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BIO 315 > Module 9: Viruses (History/Methods for Virus Stuff ) > Flashcards

Module 9: Viruses (History/Methods for Virus Stuff ) Flashcards

1
Q

What are the names of the 3 main hypotheses of viral origins?

A

1) Coevolution

2) Regressive

3) Progressive

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2
Q

What is the coevolution hypothesis?

A

Postulated that:

Viruses may have originated PRIOR TO or AT THE SAME TIME AS the primordial cell and has coevolved with these hosts

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3
Q

What is the regressive hypothesis?

A

Postulated that:

Viruses may represent a form of “life” that has LOST some of its essential features and thus has become dependent on a host

–> Viruses may have been fully functional cells that became PARASITES of other cells and gradually lost some of their essential capabilities

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4
Q

What is the progressive hypothesis?

A

Postulated that:

Viruses may have originated from existing genetical elements/material that overtime gained functions necessary for them to:

1) Be transmitted between organisms
2) Undergo aspects of replication

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5
Q

What viral origin hypothesis goes back to the “RNA world”?

How so?

A

The COEVOLUTION hypothesis

–> A self-replicating RNA molecule may have evolved to make use of protein synthesizing machinery of the primordial cell

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6
Q

What is a piece of evidence for the coevolution hypothesis?

Why?

A

The existence of Nucleocytoplasmic Large DNA Viruses (NCLDVs)

== Because these are viruses with complex DNA genome that have genes associated with TRANSLATION!

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7
Q

NCLDV

A

Nucleocytoplasmic Large DNA Virus

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8
Q

What are the 2 pieces of evidence for the regressive hypothesis?

A

1) Endosymbiosis of mitochondria + chloroplasts

== could have been an endosymbiosis of bacteria into eukaryal cells that lost so much functionality they became viruses

2) Chlamydia

== b/c it is missing key ETC genes (may have lost metabolic activities overtime)

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9
Q

What is evidence AGAINST the regressive hypothesis?

A

That the animal virus genomes have greater similarity to their HOST cell genome!

(Likely not an endosymbiosis event of bacteria into eukaryal cells because if this were the case, viruses would resemble the bacterial genome instead)

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10
Q

What is the progressive hypothesis also referred to as?

A

The “Escape” hypothesis

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11
Q

Based on the progressive hypothesis, where did bacteriophages potentially arise from?

A

PLASMIDS!

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12
Q

Based on the progressive hypothesis, where did retroviruses potentially arise from?

A

From eukaryal RETROTRANSPOSONS

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13
Q

Retrovirus

A

An RNA virus that uses REV. T to make a DNA copy of its RNA genome to be inserted into the host cell genome and undergo replication

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14
Q

Retrotransposon

A

Piece of DNA that is converted to RNA, moves to a new locaiton in genome, converted BACK to DNA by REV. T, and reinserted into genome

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15
Q

What is the process of retrotransposon replication?

A

1) RNA polymerase produces mRNA copy of the retrotransposon from its original location in genome

2) A dsDNA copy is produced from the mRNA retrotransposon template using REV.T

3) A new region of the chromosome is CUT (dsDNA break)

4) The dsDNA copy of the retrotransposon is inserted into the break to “fix” the cut with an insertion

–> Retrovirus is now in TWO locations in the genome!

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16
Q

How are retrotransposons possible evidence of the progressive hypothesis?

A

Because retrotransposons have a similar step of RNA to DNA conversion via REV. T that retroviruses also use!

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17
Q

What are the 2 main issues with studying viruses?

A

1) Cultivation of viruses requires cultivating the host cells (which may not always be the easiest to cultivate)

2) The small size of viruses makes them difficult to purify and count!

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18
Q

Overall, how are viruses cultivated?

A

By inoculating appropriate host cells with the virus of interest and then harvesting the viral progeny from the host cells

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19
Q

How are bacteriophages cultivated?

A

By inoculating liquid culture of GROWING bacteria with a small amount of phage and incubating

and THEN:

Solution is filtered to remove any remaining bacteria and cell debris to isolate the viral progeny

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20
Q

During the incubation period when cultivating bacteriophages, what changes in the appearance of the solution?

WHY?

A

The turbidity: The solution goes from turbid to clear!

–> b/c as more infection occurs, the # of bacterial cells in the culture decreases (as they die)

–> With less bacteria, the solution becomes less turbid

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21
Q

How are animal viruses cultivated?

A

By adding a small amount of virus to appropriate host cells growing in a flask

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22
Q

During the incubation period when cultivating animal viruses, what changes in the appearance of the cell culture?

WHY?

A

As more time passes a greater amount of CPE is observed

–> As greater infection occurs, more cells are damaged = CPE

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23
Q

What is CPE used as a marker for?

