The Infectious Cycle Flashcards
What are steps of the infectious cycle
Attachment Entry Uncoating Translation of proteins Genome replication Assembly Release
How do viruses evade the initial barriers of the immune system?
Skin has tight junctions so difficult to permeate
Intact skin – virus progeny can move cell-cell through basal lamina, and bypass cellular tight junctions
Broken skin – direct route of invasion
GI tract/ Resp – lymphoid tissue can transport into circulation. E.g M cells transport intestinal content to Peyer’s patches by mechanism known as transcytosis e.g polio
What is a “lipid raft”?
“lipid rafts” are small, dynamic membrane domains enriched with cholesterol and sphingolipids present in the plasma membrane, as well as in intracellular membranes and extracellular vesicles
They form a small part of the overall plasma membrane. Most of the plasma membrane is impermeable, but proteins/ lipids can be transported into cells via the lipid raft
Can be used by viruses such as ebola, to enter cells
Although the genomes of viruses come in a number of configurations, they share a common requirement: they must
be efficiently copied into mRNAs for the synthesis of viral proteins and progeny genomes for assembly. The synthesis
of RNA molecules in cells infected with RNA viruses is a unique process that has no counterpart in the cell.
Which enzymes are important in RNA and DNA virus mRNA synthesis?
to produce mRNA:
- RNA viruses - encode RNA-dependent RNA polymerase I
- DNA viruses/ retroviruses - encode DNA-dependent RNA polymerase II
RNA polymerase (Pol) II catalyses DNA-dependent RNA synthesis during gene transcription.
Cell culture can be used to grow viruses for study, and for production of vaccinations
What cell types are used for polio vaccine production?
live attenuated poliovirus - vaccine strains may be propagated in primary monkey kidney cells
Sometimes type of cell does not matter - as virus can multiply in variety of tissue.
However, specific viruses can only bind to, and replicate in specific viruses
immortal cell lines may be used to viral culture
What are the benefits?
What are the draw backs?
Benefits
- immortal cell line is usually a mutated tumour cell, which can multiply indefinitely e.g HeLa - Henrietta Lacks
- can produce large numbers of cells, quickly
Draw backs
- risk of tumour genes being transferred if cell culture used for vaccine production
- immortal cells do not resemble to original tissue of origin - lack structure and biochemical function
What do these terms mean?
- in vitro
- ex vivo
- in vivo
- In vitro means “in glass” and refers to experiments carried out in an artificial environment, such as a glass test tube.
Unfortunately, the phrase “experiments performed in vitro” is used to designate not only work done in the cell-free environment of a test tube but also work done within cultured cells. This is incorrect usage of this term
- in vitro - work done in test tube or plate (not using cultured cells)
- ex vivo - work done on cells in culture
- in vivo - research done in animals
Assays to detect viruses are either biological e.g plaque reduction or physical e.g EM
How does a plaque assay work?
Aims to determine titers of virus
monolayers of cultured cells are incubated with a preparation of virus, to allow adsorption to cells
After removal of the inoculum, cells are covered with nutrient medium
Original infect cells then release progeny particles. Gel restricts spread of viruses to neighbouring uninfected cells
As a result, each infectious particle produces a circular zone of infected cells, termed a plaque
If the infected cells are damaged, the plaque can be distinguished from the surrounding monolayer
Only viruses which cause visible damage to cells, can be cultured in this way
Most viruses have a linear relationship between number of virus particles, and plaque count
Plaque assay
When one infectious virus particle initiates a plaque, the viral progeny within the plaque are clones, and virus stocks prepared from a single plaque are known as plaque purified.
What is a benefit of this?
To produce clonal viral stocks
Plaque assay allows the calculation of plaque-forming units per ml, which tells you the titre of a virus stock
How is this performed?
