Topic 5: Virsuses

Question 1

Dimitri Ivanosky

Answer 1

-studied infectious tobacco mosaic virus
-he extracted the leaves and passed it through a filter that would usually catch bacteria and sterilizes the fluid but the virus stayed in it, showing that viruses and bacteria were different

Question 2

Walter reed

Answer 2

Studie yellow fever, which was prevalent in the spanish war,
proved that it was a virus transmitted by mosquitos, in 1901

Question 3

Felix D’Herelle

Answer 3

-Discovered bacteriophages
-bacteria eaters
-coined turned plaque

Question 4

Structure of viruses

Answer 4

-Oligate, intracellular parasites
(need to be inside another thing
-typixally between 10 and 100 nm
-genome few thousand to 200,000 nucleotides long, 1 gene is 1000 bases long, only has 200 genes, SMALL only has genes for replicating at that is IT!

Question 5

Exceptions to size?

Answer 5

FOUND IN AMOEBASM GARABAGE COLLECTORS OF THE WORLD

1)Megavirus
2)mimivirus (mimics bacteria, looks like gram pos)
3)pandora virus

Question 6

VIRUSES STRUCTURE

Answer 6

-Single or double stranded DNA or RNA (linear or circular)
-protein coat (capsid) around genome, made up of capsomere proteins
-possible envelope (cell derived membrane around caspid)

Question 7

Nucleocaspid

Answer 7

Capsid + genome = nucleocaspid

Question 8

What is the capsid made up of

Answer 8

capsomere proteins

Question 9

What is viral diversity dependent on

Answer 9

Host diversity, for every host there is out there there is a virus that infects it

Question 10

Capsids

Answer 10

-exhibit helical or icosahedral structure (20 side triangle)
-can also be irregular or complex shapes (think bacteriophage) have tail and tail fibers, syringe to inject dna into its host

Reason helical:
-capsid proteins are just coating the genome amd tjat is it
-so size is relative to genome

Question 11

Viral Envelope

Answer 11

-either have or dont
-get envelope from host, membrane comes from the hosts cytoplasmic membrane, gogli, nuclei, or endo rec (DIFFERENT VIRUSES USE DIFERENT MEMBRANES OF THE HOST TO BUILD THEIR OWN)

either
1)enveloped virus
plasma mem surrounds nucleocaspid
2) Naked virus
(no plasma membrane)

Question 12

what is a naked virus

Answer 12

has no plasma membrane

Question 13

why is washing hands effective

Answer 13

inactivates the virus by washing away membrane if it has it

Question 14

Viral Replication General Steps

Answer 14

1) Adhere
-stick to host cell due to rceptors (HIV AND CD4)
2) Enter
3) Uncoat
-release genome
4)Synthesis
-expression and replication of virus in host
-viral proteins created
5) Assembly
-new virus particles creqated from the viral proteins
6) Exit cell
-once assembled new virus, leave cell

Question 15

Entering the target cell! How?

Answer 15

Example using the virus HIV
-most important part of replication cycle
-H1v and CD4 receptors on the target cell
-attachment proteins on the virus bind to these receptors
-mechanisms vary depending on the host cell
-receptors are typically a protein or a glycoprotein
-animal viruses dont have to deal wiuth the cell wall that plant cells have

Question 16

How do you enter a animal virus?

Answer 16

NON-ENVELOPED VIRUSES

1)Endocytosis of NON-Enveloped
ex: rhinovirus
-virus binds to receptor
-membrane engulfs virus (endocystosis is initiated
-endosome forms
-nucleocaspid escapes to the cytoplasm and uncoats to release genome

ENVELOPED VIRUSES
ex: hiv
1)Endocytosis
-virus attaches to cell receptor
-endocytosis initiated
-endosome formed
-low pH of endosome causes fusion of viral envelope to endosome membrane
-nucleocaspid is released

2)Membrane fusion
ex: influenza
-virus attaches to cd4 receptor
-conformational change occurs in the attachement protein and bound receptor intiates membrane fusion
-viral envelope fuses with plasma membrane
-nucleocaspid enters the cytoplasm and uncoats genome

Question 17

How do viruses enter plant cells?

Answer 17

HAS CELL WALL so depends on damage to plant tissue to open a spot in cell wall

Damage caused by:
-insects feeding
-wind damage
-hail/rain damage
-fire damage
-human induced damage

Question 18

How do viruses enter bacteria cells

Answer 18

BACTERIOPHAGE

1)tail fibers attach to receptors
2) conformational change causes the tail fiber to bring the base of the tail into contact with the host cell surface
3)rearrangement of tail proteins allows inner core of tube proteins to extend into cell wall
4) contact with plasma membrane initiates transfer of dna through a pore formed in the lipid bilayer

Question 19

Bacteriophage replication

Answer 19

2 types!

1) Lytic Cycle (“LYTIC PHAGE”)
-virus enters, replicate, and lyse host cell
-lytic viruses ALWAYS DESTROY HOST

2) Lysogenic cycle (“TEMPERATE PHAGE”)
-bacteriophage intergrates their genome into host genome, becoming a PROPHAGE
-PROPHAGE just waits and lets host continue living
-PROPHAGE genome is replicated with the host cell too until … STRESS
STRESS CAUSES PROPHAGE TO ENTER LYTIC CYCLE AND BECOME LYTIC PHAGE

Host that has prophage in it is called LYSOGEN

Note: temperate phage can be both lytic or exist as prophage (“lysogeny”)

Question 20

Lysogen?

