Multiplication of Viruses Flashcards
What is the morphological characterisitic exhibited in measles infected HEp-2 cells?
multinucleated cells
Where in the embryonated chick egg can we innoculate the vaccinia virus (a non-pathogenic virus of the Poxvirus family) that can be used in a Pock assay?
chorioallantoic membrane
this layer of membrane can act as a platform for the innoculation of vaccinia virus
What is the Pock assay?
- semi-in vitro in vivo assay
- first method for assaying viral infectivity
- you can count foci or “pocks” that each were initiated by the infection of a single cell; this is a proliferative response to a viral infection by the vaccinia virus
What is the disadvantage to using the Pock assay to determine viral infectivity?
- it’s messy and requires thousands of eggs
- requires incubations and technicians who will manipulate and handle the embryonated eggs
- most severe problem: this system does not support the multiplication of other human viruses (only vaccinia virus and maybe herpes simplex virus)
What is the most accurate assay of viral infectivity available? Why?
Plaque assay
This is the most accurate assay for viral infectivity because we can manipulate condition in such a way that the probability that a cell will be infected by more than one virus particle is essentially zero.
How does the Plaque assay work?
- monolayer of HEp-2 cells are grown at the bottom of a petri dish (or other cel line that would support the growth of a virus)
- cell cultures are incubated until they become confluent
- growth medium is removed
- inoculate cultures with constant volume of dilutions of viral suspensions
- allow virus to attach and add agar which prevents diffusion of the virus (progeny virions are localized or restrained from movement from the cell they infected)
Measure the number of clear spots AKA plaques:
- presence of red color is due to the neutral red that is taken up and retained by viable cells
- when a cell dies it releases the neutral red dye
- clear area or plaques is the viral infected cell that was killed
The presence of more red color than clear spots in a plaque assay is indicative of what in terms of viral infectivity?
Neutral red stains only live cells, so plaques appear as clear areas against a pink background.
Viral infectivity is low as the number of plaques (clear spots) is low.
These assays are based on CPE and virion spread, however the range of the spreading is limited by agar or agarose or by specific antibody.. What is this assay? What important relationship in virology can we learn from it?
Plaque Assay
In a one-hit system, each plaque is initiated by a plaque-forming unit (pfu) of virus. These assays yield useful information when the multiplicity of infection (M.O.I) is low, i.e. between 10^-2 and 10^-5. M.O.I is defined as the ratio of virions (pfu) to viable cells: #PFU/#cells. For example, assume that the number of cells in each dish is 1X10^6. We desire to titrate a viral stock whose titer (expressed in PFU/mi.) is expected to be in excess of 10^-2. How can we prove this using a plaque assay?
What is titer?
Viral titer is the lowest concentration of virus that still infects cells.
Titer (PFU)= average # of plaques x reciprocal of dilution
Titer value denotes that in the experiment Dr. Roane designed, 1 mL of the undiluted original viral suspension contained 3x10^9 infectious particles
Titer is equivalent to the viable cell count you see.
It is the inverse of the greatest dilution.
What is the pattern of plaque formation exhibited by most human viruses?
one hit kinetics: a single infectious particle can infect a single cell and induce a plaque
Plaque consists of hundreds of cells that have been killed during sequential rounds of multiplication. but the initial event was the infection of one cell by one virus particle.
There are nonhuman viruses in which the pattern of plaque formation is 2 hit, 3 hit, and even 4 hit. What is going on when you have a 2 hit system?
The slopes of the curves are less than 1. the curve that depicts the formation of plaques when the plaque formation is a 2 hit system.
In order for a virus to multiply successfully, all of its essential genes must be functioning.
In the case of human viruses, all of the genes required to induce a plaque for a given virus are located on a single genome.
For the genes to be 2 hit kinetic the genes are distributed among 2 different particles. Particle A has 6 of the required genes and particle B has 4 required genes. Mixture of AB particles, they will interact with cells on a random basis. some cells will have 2A particles of some will have 2B particles. In these cases, plaques will not form. So it is a chance phenomenon.
What is the significance of knowing about 2-hit system?
the importance and significance of viral genes which encode essential proteins for viral multiplication
the viral genes must be introduced into a cell for viral multiplication to ensue
What is Multiplicity of Infection (M.O.I)?
In microbiology, the multiplicity of infection or MOI is the ratio of agents (e.g. phage or more generally virus, bacteria) to infection targets (e.g. cell).
It allows insight on what you can do with the viral suspension you are using. It is the ratio of plaque forming units of virus per cell. If you inoculate cultures with low dilution, there will be large # of virus per particle. An MOI of 300 means that for every viable cell there were 300 viral particles. 0.0003 means the probability that the cell will be infected by more than one viral particle is essential zero.