To calculate the titer of a virus in plaque-forming units (PFU) per milliliter, 10-fold serial dilutions of a virus stock are
prepared, and 0.1-ml aliquots are inoculated onto susceptible cell monolayers
After a suitable incubation period, the monolayers are stained and the plaques are counted. To minimize error in calculating the virus titer, only plates containing between 10 and 100 plaques are counted, depending
on the area of the cell culture vessel. Plates with 100 plaques are generally not counted because the plaques may overlap, causing inaccuracies. According to statistical principles, when 100 plaques are counted, the sample titer varies by 10%.
Virus titer obtained by multiplying the number of plaques by the dilution factor
In plaque assays
What is:
one-hit kinetics
two-hit kinetics
one-hit kinetics - number of plaques directly proportional to the amount of initial virus inoculated. Linear association. e.g if double the inoculum, double the the number of plaques
two-hit kinetics - the number of plaques is directly proportional to the square of the concentration of the virus inoculated - parabolic curve. This can occur because some viruses require two RNA strands for infectivity, so plaque can only form if both are there
Fluorescent-focus assay is a modification of the plaque assay
How does it differ?
The fluorescent-focus assay, a modification of the plaque assay, can be done more rapidly and is useful in determining the titers of viruses that do not form plaques. Th e initial procedure is the same as in the plaque assay. However, after a period sufficient for adsorption and gene expression, cells are made permeable and incubated with an antibody raised against a viral protein.
A second antibody, which recognizes the first, is then added. Th is second antibody is usually conjugated
to a fluorescent molecule. The cells are then examined under a microscope at an appropriate wavelength. The titer of the virus stock is expressed in fluorescent-focus-forming units per milliliter. When the gene encoding a fluorescent protein is incorporated into the viral genome, foci may be detected without the use of antiviral antibodies.
Infectious-Centers assay is a modification of the plaque assay
How does it differ?
Another modification of the plaque assay, the infectious-centers assay, is used to determine the fraction of cells in a
culture that are infected with a virus. Monolayers of infected cells are suspended before progeny viruses are produced.
Dilutions of a known number of infected cells are then plated on monolayers of susceptible cells, which are covered with an agar overlay. The number of plaques that form on the indicator cells is a measure of the number of cells infected in the original population. Th e fraction of infected cells can therefore be determined. A typical use of the infectious-centers assay is to measure the proportion of infected cells in persistently infected cultures.
What is a transformation assay used for?
How does it work?
Th e transformation assay is useful for determining the titers of some retroviruses that do not form plaques. For example, when Rous sarcoma virus transforms chicken embryo cells,
the cells lose their contact inhibition (the property that governs whether cultured cells grow as a single monolayer
[see Volume II, Chapter 6]) and become heaped up on one another. Th e transformed cells form small piles, or foci , that can be distinguished easily from the rest of the monolayer (Fig. 2.12). Infectivity is expressed in focus-forming units per milliliter.
What is the process of an end-point dilution assay?
It provided a measure of virus titre before the development of the plaque assay. Still remains useful for measuring titers of certain viruses, that do not form plaques, or for determining the virulence of a virus in animals
Serial dilutions of a virus stock are inoculated into replicate test units (typically 8 to 10), which can be cell cultures, eggs, or animals. The number of test units that have become infected is then determined for each virus dilution. When cell culture is used, infection may be determined by the development of cytopathic eff ect; in eggs or animals, infection is gauged by death or disease.
An example of an end-point dilution assay using cell cultures is shown in Box 2.6. At high dilutions, none
of the cell cultures are infected because no infectious particles are delivered to the cells; at low dilutions, every culture is infected. The end point is the dilution of virus that affects 50% of the test units. Th is number can be calculated from the data and expressed as 50% infectious dose (ID 50 ) per milliliter. The first preparation illustrated in Box 2.6 contains 10 5 ID 50 per ml. This type of assay is suitable for high-throughput applications.
When the end-point dilution assay is used to assess the virulence of a virus or its capacity to cause disease (Volume II, Chapter 1), the result of the assay can be expressed in terms of 50% lethal dose (LD 50 ) per milliliter or 50% paralytic dose (PD 50 ) per milliliter, end points of death and paralysis, respectively. If the virus titer can be determined separately by plaque assay, the 50% end point determined in an animal host can be related to this parameter. In this way, the effects of the route of inoculation or specific mutations on viral virulence can be
quantified.