Answer 20

Cell containing prophage

Question 21

Historically, where did viruses come from? Hypothesis

Answer 21

1)Co-evolution
2) Regression hypothesis
3)Progressive hypothesis

Question 22

CO-Evolution Hypothesis

Answer 22

-the idea that viruses originated at the same time as other microbes, and have been CO-EVOLVING with them

Question 23

Regression Hypothesis

Answer 23

-idea that viruses were previously alive organisms that evolutionarily REGRESSED into host-dependent cells

Question 24

Progressive hypothesis

Answer 24

-idea that viruses originated from genetic material that gained the ability to replicate and be transmitted semi-autonomously

Question 25

Cultivating bacteriophage

Answer 25

this is harder to work with than bacteria, since they need their specific hosts

1) add host cells to phage sample
2)add agar to this and mix
3)pour onto nutrient agar plate
4)incubate and plaque will form

Question 26

What are plaques?

Answer 26

CLEARING ZONES, areas on bacterial lawn where it is clear cuz the bacteria died (did not grow) due to virus

Felix d’herelle coined this term

Question 27

Cultivating animal viruses

Answer 27

NEED ANIMAL VIRUS CELLS

-tissue cultures of host cells used to grow targets for viruses or embryonated chicken or duck eggs, eggs is expensive, one egg for one vaccine
-for eggs, inject egg with virus, fertilize the egg and let virus propogate
-must be kept sterile and bacteria free
-modern virology couldnt exist without it
-mant delveoped from first human cell line, known as heLa cells

Question 28

Viral purification

Answer 28

-needs to be done to seperate the now grown virus from the host cell

-usually starts with simple diffusion to remove large cells and cellular debris, and then they are purified and concentrated with centrifugation

Question 29

Two types of centrifugation

Answer 29

Viral purification techniques

1)Differential centrifugation
-slow speed (cells and virus suspension seperates , pellets of whole and broken cells at bottom)
-Transfer supernatant and centriguge at medium speed (pelle of nuclei and other large organelles
-Transsupernatant and centrifuge at high speed (ultracentrifugation) -> pellet of viruses at bottom

2) Gradient centrifugation
depends on densities of viral components
one spin-put highest conc on bottom, layer on top until u get a bunch of decreasing layers of sucrose
-add suspension of virus on top
-centrifugate it once (spin it once)
-the contents of the suspension will move down until it reaches a density that matches its contents, this will cause the seperation

viral particles will typically be lower down than cells as they have a higher density, stick syringe in to withdraw viral particles

Question 30

Titer means

Answer 30

a count (viral quantification)

Question 31

Viral Quantification methods

Answer 31

1) Direct Count
-electron microscope used to count, numbers are scaled up to determine titer
-doesnt differentiate between infectious or non-infectious viral particles

2) Hemagglutination assay
-exploits animal virus ability to stick to red blood cell, due to sailic acid sugar residues in membrane, causing it the blood cells to combine together in a network, forming a shield well (floats to top)
-lots of virus means more blood networked together, the better the shield, the more the virus there is
-doesnt differentiate between infectious and non-infectious, doesnt give a number, some virus dont do this

3)Plaque Assay
-virus diluted and placed on target cell
-plaques counted to determine Plaque forming units titer of og sample
-useful for phages and plant viruses
-determines how many are infectious for host

4) Endpoint assay
-endpoint means cell with evidence of infection
-TCID50: amount of virus needed to induce a CPE (cytoplasmic effect or cell damaging) in 50% of cultured cells, can graph this

-Lethal Dose (LD50): amount of virus needed to kill 50% of animal subjects (acctually need to inject animals so tcid50 better)
-can calculate how many infectious

Question 32

ICTV Follows taxonomy based on…

Answer 32

classify viruses based on order, family, subfamily, genus and species

-morphology, biological features, envelop, genome structure, disease caused, genomes

Question 33

Baltimore classification system

Answer 33

based on mRNA production methods/ GENOME
seperates viruses into 7 classes

-doublse stranded or single stranded
-rna or dna
-and how genome gets translated

Question 34

Virus Identification

Answer 34

1) Using electron microscope
-visual observations of viral morphology are made to identify

2)Nucleic acid analysis
Pcr and reverse transcriptase pcr
-used to identify genome sequence and study evolutionary pattern

Question 35

viroids

Answer 35

consist only of naked rna
-virus like, but they are just nucleic acids that do funny things due to high degree of internal complementary
-resistant to ribonucleases
-

Question 36

Prions

Answer 36

Virus like particles but NOT
“Proteinaceous infectious particles”
=proteins that are responsible for some diseases such as Mad cow disease (transmissible spongiform excephalopathies)
-membrane proteins that dont fold properly that cause neighburing proteins to not fold proerply leadingto issues
-infected prions cause conformational change in normal proteins, converting it to a prion, these prions form fibrisl that lead to disease

Question 37

REview virology today and crispr