What is the poisson distribution?
Poisson distribution is used by scientist to know how much virus is going to infect a cell under the conditions it is using.
The power of the plaque assay relies on what relationship?
Increased # of viruses that can induce plaques is linear.
There’s a direct relationship between input of virus with number of plaques (slope of curve is one).
Viral genes encode essential proteins for viral multiplication. It needs to be introduced in a cell for multiplication to ensure.
Small # of gene –> polio ~10 gene
Large # of gene –> Poxvirus ~200 gene
When a virus multiplies, each virus particle it produces is infectious. True or false?
FAALLLSEEE!!
When virus multiplies, not every [article that they produce is infectious. For example, in the papillomavirus, for every single infectious particle, there is 10,000 non-infectious particles produced. For herpes on the other hand, for every single infectious particle there are 50-200 non-infectious particles produced.
What is endpoint titration?
It is widely used but not as accurate as the plaque assay. You can assess viral infectivity via the production of CPE in culture. You can microscopical detect CPE in infected cultures. The presence or absence of an endpoint at a particular viral suspension dilution can help determine viral infectivity. This can be used IN VIVO.
What are TCID50 and LD50?
They are endpoint dilutions that quantify the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells.
Tissue culture Infective Dose (TCID50) is the measure of infectious virus titer in cell culture.
Lethal Dose (LD50) is the measure of infectious virus titer in animals.
Varivax, Zostavax, and MMRII are what type of vaccines and what do they each prevent?
they are all live attenuated vaccines meaning that their preparations are not pathogenic but since they are live they can multiply in cells.
Varivax prevents chickenpox
Zostavax prevents shingles
MMRII prevents measles, mumps, and rubella
What viral infectivity assay is used heavily in vaccine production?
Plaque assay
What are criteria must vaccines have to be approved by FDA before being used in humans?
- a standardized dose of live attenuated virus used in each vaccine
- ensure that the standardized does does indeed produce immunity
- vaccine is safe
Varivax: the minimum amount of virus present in a standard 0.5 mL dose is 1,350 PFU (plaque forming units).
Zostavax: the minimum amount of virus present in a standard 0.5 mL dose is 19,400 PFU
MMR, in each 0.5 mL dose contains not less than 1,000 TCID50 of measles virus, 20,000 TCID50 of mumps virus, and 1,000 TCID50 of rubella virus.
What is the point of endpoint titration in vaccines?
This endpoint titration, TCID50, this assay is used to standardize the concentration of infectious virus in an FDA approved vaccine, making it a valid assay.
Endpoint titration can be used in vivo conditions and in vitro conditions. True or false?
True
What are two characterizing examples of “endpoint” in endpoint titrations?
The virus can either kill the mouse or produce neurotropic damage in mice (eg. mice chasing its own tail). You have no problems distinguishing between the unaffected and affected mice.
Compare the Pock assay, plaque assay, and endpoint titration.
Pock assay: 1st assay historically, not widely used
Plaque assay: most wide used, most accurate assay available
Endpoint titration: not as accurate as plaque assay, no effort is made to restrict the movement or dispersion of newly produced virus particles (no agar added); considered the most sensitive assay but not the most accurate
Which assay is widely used in studies of influenza virus for quantifying the relative concentration of the virus? How does the assay work?
Hemagglutinin assay
- does NOT measure viral infectivity
- is the basis of measuring antibodies against viruses specifically called hemagglutinin inhibition test
- the antibody that will inhibit agglutination have to be specific for that virus (the antibody binds to the virus inhibiting agglutination of RBCs by the virus and so the amount of viral particles can be INDIRECTLY measured)
- it all about how far we can dilute a viral suspension and still detect agglutination
Analysis of the replication cycles of the large human DNA viruses (Papova-Adeno-Herpes-and Pox-) reveal the existence of fairly distinct phases. What is the early phase, eclipse, late phase, and latent period?
During the early phase, the virus recognizes and attaches to its target cell. These steps are followed by penetration and uncoating of the viral genome. Following transport of the genome to correct intracellular site in the cytoplasm or the nucleus, an early round of mRNA and protein synthesis commences. The gene products (viral proteins) that appear at this time are either enzymes that catalyze the synthesis of precursors of viral DNA or the synthesis of DNA itself. Regulatory proteins may also be synthesized during the early phase. The late phase actually begins with the onset of viral DNA synthesis. This event triggers the expression of late viral genes which encode structural proteins. Assembly maturation and release also occur during the late phase. The eclipse period corresponds to that period that follows uncoating and precedes the appearance of intracellular progeny virions. The latent period corresponds to that period that ensures between the end of the eclipse period and the appearance of extracellular virus. You should understand that a sinlge infected cell may produce 100,000 virions.