What is the particle-to-plaque-forming unit ratio (P:PFU ratio)
A lesser known measure of infectivity is the particle to plaque-forming unit ratio
The P:PFU measures the fraction of viral particles able to infect susceptible cells in tissue culture under idealized in vitro conditions
What does a P:PFU ratio of 1 represent?
That 1 virus will cause 1 plaque to form
Indicates very high infectivity. Typically seen with bacteriophage
Normal values may range from 1 to 10000
What is the significance of a high P:PFU ratio
May indicate lower infectivity as a raw marker
Other explanations
- mutated virus occurs during growth/ purification
- all viruses are capable of initiating infection, but not all succeed due to complexities of infectious cycle
- high P:PFU ratio indicates not that most particles are defective, but rather, that they failed to complete the infection
Which viruses can be detected by haemagluttination?
Adenoviridae
Orthomyxoviridae - influenza hemagglutinin binds to N -acetylneuraminic acid-containing glycoproteins on erythrocytes.
Paramyxoviridae
Contain proteins that can bind to erythrocytes (red blood cells); these viruses can link multiple cells, resulting in formation of a lattice. This property is called hemagglutination
How is a haemagluttination test prepared and performed?
In practice, 2-fold serial dilutions of the virus stock are prepared, mixed with a known quantity of red blood cells, and added to small wells in a plastic tray (Fig. 2.14). Unadsorbed red blood cells tumble to the bottom of the well and form a sharp dot or button. In contrast, agglutinated red blood cells form a diff use lattice that coats the well. Because the assay is rapid (30 min), it is often used as a quick indicator of the relative quantities of virus particles. However, it is not sufficiently sensitive to detect small numbers of particles.
Viruses such as retroviruses to not transform cells or form plaques.
How can we use fluorescence. radioactivity to measure viral enzyme activity?
Some animal virus particles contain nucleic acid polymerases, which can be assayed by mixing permeabilized
particles with radioactively labeled precursors and measuring the incorporation of radioactivity into nucleic acid.
The reverse transcriptase incorporated into the virus particle is assayed by mixing cell culture supernatants with a mild
detergent (to permeabilize the viral envelope), an RNA template and primer, and a radioactive nucleoside triphosphate.
If reverse transcriptase is present, a radioactive product will be produced by priming on the template. Th is product can be
detected by precipitation or bound to a filter and quantified.
Because enzymatic activity is proportional to particle number, this assay allows rapid tracking of virus production in the
course of an infection. Many of these assays have been modified to permit the use of safer, nonradioactive substrates. For
example, when nucleoside triphosphates conjugated to biotin are used, the product can be detected with streptavidin (which
binds biotin) conjugated to a fluorochrome. Alternatively, the reaction products may be quantified by quantitative real-time
PCR
What are examples of serological testing for viruses?
virus neutralisation
haemagluttination inhibition
immunostaining
enzyme immunoassays
Serological testing
How does virus neutralisation work?
When a virus preparation is inoculated into an animal, an array of antibodies is produced. These antibodies can bind to virus particles, but not all of them can block infectivity ( neutralize ), as discussed in Volume II, Chapter 4.
Virus neutralization assays are usually conducted by mixing dilutions of antibodies with virus; incubating them; and assaying for remaining infectivity in cultured cells, eggs, or animals. The end point is defined as the highest dilution of antibody that inhibits the development of cytopathic effect in cells or virus replication in eggs or animals.
Some neutralizing antibodies define type-specific antigens on the virus particle. For example, the three serotypes of
poliovirus are distinguished on the basis of neutralization tests: type 1 poliovirus is neutralized by antibodies to type 1
virus but not by antibodies to type 2 or type 3 poliovirus, and so forth.
The results of neutralization tests were once used for virus classification, a process now accomplished largely by comparing viral genome sequences. Nevertheless, the detection of antiviral antibodies in animal sera is still extremely important for identifying infected hosts. These antibodies may also be used to map the three-dimensional structure of neutralization antigenic sites on the virus particle (Box 2.7).
