Exam One

Question 1

What is the purpose of Kosh’s Postulates?

Answer 1

1. link agent to disease; pathogen to etiologic agent

Question 2

List the postulates that consist of Kosh’s Postulates:

Answer 2

1. Same microorganisms are present in every case of disease.
2. The microorganisms are isolated from the tissues of dead animal in a pure culture can be prepared.
3. Microorganisms of the pure culture are inoculated into a healthy animal. Disease is reproduced.
4. The identical microorganisms are isolated and recultivated from the tissue specimens of the experimental animal.

ADDITIONAL:
1. Proof of filterability
2. Detection of specific immune response to the virus by host (T cells of antibodies and other immune proteins)

Question 3

How is filterability a demonstration that a disease is microbial based?

Answer 3

Virons will pass through filters due to their small size (nm).

Bacterium will not. They will be retained.

Question 4

What does a limiting dilution experiment show about virons?

Answer 4

Microbes can multiply but only in living tissue.

Question 5

Why do microbes need a host?

Answer 5

to perform biological responses using the host’s proteins, ATP consumption, enzymatic reactions

Question 6

In terms of size bacterium are on the [] scale while viruses are on the [] scale.

Answer 6

micro; nano

Question 7

Can a light microscope see a virus?

Answer 7

No, it’s range does not extend to the size of a virus

Question 8

Plaque:

Answer 8

Plaque: area of cells that have been killed by a virus infection.

Question 9

What is evidence of infection at the macroscopic level?

Answer 9

immune response of skin changes such as warts, skin lesions, or rashes

Question 10

Can viruses be grown in nutrient broth or agar plates? If not, where can they grow?

Answer 10

No only bacteria

Grown in a cell culture as they’re intracellular parasites

Question 11

New viruses are formed by a process of [].

Answer 11

assembly

Question 12

Explain the general principle of the “germ theory”.

Answer 12

Some infectious diseases that plague our world are caused by unseen microbes.

Question 13

Explain how early experiments with tobacco mosaic virus helped scientists develop a concept of a virus.

Answer 13

Early model for microbial based disease.

1. Transmissible: agent was transmissible from a disease sap to a healthy plant.
2. Filterability: agent was filterable, passed through due to small size. Removed bacteria but not virus.
3. Amplification: signals microbes.
4. Small: first viewed virus

Question 14

Understand the definition and properties of a “filterable agent”.

Answer 14

The agent is able to pass through a filter due to its small size (nm).

Question 15

Explain why viruses are not considered to be living organisms.

Answer 15

Self-propagating, however, don’t demonstrate properties of life.

Inert no biological processes occurring while outside a cell. Intracellular allows RNA/DNA/protein synthesis, replication, ATP consumption.

Needs support of another cell.

Question 16

Explain the meaning of an “obligate intracellular parasite” and explain why viruses are described as such.

Answer 16

All viruses specified processes happen in the cell.

If you can exist and replication only within the cell.

Outside viruses are inert. Cannot live outside the cell.

Question 17

Explain how the process of virus assembly fundamentally differs from the processes of mitosis and binary fission.

Answer 17

Eukaryotic cells make more through cellular division.

These have more complex and similar processes.

Virions amplify their number by synthesizing their structural components within the host’s cell and then assemble

Question 18

Explain why early scientists to fulfill Koch’s postulate for virus-based diseases.

Answer 18

Technological Issue:

Failed to successfully complete postulate two (isolate in a pure culture).

Viruses do not grow on agar plates or broth medium like bacteria.

Don’t grow outside of the cell.

Question 19

Explain the meaning of a cytopathic effect and be able to identify/describe the different forms of CPE that can arise during virus infection of a cell.

Answer 19

CPE is the consequences of a disease affecting the cell.

These characteristics can be visualized in infected cells but absent in uninfected cells.

1.plaque/cell lysis
2. inclusion bodies
3. proliferation of the nuclear membrane
4. vacuoles in the cytoplasm
5. rounding of cells
6. cell detachment from monolayer
7. cell fusion

Question 20

Explain how a virus plaque forms on a cell monolayer.

Answer 20

1. cell monolayer grown in a liquid environment
2. liquid environment removed and antigen placed
3. agar nutrient overlayed
4. antigen placed and infects one cell
5. neighboring cells are infected and plaque grows

Question 21

Explain the meaning of an inclusion body and be able to identify/describe the different forms of inclusion bodies that can form during virus infection of a cell.

Answer 21

Intracellular abnormalities (new structures) are a type of CPE. They can be found in the nucleus or the cytoplasm.

