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BSCI437: General Virology > Lecture 5-7: Working with Viruses > Flashcards

Lecture 5-7: Working with Viruses Flashcards

1
Q

Propagating plant viruses

A

Plants could be grown in the lab and transmission of the virus can be achieved by applying extracts of an infected plant to a scratch made on a healthy plant

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2
Q

Symptoms of viral infections in plants

A

-Growth retardation
-Distortion
-Mosaic patterning on leaves
-Yellowing
-Wilting

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3
Q

Pros of propagating plant viruses

A

High yield & inexpensive

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4
Q

Propagating animal viruses

A

Before 1900’s without cell cultures or fridges, they had to be continuously passaged in animals

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5
Q

Embryonated Eggs

A

Inoculation can occur at many sites such as yolk sac or amniotic cavity

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6
Q

Cons of passage through animals

A

-Expensive
-Low recovery
-Often results in adaptation of the virus (Virus attenuation) to become more virulent

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7
Q

Pros of using embryonated eggs

A

Generates large quantity of virus and used for vaccine production

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8
Q

Progress in propagating animal viruses

A

-1949: Enders, Weller, and Robbins grew poliovirus in cultured cells marking a major breakthrough
-Enabled discovery of new viruses and large scale vaccine development
-Basic technology for molecular and cellular biology
-Enables growth of large amounts of pure virus, making studying on virus composition and structure possible

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9
Q

HeLa Cells

A

-Vital for the development of polio vaccine, cloning, gene mapping, in vitro fertilization, and most recently for vaccines of HPV that causes cervical cancer

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10
Q

Primary culture

A

-5 to 20 cell divisions
-Normal chromosome number
-Contact inhibition
-Need constant source

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11
Q

Propagating viruses in flasks with a chemically defined media supplemented with serum (Three Steps)

A
  1. Find a cell line the virus replicates in
  2. Grow the cells and infect them with a small amount of material containing virus
  3. Let the virus propagate until high amounts can be purified from cell media
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12
Q

Continuous cell lines (Immortalized)

A

Single cell type that can be propagated indefinitely
-Adherent: Grow as monolayer on plastic dishes
-Suspension. Sources can be from tumor tissue or immortalized

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13
Q

Primary Cell Culture

A

Better for understanding the biology of a virus
-5-20 cell divisions
-Contact inhibition
-Needs constant source

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14
Q

Aneuploid

A

Abnormal in chromosome morphology and number. Can also grow rapidly

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15
Q

Means of detecting virus components: Virus Infectivity

A

Multiplication in a suitable host (cytopathic effect)

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16
Q

Means of detecting virus components: Virions

A

Electron microscopy

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17
Q

Electron microscopy

A

-Allows visualization of single virus particles
-Allows resolution up to nanometer range
-Electron scattering principle: Beam of electrons is focused on a sample and electrons in the specimen will scatter the electron beam
-Scanning EM v. Transmission EM

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18
Q

Means of detecting virus components: Viral Antigens

A

-Western Blot
-Immunofluorescence assay (IFA)
-ELISA (Detection & Quantification)

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19
Q

Means of detecting virus components: Viral Nucleic Acid

A

PCR

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20
Q

Agarose Gel Electrophoresis

A

Must isolate RNA/DNA from virus
-Separation of nucleic acid by size
-Detected by using dyes to bind to DNA & RNA such as ethidium bromide
-Addition of Urea or Formamide ill denature sample

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21
Q

Detection of nucleic acids by autoradiography

A

Indirect detection of viral nucleic acid
-Technique: Detects specific nucleic acids within a sample
-Southern Blot & Northern Blot

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22
Q

Steps of autoradiography

A

-Separate nucleic acid on gel
-Transfer to solid phase
-Probe with labeled nucleic acid

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23
Q

What northern blot detects

A

RNA

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24
Q

What southern blot detects

A

DNA

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25
Q

Method for the indirect detection of viral nucleic acids

A

PCR Based assays

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26
Q

PCR Based assays

A

-Directly amplifies nucleic acids from sample
-Highly sensitive
-Can detect primary tissue samples - do not have to culture the virus
-Used diagnostic in clinical virology

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27
Q

Means of detection of viral proteins

A

SDS-Page (Separation of proteins by size)

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28
Q

Require the use of antibodies to detect viral proteins

A

-Western Blot
-Immunofluorescence Assay
-ELISA

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29
Q

Ways antibodies can be made

A

-Whole virus or recombinant proteins injected into animals

30
Q

Western blot

A

Can be used to detect viral proteins in crude lysates - requires antibodies
-Lyse Cells
-Separated proteins by SDS-PAGE
-Transfer to membrane
-Probe for viral protein

31
Q

Immunofluorescence Assay

A

Can be used for detection of virus in tissue samples, biopsies, cell cultures

32
Q

MOI

A

Multiplicity of Infection (How many infectious units/cell)

33
Q

ELISA-Enzyme Linked Immunosorbant Assay

A

Often used in diagnostic tests for various viruses to detect…
-Viral protein in sample
-Antibodies to viral proteins
-Viral particles

34
Q

Hemagglutination Assay

A

Ability to bind and cross link erythrocytes

35
Q

Physical Methods

A

Detection of the number of virus particles in your sample
-Electron Microscopy
-Hemagglutination assay
-ELISA

36
Q

Functional Methods

A

The number of infectious particles in your sample
-Plaque Assay
-Endpoint dilution assay

