Lecture 2 Flashcards
1
Q
how were viruses first detected and studied
A
- infection of intact organisms, take cell or plant extract and try to infect organisms with it
- expensive
- time consuming
- unethical
- animal models are commonly used
2
Q
plaque assay for detection and measurement of viruses
A
- allows fo quantitation of viruses (how many viruses present in a given volume)
- bacterial growth can be measured with a spectrophotometer, intact bacteria diffract visible light, dense bacterial cultures look cloudy
- bacteriophages lyse their hose cell which causes a loss of diffraction which leads to clearing of bacterial culture
- phage binds to bacterial cells, replicates and releases progeny phage particles that are then taken up by surrounding cells, cycle repeats
- repeated cycles lead lysing of cells in area surrounding initial infection which is observed as a clear area (plaque) against uninfected cells
- reported as plaque forming units (PFU) which allows us to count number of infectious virus particles in a suspension
- have to dilute viral suspension because it would be too concentrated if you used original viral culture
3
Q
why are serial dilutions used
A
it gives exponential dilutions without need for very large volumes
4
Q
in-vitro cultures of eukaryotic cells for detection and measurement of viruses
A
- eukaryotic cells are stained
- dead cells do not stain well
- virus will kill some of the cells
- plaques where the dead cells are will appear clear
- a higher dilution makes it easier to count the viruses
5
Q
RBCs and hemagglutination assays for detection and measurement of viruses
A
- binding of excess of virus with red blood cells results in agglutination (clumping)
- virus particles form “bridges” between adjacent cells which forms clumps
- measured in hemagglutinating units (HAU)
limitations - sensitive to conditions
- some viruses only cause agglutination in particular mammalian or avian species
6
Q
why are red blood cells commonly used for assaying viruses
A
- visible due to colour
- can be isolated and stored easily
- have carbohydrate-containing receptors on their surface that a number of animal viruses bind to
7
Q
hemagglutinating units (HAU)
A
highest dilution of virus that agglutinates a given aliquot of cells is considered 1 HAU
- minimum number of viruses that can cause agglutination
= approx 10^5 virus particles
- approx 1 virus per RBC
8
Q
electron microscopy for detection and measurement of viruses
A
- shoots beam of electrons into the sample
- electrons bounce off electron dense materials and just go through areas without electron dense materials
- virus particles are mixed with an electron dense stain and the viruses do not take it up
- the virus is observed as a light image against a dark background (negative staining)
- add a measure aliquot of diluted virus and count the number of virus particles in a given area
Limitation: - cannot distinguish between infectious and non-infectious particles
- it looks at both at once
9
Q
how to get ratio of infectious particles
A
- combining plaque assays and electron microscope observations
- usually combined with inert, uniformly sized beads to establish absolute number of virus particles per unit volume
- ratio of physical virus particles to infectious particles can be much greater than one due to defective particles
10
Q
multiplicity of infection (MOI)
A
- number of infectious virus particles per susceptible cell
- ratio of infectious agents (like viruses, bacteria, or phages) to infection targets (usually host cells)
- PFU/cell
MOI = infectious agents/infectious targets
or
MOI = viral particles/target cells
or
MOI = virus titer (concentration) x virus volume/total cell number
11
Q
study of virus replication cycles
A
- studied by infecting 10^3 - 10^6 cells to get enough amount of viral genetic material and proteins
- all cells must be infected at the same time to synchronize the events of replication
- infect with excess of virus to ensure that each cell receives at least one infectious particle
- use MOI to figure this out
- difficult to study because steps in the replication cycle overlap
12
Q
What can be used to study virus replication pathways
A
- analysis of viral macromolecules (proteins, mRNAs, genomes)
can be done with: - radiotracers
- antibodies against specific proteins
- molecular hybridization (labelled DNA or RNA probes)
- PCR (look for DNA and RNA, easy and efficient)
- gel electrophoresis
- microscopy
13
Q
Common features of virus replication cycles
A
- attachment or viral absorption: binding to host cell
- entry into host cell
- genome replication and gene expression
- assembly and morphogenesis
- release and exit
14
Q
Binding to cell receptor (Step 1 in virus replication cycle)
A
- some virus surface proteins bind to specific molecules on the cell surface like glycoproteins or glycolipids that are on many cells types
- some viruses bind to surface proteins only present on surface cell types
generally: non-specific primary receptor followed by more specific secondary receptor
15
Q
Entry and uncoating (Step 2 in the virus replication cycle)
A
- virion or viral genome enters the cell
- bacteriophages drill holes in cell walls and membranes to inject genetic material
- enveloped viruses fuse lipid envelope with host plasma membrane and release capsid or genetic material inside the cell
- endocytosis, releases virion or genome in the cytoplasm