Can show with immunofluorescence (antibody with fluoro tag bound to viral proteins in this structure)

Negri bodies are found in rabies virus.

Virus assembly areas. That can be typically found near nucleocapsids.

Question 22

Explain what a syncytium is and explain how virus infection can cause syncytium to form.

Answer 22

CPE: a consequence of multi-fusion of cells: multinucleated cells

HOW: pH independent binding. Mediates membrane fusion. Infected virion has glycoproteins on the surface and can bind to neighboring cells.

Question 23

Explain in general terms how virus alteration of the cell cycle can result in the formation of tumors.

Answer 23

virus transforms cells into CA cells

certain proteins take the resting cell (majority of cells are in this state as they’re highly specialized) and initiate cell division

cells never go back to G0 state

Question 24

Explain key phenotypic differences between transformed and non-transformed cells.

Answer 24

rounded up cells = infection center

1. loss of contact inhibition
2. anchorage-independence
3. loss of serum-dependence

Question 25

Understand that apoptosis is a common cellular response to virus infection (sensed as a cellular stress).

Answer 25

DNA laddering is a sign of apoptosis

Question 26

Understand that apoptosis is not in the interest of virus survival and many viruses have evolved strategies to prevent or delay the apoptosis process.

Question 27

Explain in general terms (not the actual molecular mechanisms) how virus infection can influence gene expression by the host cell and explain how the virus alteration of host cell gene expression.

Answer 27

virus needs access to the host cells resources: competition shut off gene expression for host cell proteins and turn on selective translation of viral proteins

Question 28

Explain the primary functions of the virus particle (viron/virus):

Answer 28

1. package the virus genome 2. protect the virus genome (from enzymes, acids, bases) 3. transport the virus genome between cells or hosts

Question 29

Describe the many different forms of virus genomes.

Answer 29

1. DNA or RNA 2. Single stranded or double stranded 3. polarity (sense) of single stranded genome is positive, negative, or ambisense 4. linear or circular 5. monopartite (single molecule) or segmented/mutipartite (multiple gene segments)

Question 30

Explain the difference between a positive-sense and negative-sense RNA.

Answer 30

Positive: RNA/DNA message sense order, sequence is translatable Negative: RNA/DNA genome that is not translatable; complement of positive; template to create complementary strand results in a translatable strand

Question 31

Explain the fundamental properties of capsid, nucleocapsid, matrix, tegument.

Answer 31

Capsids: protein shells that encase or encapsidate the viral genome. External. Made by 4 repeated proteins: VP1-4. Internal. CA protein. Can see on scope as a dark region circular (front) or bull horn (side) Nucleocapsid: protein shell that is intimately associated with the virus genome. External: TMV Internal: Rabies Matrix: many enveloped virus structures have internal protein structure right underneath the lipid envelope. Composed of repeating M protein that functions in genome packaging and virus maturation.

Question 32

Explain the fundamental protein: protein interactions that maintain the structure of the virion (H bonds or electrostatic interactions)

Answer 32

covalent binding helps maintain virions structure hydrogen electrostatic attractions hydrophobic van der waals

Question 33

Explain the general structural properties of glycoprotein spikes.

Answer 33

Different subunits: dimer, trimer, tetramer Alike or different subunits: homo or hetero

Question 34

Explain the difference between a structural protein and a non-structural protein.

Answer 34

NSP: viral produced proteins that don't affect the assembly process; are not part of the virion SP: affect the assembly process

Question 35

Explain the structure (what is it made of) and origin (where did it come from) of the virus envelope.

Answer 35

lipid bilayer (hydrophobic interior, hydrophilic exterior) with glycoproteins derived from the cellular membrane, not produced by the virus

Question 36

Explain the fundamental forms of virus symmetry and be able to identify symmetry when viewing a virus particle.

Answer 36

1. helical symmetry 2. icosahedral symmetry (2-3-5 fold) 3. complex: draw line and mirror image reflects the other 4. pleomorphic: no symmetry but many different shapes

Question 37

Explain the three main criteria for placing a virus in its proper taxonomic classification.

Answer 37

1. virion morphology and composition 2. genome structure and composition 3. gene expression and replication strategies

Question 38

Provide a general description of the sequential steps of the generic virus replication cycle.

Answer 38

1. Bind to cell receptor 2. entry and uncoating: release genome 3. early gene expression: make proteins 4. replication 5. late gene expression 6. assembly 7. exit

Question 39

Explain the biochemical basis of virus/receptor binding (physical fit and formation of non-covalent bonds).

