Lecture 5-7: Working with Viruses Flashcards
Propagating plant viruses
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
Symptoms of viral infections in plants
-Growth retardation
-Distortion
-Mosaic patterning on leaves
-Yellowing
-Wilting
Pros of propagating plant viruses
High yield & inexpensive
Propagating animal viruses
Before 1900’s without cell cultures or fridges, they had to be continuously passaged in animals
Embryonated Eggs
Inoculation can occur at many sites such as yolk sac or amniotic cavity
Cons of passage through animals
-Expensive
-Low recovery
-Often results in adaptation of the virus (Virus attenuation) to become more virulent
Pros of using embryonated eggs
Generates large quantity of virus and used for vaccine production
Progress in propagating animal viruses
-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
HeLa Cells
-Vital for the development of polio vaccine, cloning, gene mapping, in vitro fertilization, and most recently for vaccines of HPV that causes cervical cancer
Primary culture
-5 to 20 cell divisions
-Normal chromosome number
-Contact inhibition
-Need constant source
Propagating viruses in flasks with a chemically defined media supplemented with serum (Three Steps)
- Find a cell line the virus replicates in
- Grow the cells and infect them with a small amount of material containing virus
- Let the virus propagate until high amounts can be purified from cell media
Continuous cell lines (Immortalized)
Single cell type that can be propagated indefinitely
-Adherent: Grow as monolayer on plastic dishes
-Suspension. Sources can be from tumor tissue or immortalized
Primary Cell Culture
Better for understanding the biology of a virus
-5-20 cell divisions
-Contact inhibition
-Needs constant source
Aneuploid
Abnormal in chromosome morphology and number. Can also grow rapidly
Means of detecting virus components: Virus Infectivity
Multiplication in a suitable host (cytopathic effect)
Means of detecting virus components: Virions
Electron microscopy
Electron microscopy
-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
Means of detecting virus components: Viral Antigens
-Western Blot
-Immunofluorescence assay (IFA)
-ELISA (Detection & Quantification)
Means of detecting virus components: Viral Nucleic Acid
PCR
Agarose Gel Electrophoresis
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
Detection of nucleic acids by autoradiography
Indirect detection of viral nucleic acid
-Technique: Detects specific nucleic acids within a sample
-Southern Blot & Northern Blot
Steps of autoradiography
-Separate nucleic acid on gel
-Transfer to solid phase
-Probe with labeled nucleic acid
What northern blot detects
RNA
What southern blot detects
DNA
Method for the indirect detection of viral nucleic acids
PCR Based assays
PCR Based assays
-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
Means of detection of viral proteins
SDS-Page (Separation of proteins by size)
Require the use of antibodies to detect viral proteins
-Western Blot
-Immunofluorescence Assay
-ELISA
Ways antibodies can be made
-Whole virus or recombinant proteins injected into animals
Western blot
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
Immunofluorescence Assay
Can be used for detection of virus in tissue samples, biopsies, cell cultures
MOI
Multiplicity of Infection (How many infectious units/cell)
ELISA-Enzyme Linked Immunosorbant Assay
Often used in diagnostic tests for various viruses to detect…
-Viral protein in sample
-Antibodies to viral proteins
-Viral particles
Hemagglutination Assay
Ability to bind and cross link erythrocytes
Physical Methods
Detection of the number of virus particles in your sample
-Electron Microscopy
-Hemagglutination assay
-ELISA
Functional Methods
The number of infectious particles in your sample
-Plaque Assay
-Endpoint dilution assay
Why is it important to distinguish between physical and functional assay
Produce a particle to PFU ratio
Plaque Forming Unit
Virus particle able to initiate a productive infection
Plaque Assay
Titer-concentration of a virus in a sample (PFU/mL)
Plaque
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
Steps of Plaque Assay
- Serial Dilution of sample
- Add diluted virus to cell monolayer
- Incubate cultures at 37 degrees to allow adsorption of virus
- Remove inoculum
- Add overlay to culture
- Plaque results from a single virus
Endpoint Dilution Assay
50% tissue culture infective dose
Centrifugation
-Used for characterization of viral proteins
-Main technique used to isolate/purify virus particles or proteins
Ultracentrifugation
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
What the ultracentrifuge is used for
- Characterize and separate macromolecules
- Determine the sedimentation coefficient
Sedimentation coefficient
Rate at which a macromolecule sediments under a defined gravitational force
Ways centrifugation is influenced
- Molecular Weight
- Density
- Size & shape of a macromolecule
Why is centrifugation such a good technique to purify viruses
Behavior of viruses and other cellular material during centrifugation is based on density and sedimentation coefficient
Types of Sedimentation Mediums
-Aqueous Buffer
-Sucrose or glycerol gradients or cushions (Rate Zonal)
-CsCl gradient centrifugation-isopycnic
Aqueous buffer
Used to separate molecules with widely different S values for harvesting cells or producing crude subcellular fractions
Sucrose or glycerol gradients or cushions (Rate Zonal)
-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
CsCl gradient centrifugation-isopycnic
-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
Buoyant density
Point where forces of buoyancy and sedimentation are balanced
Only system for studying pathogenesis & immune response
Whole organism system
Pathogenesis
Process of an agent causing tissue
Mouse model advantages
-In-breed strains reduce genetic variability
-Genetics are well understood
-Introduce, mutate, or inactivation specific genes thought to control the immune response
Disadvantages of mouse model
-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
Transgenic mouse models
Express new genes in a mouse model to allow the study of viruses that do not normally infect mice
Code of respect for animals
-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
The Institutional Animal Care and Use Committee (IACUC)
-Members
-Reviews animal study proposals
-Reviews animal programs & facilities
-Informs IO (Institutional official)
-Can suspend animal activities
-Responds to animal welfare concerns
Contributions of animal research
-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
Infectious Dose 50 (ID50)
Dose required to infect 50% of the inoculated animals. With most viruses several PFU are required to infect an animal
Lethal Dose 50 (LD50)
-The dose required to kill 50% of the inoculated animals.
Means by which LD50 and ID50 may vary
- Mouse strains
- Route of inoculation
- Animal Model
- Different viruses
Incubation Period
Time between the initial infection and the onset of disease symptoms
Biosafety levels
Categories of protection and control for viral contaminants. Level 1 is lowest risk while level 4 is high risk
How BSL levels are determined
-Infectivity
-Severity of disease
-Transmissibility
-Nature of the work conducted
BSL-3
-Personal protective equipment
-All work performed in a biological safety cabinet
-Laboratory is under negative pressure (Air in hall is sucked INTO lab)
BSL-1
-No contaminent
-Defined organisms
-Unlikely to cause disease
BSL-2
-Containment
-Moderate risk
-Disease of varying severity
BSL-4
-Maximum containment
-“Exotic” high risk agents
-Life threatening disease
