmission
Direct-Contact Transmission
Droplet Transmission: Transmission of virus in droplet nuclei (saliva or mucus) that
travel less than 1 meter from the source to the susceptible host.
Sneezing, coughing, etc.
Due to short distance travel not considered as air-borne transmission.
Indirect-Contact Transmission
Indirect-contact transmission occurs via fomites, such as shared eating containers, bedding, dander, restraint devices, vehicles, clothing, improperly sterilized surgical equipment, or improperly sterilized syringes or needles.
Fomite
An inanimate object or substance that is contaminated with the infectious agent and is capable of transmitting infectious organisms from one individual to another.
Airborne transmission
Contact Transmission
Indirect-Contact Transmission:
Airborne transmission: Spread of infectious agents by droplet nuclei in dust that
travel more than one meter, sometimes for miles, from the infected to the susceptible host.
Droplets are small enough to remain in air for prolonged periods.
Vector Transmission
Also known as Arthropod-borne transmission. Arthropod vectors carry the viruses from the infected host to susceptible host.
Fowlpox virus
Mosquitoes and other biting insects may serve as mechanical vectors for Fowlpox virus
Biological Transmission
The arthropod vector acquires virus by feeding on the blood of a viremic animal. Replication of the ingested virus, initially in the insect gut, and its spread to the
salivary gland take several days. This period is known as extrinsic incubation period.
Virions in the salivary secretions of the vector are injected into new animal hosts during blood meals.
Helps virus to cross species barriers.
Have you wondered why?
When arthropods are active, arboviruses replicate alternately in vertebrate and invertebrate hosts.
What happens to these viruses during the winter months in temperate climates when the arthropod vectors are inactive.
Overwintering”
The survival of the virus from one ‘vector season’ to the next (period during which arthropods hibernate) is called “Overwintering”
Important mechanisms for “overwintering” are transovarial and trans-stadial transmission.
Transovarial transmission
The virus is transmitted from the mother tick through infected eggs to next generation of ticks.
Trans-stadial transmission:
The virus is transmitted from larva or nymph to next stage of development (nymph or adult). But not transmitted vertically (from mother tick to eggs and next generation).
Example: tick-borne flaviviruses
Arboviruses
Typical Mechanisms of Transmission of Arboviruses
Arboviruses: A class of viruses transmitted to humans by arthropods such as mosquitoes and ticks. The first two letters of the words arthropod’ andborne, make up the ‘arbo’ that now designates this group of viruses as arthropod-borne.
Arboviruses are maintained in complex life cycles involving nonhuman primate/ vertebrate hosts and primary arthropod vectors.
Typical Mechanisms of Transmission of Arboviruses
Types of Transmission Cycles:
Enzootic cycle (Sylvatic or Jungle cycle): The natural transmission of virus between
wild animals/birds (vertebrate hosts) and primary insect vectors.
Epizootic cycle (rural cycle): The virus is transmitted between non-wild or domestic animals and the primary or accessory insect vectors.
Urban cycle: The virus cycles between humans and insect vectors
Amplifying host
Amplifying host is in which the level of virus can become high enough that an insect vector such as a mosquito that feeds on it will probably become infectious.
Dead-end host or incidental host
Dead-end host or incidental host: a host from which infectious agents are not transmitted to other susceptible hosts. They do not develop sufficient viremia to be picked up by the insect vectors.
Bridge vector
Bridge vector is an arthropod that acquires virus from an infected wild animal and subsequently transmits the agent to human or secondary host.
Western Equine Encephalitis Virus (WEEV) Transmission
Enzootic and epizootic/epidemic transmission cycles of Western equine encephalitis virus (WEEV). WEEV is maintained in an enzootic cycle between passerine birds as reservoirs and its specific mosquito vector, C. tarsalis. Domestic and wild birds are considered important reservoir and epizootic amplifying hosts. It has also been suggested that lagomorphs and rodents can serve as amplification hosts. Other mosquito vectors include Aedes melanimon, C. stigmatosoma , Ae. vexans, Ae. dorsalis
Common-Vehicle Transmission
Common-vehicle transmission includes fecal contamination of food and water supplies (fecal–oral transmission) and virus-contaminated meat or bone products [e.g., for the transmission of vesicular exanthema of swine, classical swine fever (hog cholera) and bovine spongiform encephalopathy].