Answer 39

PHYSICAL: key and lock accommodation between interaction CHEMICAL: noncovalent bonds that establish the binding

Question 40

Explain the fundamental steps involved in the process of receptor-mediated endocytosis.

Answer 40

NON-ENVELOPE VIRUS 1. bind initiates coated pit 2. uncoated vesicles will fuse with endosome 3. acidification: drop in pH triggers conformation change and disassembly 4. protein VI ruptures endosome ENVELOPE 1. bind and entry 2. pH drop and conformational change 3. fusion peptide and protein triggered; inserts into hydrophobic bilayer 4. pore virion leaves ENVELOPE and PH INDEPENDENT 1. binding 2. conformational change of fusion peptide 3. membrane fusion 4. uncoating

Question 41

Explain the meaning of metastability, and understand the important role that metastability plays in cell entry by a virus.

Answer 41

intermediate stability in order to enter the cell without losing its structural integrity

Question 42

Explain the role of acidic pH in the process of pH-dependent cell entry (by both enveloped and non-enveloped viruses).

Answer 42

pH triggers conformational changes that virus' have evolved to accomplish cell entry NON-ENVELOPE VIRUS 1. bind initiates coated pit 2. uncoated vesicles will fuse with endosome 3. acidification: drop in pH triggers conformation change and disassembly 4. protein VI ruptures endosome ENVELOPE 1. bind and entry 2. pH drop and conformational change 3. fusion peptide and protein triggered; inserts into hydrophobic bilayer 4. pore virion leaves

Question 43

Explain the general mechanism of membrane fusion (understand how the virus glycoproteins mediate the process).

Answer 43

low pH again triggers a conformational change, the

Question 44

Explain how non-enveloped viruses such as poliovirus and adenovirus enter the host cells.

Answer 44

1. receptor bind down to canyon 2. major rearrangement: V4 is externalized, and the hydrophobic VP1 N terminus inset into the membrane 3. form a channel 4. viral DNA can now pass ** happens to all 5 sections of VP1

Question 45

Explain the general process of virion uncoating (what does this term mean?).

Answer 45

disassembly of virus coating; release of the viral genome into appropriate compartment of the cell (cyto or nucleus)

Question 46

Explain how the principle of metastbility facilitates virion uncoating.

Answer 46

intermediate stability allows for the outer coat to be disassembled and even internal capsids to be taken down by certain cellular components EX. evolve proteins to be disrupted by certain biological cues ribosomal subunit affinity histone proteins cause final disruption to release DNA

Question 47

Explain what is generally involved in the process of virus assembly (what does the term virus assembly actually mean?).

Answer 47

after gene expression; proteins are produced to assemble new virions budding

Question 48

Explain the general process of virion budding. Where does it occur, what does it achieve?

Answer 48

1. capture genetic material 2. close contact with plasma membrane (M/matric proteins or capsid proteins) 3. free energy used from binding drives fusion and pinch off hosts' membrane

Question 49

Describe the general process of exocytosis as it applies to a virus that buds intracellularly, such as budding into the RER or the Golgi.

Answer 49

vesicles go to membrane and fuse with membrane and release their contents

Question 50

Understand how to interpret a one-step virus growth curve.

Answer 50

NON-ENVELOPE has an additive cell-associated virus meaning that virions have not left the cell cell-free virus is cells that have been released from a lyse cell ENVELOPE these two happen simultaneously; completed replication is termed cell-free

Question 51

Describe the types of viral components that function and pathogen-associated molecular patterns (PAMPs).

Answer 51

Recognize the dangerous non self with expression of our receptors. Molecular feature not found on our own cells. dsDNA located in the cytoplasm ssDNA dsRNA ssRNA LPS Flagellum Improperly methylated CAP ends No CAP on genetic material

Question 52

Describe the cellular PAMP-receptors that function in recognize cellular invasion by viruses.

Answer 52

RIG-1 binds to dsRNA and ssRNAs that possess 5' triphosphates. Mda5 binds dsRNA and mRNAs lacking 2 methyl groups

Question 53

Explain the general principles of signal transduction. Conceptually, how does the interaction between a PAMP receptor and PAMP lead to the activation of the type I interferon genes?

Answer 53

Domino cascade 1. virus detection via PAMP and PRR 2. Expression of IFN produces a cellular response 3. terminate in nucleus turn on interferon and inflammatory cytokines

Question 54

Explain what type I interferons are. What are their basic properties, what do they do?