Virus neutralising antibodies are used to create monoclonal antibodies used for treatment.
Monoclonal antibodies bind specifically to a short amino acid sequence (8 to 12 residues) that fits into the antibody-combining site. This amino acid sequence, which may be linear or nonlinear, is known as an epitope
What is epitope mapping, and how is this used to produce a monoclonal antibody?
The use of monoclonal antibodies (antibodies of a single specificity made by a clone of antibody-producing cells) in neutralization assays permits mapping of antigenic sites on a virus particle, or of the amino acid sequences that are recognized by neutralizing antibodies.
In contrast, polyclonal antibodies comprise the repertoire produced in an animal against the many epitopes of an antigen.
Antigenic sites may be identified by cross-linking the monoclonal antibody to the virus and determining which protein is the target of the antibody.
Epitope mapping may also be performed by assessing the abilities of monoclonal antibodies to bind synthetic peptides representing viral protein sequences. When the monoclonal antibody recognizes a linear epitope, it may react with the protein in Western blot analysis, facilitating direct identification of the viral protein harboring the antigenic site. The most elegant understanding of antigenic structures has come from the isolation and study of variant viruses that are resistant to neutralization with specific monoclonal antibodies (called monoclonal antibody-resistant variants). By identifying the amino acid change responsible for this phenotype, the antibody-binding site can be located and, together with three-dimensional structural information, can provide detailed information on the nature of antigenic sites that are
recognized by neutralizing antibodies (see the figure).
Immunostaining uses antibodies to visualise viral proteins in infected cells or tissues
what is the process of direct immunostaining?
In direct immunostaining , an antibody that recognizes a viral protein is coupled directly to an indicator such as a fluorescent dye or an enzyme
- Virus infected cells incubated with polyclonal or monoclonal antibodies against viral antigen
- excess antibody washed away
- cells examined by microscopy for fluorescence
A more sensitive approach is indirect immunostaining, in which a second antibody is coupled to the
indicator.
Immunostaining uses antibodies to visualise viral proteins in infected cells or tissues
what is the process of indirect immunostaining?
- Virus infected cells incubated with polyclonal or monoclonal antibodies against viral antigen
- excess antibody washed away
- second antibody added, which binds to the first polyclonal/ monoclonal antibody
- excess antibody washed away
- cells examined by microscopy for fluorescence
More sensitive than direct staining. Also means sample does need any purification before testing
What are examples of fluorescent indicators used in indirect immunostaining?
Fluorescein - green light emitted
Rhodamine - red light emitted
Both fluoresce on exposure to UV light
Use an eye piece to filter blue/ UV light, leaving a dark field with green/ red fluorescence
what is the difference between immunostaining, and immunohistochemistry?
Both same process
immunostaining may use fluid such as a nasal swab for influenza
immunohistochemistry may use tissue
Recent improvements in microscopy have led to improvements in resolution and contrast - evening enabling 3D visualisation
what is super resolution microscopy?
Super-resolution microscopy combines the advantages of fluorescent imaging (multicolor labeling and live-cell imaging)
with the high resolution of electron microscopy. While conventional fluorescent microscopy has a resolution of 200
to 500 nm, single-molecule localization microscopy can achieve resolution below 1 nm. Th is resolution is achieved
by combining sequential acquisition of images with random switching of fluorophores on and off. From several hundred
to thousands of images are collected and processed to generate a super-resolution dataset that can resolve cellular
ultrastructure.
Enzyme immunoassay enable detection of viral antigens of antiviral antibodies
What is the process?
- serum sample added to solid support.