Iatrogenic transmission
Iatrogenic transmission: Infection that is transferred during medical or surgical practice.
This can happen in two ways:
Introduction of pathogens by contaminated instruments (non-sterile surgical
instruments, syringes), or contaminated body surface (inadequate handwashing). Example: Spread of equine infectious anemia virus via multiple-use syringes and needles.
Introduction of pathogen through contaminated prophylactic or therapeutic preparations.
Example: Presence of porcine circovirus genome in Rotavirus vaccine.
Nosocomial Transmission
Nosocomial transmission occurs while an animal is in a veterinary hospital or clinic. Also known as Hospital Acquired Infection.
Example: During the peak of the canine parvovirus epidemic in the 1980s, many puppies became infected in veterinary hospitals and clinics.
Vertical Transmission
The term “vertical transmission” is usually used to describe infection that is transferred from dam to embryo, or fetus, or newborn before, during, or shortly after parturition (colostrum, milk, or fecal contamination of teats)
Vertical transmission of a virus may cause:
Early embryonic death or abortion (e.g., several lentiviruses)
Or may be associated with congenital disease (e.g., bovine viral diarrhea virus,
border disease virus, porcine entero-virus),
Or the infection may be the cause of congenital defects (e.g., Akabane virus,
bluetongue virus, feline parvovirus).
Examples of Vertical Transmission:
Certain retroviruses are transmitted vertically via the integration of proviral DNA
directly into the DNA of the germ line of the fertilized egg.
Cytomegaloviruses are often transmitted to the fetus via the placenta, whereas other herpesviruses are transmitted during passage through the birth canal.
Viruses are transmitted via colostrum and milk (e.g., caprine arthritis-encephalitis virus and maedi-visna virus of sheep).
Mechanisms of Survival of Viruses
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
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.
Hemagglutination & Hemagglutination inhibition test:
The hemagglutination & Hemagglutination inhibition method relies on the property of some pathogens (mainly viruses) to nonspecifically agglutinate erythrocytes.
Examples: Hemagglutination inhibition test has been used extensively for detection of serotype specific antibodies against
Examples: Hemagglutination inhibition test has been used extensively for detection of serotype specific antibodies against avian influenza & peste des petits ruminants (PPR).
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:
Hemadsorption
Glycoproteins are inserted into host cell membrane at sites of budding of enveloped viruses. This allows monolayer cells to adsorb erythrocytes on their cell membranes. This phenomenon is known as hemadsorption.
Hemadsorption-Inhibition Assay
• Infected monolayer cells are incubated with known specific antibody
Antibodies bind to viral glycoproteins [spikes] in cell membrane
• Attempts are made to wash away anti- bodies
Antibodies remain bound to viral proteins
• Pretreated monolayer cells are incu-
bated with RBCs
RBC binding is inhibited
Neutralization Assays:
Neutralization of a virus is defined as the loss of infectivity through reaction of the virus with specific antibody.
Normally, the presence of unneutralized virus may be detected by reactions such as CPE, haemadsorption/haemagglutination, plaque formation, disease in animals.
However, when Virus and Serum (Antibodies) are mixed under appropriate condition and then inoculated into cell culture, eggs or animals. There is antigen-antibody reaction.
Antibody-bound virus (Neutralized) becomes non-infectious and cannot produce desired effects in eggs, cell-lines or animals.
IgM Class-Specific Antibody Assay
Because IgM antibodies appear early after infection but drop to low levels within 1–2 months and generally disappear altogether within 3 months, they are usually indicative of recent infection.