A

Used as a marker for the EXTENT of viral reproduction when cultivating

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24
Q

CPE

A

Cytotoxic Effects

== Visible changes in cellular morphology often associated with cell damage or death

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25
Clonal Viruses
Viruses originating from a SINGLE ancestral virus
26
What is the benefit of cultivating clonal viral populations?
Clonal populations display **genetic homogeneity** and **stability** = population has more consistent properties!
27
Why are heterogenous populations likely to be LESS consistent than clonal populations?
Because with each virus producing its own progeny, there is a greater chance for recombination and mutations to occur == greater genetic diversity within the heterogenous population!
28
What is the process for cultivating CLONAL bacteriophages?
1) a SMALL volume of susceptible bacterial host cells are added to a phage sample 2) The phage/host mix is added to molten nutrient agar and mixed 3) The agar mix is then poured onto a nutrient agar base and allowed to solidify 4) The plate is incubated and the phage replicates 5) Progeny infect only neighboring cells (due to restricted movement by agar) == **Distinct plaques form** 6) Independent clonal populations collected from each plaque
29
Plaque
A localized clear area on a lawn of cells caused by cell death
30
In clonal bacteriophage cultivation, what does each plaque contain?
Each plaque contains viral progeny from a SINGLE virus! (One CLONAL population!)
31
In bacteriphage clonal cultivation: What ensures that each plaque consists of ONE clonal population?
The AGAR! == Immobilizes the viruses released from the host cells within the culture (prevents them from moving very far, so they stay localized to one distinct area)
32
What is the purpose of the molten agar in clonal bacteriophage cultivation?
Purpose is to spatially constrain the viral progeny released from host cells to keep the progeny from each virus localized to distinct areas (prevents contact so that we can isolate clonal populations)
33
What is different between cultivating clonal bacteriophage and clonal animal virus populations?
**Bacteriophages** = Host cell/phage mix is added TO the molten agar and then poured on a plate **Animal Viruses** = Host cell/virus mix is added onto a plate and molten agar is poured ONTOP
34
Simple/Crude Purification of Viruses
Method that involves simply centrifuging a viral suspension and then filtering the supernatant through a very small pore filter
35
What is the limitation of simple/crude purification of viruses method?
The filtration step allows for separation of the viral particles BUT it **DOES NOT** allow us to **CONCENTRATE** the viral sample
36
What are 2 methods to get a more concentrated purified viral sample? (compared to simple/crude purification)
1) Differential Centrifugation 2) Gradient Centrifugation
37
Differential Centrifugation
Process of subjecting a solution with a mix of different size particles to **multiple rounds of centrifugation at progressively higher speeds**, with each spin pelleting down particles that are heavier and larger than the ones in the supernatant --> Used to pellet out small particles! (like viruses)
38
What is the differential centrifugation process for purifying a viral sample?
1) Cell and viral suspension is centrifuged at LOW speed == Supernatant is collected (pellet containing whole + broken cells is discarded) 2) Supernatant is centrifuged at MEDIUM speed == supernatant is collected again (pellet containing nuclei + organelles is discarded) 3) Supernatant is centrifuged at HIGH speed in an ULTRA-centrifuge == PELLET is collected (contains viral particles!)
39
How many centrifuge steps are involved in differential centrifugation to isolate viruses from a sample?
3 main centrifugation steps! 1) LOW speed 2) MEDUM speed 3) ULTRA centrifugation (HIGH speed)
40
Viral Sample Differential Centrifugation: What are the contents of the supernatant + pellet after FIRST centrifuge step?
LOW speed = **Supernatant: Subcellular components + viral particles** Pellet: Whole + broken cells
41
Viral Sample Differential Centrifugation: What are the contents of the supernatant + pellet after SECOND centrifuge step?
MEDIUM speed = **Supernatant: Mainly virus + small particles** Pellet: Nuclei + other cell organelles
42
Viral Sample Differential Centrifugation: What are the contents of the supernatant + pellet after THIRD/LAST centrifuge step?
ULTRAcentrifuge (HIGH speed) = Supernatant: Small particles **Pellet: VIRUS PARTICLES**
43
What does gradient centrifugation separate component by?
Separates by BOTH size + density
44
Gradient Centrifugation: Process
1) Density gradient (20%-70%: top-bottom) of sucrose or salt is made in a centrifuge tube 2) Viral suspension is added ontop of the gradient 3) The tubes are ULTRAcentrifuged 4) Bands of interest are isolated
45
In gradient centrifugation, how do particles move through the tube?
Particles move down through the tube until the reach a region of the gradient that has an EQUAL density = particle STOPS in that area (no more movement) and forms a band in the tube!
46
Viral Load
Amount of virus in the BLOOD of an infected individual
47
Viral Titer
Concentration of virus in a solution (preparation)
48
What are the 2 main applications of viral quantification?
1) RESEARCH 2) CLINICAL
49
Why is viral quantification helpful in research?
Because knowing the viral titer of a given sample can allow for researchers to run multiple repeat trials with the same conditions!
50
Why is viral quantification helpful in a clinical setting?