- antiviral antibody or protein adsorbed to solid surface e.g HBsAg
- capture antibody added which binds to the antiviral antibody e.g anti-HBs. This also has a chromogenic molecule
- chromogenic molecule triggers a catal
Detect viral proteins
- Sample added to a well, which contains antibodies for specific virus immobilised on solid support e.g anti-HBs
- viral proteins in sample captured by anti-HBs
- wash to remove unbound proteins
- second antibody added, binds to first antibody, acts as a chromogenic indicator
Detect antibodies to a virus
- sample added to a well, which contains viral antigen immobilised on well surface e.g HIV
- wash to remove unbound proteins
- second antibody added, binds to first antibody, acts as a chromogenic indicator
Modification of enzyme immunoassay is a lateral flow immunochromatographic test
Can be used for rapid antigen detection
How is it different to a typical enzyme immunoassay?
Antigens in the sample react with a specific antibody, which is conjugated to a detector.
The antigen-antibody complexes move across the membrane until they are captured by a second antibody.
At this point a line becomes visible, indicating that viral antigen is present in the sample
The lateral flow immunochromatographic assay does not require instrumentation and can be read in 5 to 20 min in a physician’s office or in the field.
Molecular methods of detecting viral nucleic acids/ antigens is very common
What are examples of testing methods?
PCR
DNA microarray
High throughput sequencing aka next-generation sequencing
What are the steps of PCR?
Each cycle consists of primer annealing, extension, and thermal denaturation carried out by automated cycler machines
How does a DNA microarray work?
This approach provides a method for studying the gene expression profile of a cell in response to virus infection (Chapter 8), and can also be used to discover new viruses. In this method, millions of unique viral DNA sequences fixed to glass or silicon wafers are incubated with complementary sequences amplified from clinical and environmental samples. Binding is usually detected by using fluorescent molecules incorporated into amplified nucleic acids.
Next generation sequencing can be used to look for unknown pathogens, and document the normal virome
What are drawbacks to this?
Very sensitive, so can detect viruses which may not be pathogenic, and may instead be contaminants
After a virus infects a cell, what is the latent period?
the time it takes to replicate, assemble, and release new virus particles before lysis,
After a virus infects a cell, what is the eclipse period?
After virus enters cell, if the cell bursts, then it may not release infectious virus as virus is disassembled, and new virus has not been formed yet
Bacterial growth curve, has lag phase, then exponential phase until all nutrients are used up. Then stationary and death phase
How does a viral growth curve in cells differ in one-step growth analysis?
Eclipse period when there is no infectious particles produced
Latent phase - virions being assembled
Burst phase - cells burst releasing a high number of virions
Return to eclipse period once new cell infected
What does multiplicity of infection (moi) mean?
Multiplicity of infection is commonly defined as the ratio of infectious virions to cells in a culture.
if there are 1000 virions, and 1000 infected cells, the moi is 1
Infection depends on the random collision of cells and virus particles.
When susceptible cells are mixed with a suspension of virus particles, some cells are uninfected and other cells receive one, two, three, etc., particles. The distribution of virus particles per cell is best described by the Poisson distribution.
The yield of infectious virus per cell can be calculated from the data collected during a one-step growth experiment
(Fig. 2.19). Th is value varies widely among diff rent viruses and with different virus-host cell combinations. For many
viruses, increasing the multiplicity of infection (Box 2.10) above a certain point does not increase the yield: cells have a
finite capacity to produce new virus particles. In fact, infecting at a very high multiplicity of infection can cause premature
cell lysis and decrease virus yields.
How do viral growth curves differ between enveloped and non-enveloped viruses?
enveloped - only become infectious at plasma membrane. So non-infectious if sampled from the cell
non-enveloped - infectious virions produced in cell
What are problems that might be encountered when trying to evaluate the role of a significant viral/cellular protein, in viral pathogenesis?
• More than one protein may provide the required function (redundancy).
• The function of the protein might be essential to the cell, and mutation of the gene that encodes it (or inhibition of
protein production) could be lethal.
• Only small quantities of the protein might be required, and reducing its activity with an inhibitor, or its concentration
may be insufficient to observe a defect in viral reproduction.
• The cellular protein might provide a slight enhancement to viral reproduction that could be difficult to detect, but
may be physiologically significant.
• Synthesis of an altered cellular gene or overexpression of a normal cellular gene may produce changes that affect virus
reproduction for reasons that are irrelevant to the natural infection (artifacts).