37
Q

Why is it important to distinguish between physical and functional assay

A

Produce a particle to PFU ratio

38
Q

Plaque Forming Unit

A

Virus particle able to initiate a productive infection

39
Q

Plaque Assay

A

Titer-concentration of a virus in a sample (PFU/mL)

40
Q

Plaque

A

Circular zone of infected cells that results from a single infectious particle
Does NOT work if virus does not infect cells that can make a monolayer

41
Q

Steps of Plaque Assay

A
  1. Serial Dilution of sample
  2. Add diluted virus to cell monolayer
  3. Incubate cultures at 37 degrees to allow adsorption of virus
  4. Remove inoculum
  5. Add overlay to culture
  6. Plaque results from a single virus
42
Q

Endpoint Dilution Assay

A

50% tissue culture infective dose

43
Q

Centrifugation

A

-Used for characterization of viral proteins
-Main technique used to isolate/purify virus particles or proteins

44
Q

Ultracentrifugation

A

A centrifugation capable of generating large centrifugal fields by rotating samples at 20,000-100,000 rpm. Centrifugal forces of greater than 100,000 X gravity can be generated

45
Q

What the ultracentrifuge is used for

A
  1. Characterize and separate macromolecules
  2. Determine the sedimentation coefficient
46
Q

Sedimentation coefficient

A

Rate at which a macromolecule sediments under a defined gravitational force

47
Q

Ways centrifugation is influenced

A
  1. Molecular Weight
  2. Density
  3. Size & shape of a macromolecule
48
Q

Why is centrifugation such a good technique to purify viruses

A

Behavior of viruses and other cellular material during centrifugation is based on density and sedimentation coefficient

49
Q

Types of Sedimentation Mediums

A

-Aqueous Buffer
-Sucrose or glycerol gradients or cushions (Rate Zonal)
-CsCl gradient centrifugation-isopycnic

50
Q

Aqueous buffer

A

Used to separate molecules with widely different S values for harvesting cells or producing crude subcellular fractions

51
Q

Sucrose or glycerol gradients or cushions (Rate Zonal)

A

-Increases the density and viscosity compared to water
-Causes a decreasing sedimentation rate compared to water to prevent the sedimentation of molecules with densities less than the medium
-Controlling time and speed of centrifugation can allow a significant purification can be obtained
-Separation based on S values (Density size & shape) since macromolecules have greater densities
-Can separate molecules with relatively close S values

52
Q

CsCl gradient centrifugation-isopycnic

A

-Linear gradient of these compounds in buffer is prepared in centrifuge tube
-As concentration of compound is increased, the density of the medium increased in the tube
-Macromolecule centrifuged through will form a band at a position equal to buoyant density
-Useful for separating molecules of different densities even when the densities are very close
-Drawback: CsCl can permanently inactivate some viruses

53
Q

Buoyant density

A

Point where forces of buoyancy and sedimentation are balanced

54
Q

Only system for studying pathogenesis & immune response

A

Whole organism system

55
Q

Pathogenesis

A

Process of an agent causing tissue

56
Q

Mouse model advantages

A

-In-breed strains reduce genetic variability
-Genetics are well understood
-Introduce, mutate, or inactivation specific genes thought to control the immune response

57
Q

Disadvantages of mouse model

A

-Sometimes not infected-therefore virus has to be adapted or use a closely related surrogate virus
-Does not always cause some disease state
-Mice are not human

58
Q

Transgenic mouse models

A

Express new genes in a mouse model to allow the study of viruses that do not normally infect mice

59
Q

Code of respect for animals

A

-Treat all animals in our care with respect
-Strictly follow all applicable laws and regulations for animal treatment
-Employ alternative scientific methods to animal use
-Minimize animal discomfort

60
Q

The Institutional Animal Care and Use Committee (IACUC)

A

-Members
-Reviews animal study proposals
-Reviews animal programs & facilities
-Informs IO (Institutional official)
-Can suspend animal activities
-Responds to animal welfare concerns

61
Q

Contributions of animal research

A

-Virtually every medical achievement of the past century has arisen from research using animals
-Over the past 40 years, only one Nobel Prize in physiology or medicine did not depend on animal research

62
Q

Infectious Dose 50 (ID50)

A

Dose required to infect 50% of the inoculated animals. With most viruses several PFU are required to infect an animal

63
Q

Lethal Dose 50 (LD50)

A

-The dose required to kill 50% of the inoculated animals.

64
Q

Means by which LD50 and ID50 may vary

A
  1. Mouse strains
  2. Route of inoculation
  3. Animal Model
  4. Different viruses
65
Q

Incubation Period

A

Time between the initial infection and the onset of disease symptoms

66
Q

Biosafety levels

A

Categories of protection and control for viral contaminants. Level 1 is lowest risk while level 4 is high risk

67
Q

How BSL levels are determined

A

-Infectivity
-Severity of disease
-Transmissibility
-Nature of the work conducted

68
Q

BSL-3

A

-Personal protective equipment
-All work performed in a biological safety cabinet
-Laboratory is under negative pressure (Air in hall is sucked INTO lab)

69
Q

BSL-1

A

-No contaminent
-Defined organisms
-Unlikely to cause disease

70
Q

BSL-2

A

-Containment
-Moderate risk
-Disease of varying severity

71
Q

BSL-4

A

-Maximum containment
-“Exotic” high risk agents
-Life threatening disease

BSCI437: General Virology (14 decks)

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