Answer 54

proteins sent out when a virion is detected. a type of cytokine (cell to cell communication) that causes stimulation when bound to it. Antiviral state is initiated to prep for incoming virions. Interferon alpha and beta

Question 55

Explain the effects that type I interferon has on cells that express type I interferon receptors. How does interferon binding affect gene expression of the cell?

Answer 55

antiviral state through the production of newly gene expression interferon binds to outside dimerization, phosphorylation and confirmational changes allow TF to express interferon-stimulated genes which in turn produce antiviral activities

Question 56

Explain the antiviral activities of PKR, 2'-5' OAS, and RNase L. Explain what is meant by the term "antiviral state".

Answer 56

Antiviral state: Not conducive to supporting viral replication. PKR (PO4 and dimerization when virus is present) suppression of translation /halting protein synthesis by not allowing the structure to enter the recycling pathway through PO4 2-5 OAS and RNase L degradation of RNAs once 2-5 OAS is activated by dsRNA this will activate nuclease to degrade viral mRNA (replication and translation)

Question 57

Explain some of the strategies that viruses have evolved to evade or actively interfere with the type I interferon response.

Answer 57

NS1 protein prevent PAMP recognition by covering genetic material. NS3/4A and Vpu disrupts message, no signal transduction pathway STAT degradation causes interference with IFN signaling and no antiviral state is produced Interfere with effector molecules for the antiviral state (ex. PKR)

Question 58

Explain how primary cell cultures are produced: Explain the differences between primary cells, cell stains, and cell lines.

Answer 58

Primary: if the cell came directly from an in vivo source (euthanized mouse, harvested tissue, disassociation/enzymatic digestion) Not really used. Only if viruses thrive in this environment, and only exist with intact tissue. ============================= Cell strains: passaged for a period of months, eventually cells crashed (stop behaving and die due to telomere ends) Cell lines: cell strains that develop genetic mutation (required), causing a transformation into CA cells. The cells are immortalized that can never crash.

Question 59

Explain in general terms how cells are maintained in culture Explain why cell culture work needs to be performed within the confines of a laminar flow hood

Answer 59

MAINTAINED: growth media Under the hood to maintain sterile conditions and to protect cells from contamination with other biological microbes.

Question 60

Explain the common alternatives to cultures cells with respect to virus propagation (intact host or eggs)

Answer 60

injected into a part of an embryonic egg more labor intensive sometimes viruses require this type of environment

Question 61

What is cell lysis? Explain the general process to performing a plaque assay.

Answer 61

CPE that demonstrates cytolytic infection/cell rupture 1. create cell monolayer with liquid nutrient broth 2. remove broth, and place virus with an agar overlay 3. plaque = area of infected cells

Question 62

Analyze results of a plaque assay to determine a virus titer (quantity of virus/volume).

Answer 62

Count cells that have 20-100 plaques. X PFU * (DF for X) = X2 PFU/100microL * 1000 = X2 PFU/mL

Question 63

Explain the meaning of particle to PFU ratio Explain the reasons that can explain why there are usually many more particles in a virus sample than are determined by plaque assay

Answer 63

particle/PFU low = high number of cells capable of forming plaques high = low number of cells capable of forming plaques There is only a subpopulation that is capable of initiating and forming plaque assay. Many virions with not form plaque assay. EX: no cell receptor found bound receptor failed endosome faulty virus assembly Plaque assays do not measure the number of virus particles in a sample they only measure those particles that are capable of infecting a cell.

Question 64

Explain how hemagglutination assay is performed Why is it only useful for quantifying certain viruses How does the HA titer differ from the titer determined in plaque assay

Answer 64

Swabbed samples (up to down) Dilution of virus (L to R) * add a drop of RBC * if the virus is in a sufficient quantity you will get hemagglutination (hazy red not red dots) Commonly use to quantify influenza A virus in samples. Ability of FluA to hemagglutinate RBC as RBC has sialic acid on glycoproteins. HA titer is reported as the inverse of the dilution at which hemagglutination is lost. Point of last hazy sample.

Question 65

Explain how the virus concentration of a sample can be determined using electron microscopy Analyze data collected in this type of assay to calculate a virus concentration

Answer 65

Physical examination and physically counting virions, not infectivity. Large ping-pong = micro-latex spheres with concentration Unknown (virus in sample) mixed with known MCP (concentration) Place sample on electron grid 1. count latex beads 2. count virus particles (tiny dots) 3. complete ratio 4. ratio x known concentration = virions/mL