The viral load of an individual can be assessed to determine the health of the person with a viral infection (Ex: helpful for those with HIV)
51
What are the 4 main "dilemmas"/problems when dealing with quantifying viruses?
1) How can we "see" viruses to count them? 2) Should non-infectious viruses be counted? 3) What does infectious mean? 4) Should we only count viruses that can infect humans?
52
What are the 4 main methods of viral quantification?
1) Direct Count 2) Hemagglutination Assay 3) Plaque Assay 4) Endpoint Assay
53
What do Direct Count methods measure?
These methods measure the ABSOLUTE # of total viral particles in a sample
54
What are the main Direct Count methods? (3)
1) Electron Microscopy 2) Fluorescent Microscopy 3) Flow Cytometry
55
What is the process of direct count method using an EM?
1) Known quantity of microscopic markers are added to a given viral sample (Ex: Latex Beads) 2) Viral sample with markers is added to a grid plate 3) Grid is viewed under EM + # of viral particles/markers (beads) is counted
56
What are the drawbacks of using "Direct Count" methods for viral quantification?
1) Requires the use of expensive machinery (EM microscope) 2) ALL particles are counted = does not differentiate between infectious and non-infectious particles
57
Hemagglutination Assay
A viral quantification method that is based on the capability of most viruses to bind to RBCs and cause hemagglutination at high enough viral concentrations
58
Hemagglutination
The clumping together of RBCs
59
Process of hemagglutination assay
Serial dilutions of a viral sample are added to a constant (known) # of RBCs in a well plate --> Each dilution goes into its own well --> Multiple samples can be run at the same time in different rows of the well plate
60
When a hemagglutination assay is done, what are the 2 types of results that are observed in the wells?
1) Shield Well (AKA "Gel mat") 2) Button Well
61
What is the difference between a shield well and a button well? What do each of them tell us?
**Shield Well** = Hemagglutination occurred! (this clumping is what creates the diffuse mesh/shield visible in the well) **Button Well** = NO hemagglutination (no clumping causes the RBCs to just sink to the bottom of the well)
62
Shield Well vs Button Well: What do each mean for viral concentration?
**Shield Well** = Concentration of the given sample dilution is high enough for hemagglutination to occur (> conc. of virus) **Button Well** = Concentration of the given sample dilution is NOT high enough for hemagglutination to occur (< conc. of virus)
63
In a hemagglutination assay, what results would NOT allow us to determine a hemagglutination titer?
When ALL tested dilutions DO NOT yield hemagglutination!
64
Hemagglutination Titer
The MAX dilution of a viral sample that still results in complete hemagglutination (shield/mat)
65
What EXACTLY in hemagglutination assay measuring? (Hint: NOT viral conc.)
Hemagglutination Titer
66
What are drawbacks to hemagglutination assay? (3)
1) Does **not allow for differentiation** between infectious + non-infectious viral particles 2) Hemagglutination titer **does NOT reflect the total # of viral particles** in a sample 3) **Potential inaccuracy**: Other components in a given sample (like viral proteins alone) may cause hemagglutination (so the H. titer may not be reflective of the viral conc.)
67
What are the two main benefits of hemagglutination assay?
1) Simple + affordable 2) Provides useful virus concentration info when COMPARING titers among samples
68
Plaque Assay
A method for quantifying only INFECTIOUS viral particles by infecting host cells and counting plaque forming units (PFUs)
69
What does a plaque assay measure?
Infectious Titer (concentration of infectious viral particles)
70
What is the process of a plaque assay?
1) Plates are prepared with appropriate host cells 2) Plates are inoculated with serial dilutions of a viral sample 3) Nutrient agar is added to the cell plates (or some gel-like medium) 4) Incubation 5) Plaques are counted on each plate
71
What is the role of agar in plaque assays?
To constrain the movement of viruses = each virus forms a distinct plaque IMPORTANT! b/c it allows us to use the plaque count as a count of the infectious viral particles!
72
PFU
Plaque Formung Unit
73
What is a drawback to the plaque assay?
The assay depends on the tested virus being able to LYSE cells! == Will not work for quiescent phages or other non-lysing viruses
74
What is the process of the plaque assay variant method used for plant viruses?
1) Serial dilutions of a plant virus sample are created 2) The serial dilutions are applied to scarified/scratched leaves 3) Incubation 4) # of LESIONS is counted!
75
Lesion
Localized area of cell death
76
What is an endpoint assay?
A viral quantification method used to quantify particles that: DO NOT form plaques + DO NOT agglutinate RBCs BUT **DO have an OBSERVABLE effect** on inoculated cells / animals
77
What feature is required of the virus being tested when using an endpoint assay?
The virus MUST produce some observable effect on the inoculated host! (so that we can measure its quantity!)
78
What is the process of an endpoint assay?
1) Host cells/organisms are inoculated with a given virus 2) The inoculated cells/organisms are observed for any viral effects
79
LD50
Lethal Dose 50 == The viral dilution that causes 50% of inoculated host organisms to die
80
What is a drawback of using LD50? What is an alternative?
LD50 requires the use of a large # of animal hosts Alternative = TCID50
81
TCID50
Tissue Culture Infectious Dose 50 == The viral dilution that causes 50% of inoculated cells to display CPE

BIO 315 (25 decks)

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