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Lecture 2

Microbiology and Immunology 2500A/B Lecture Notes - Lecture 2: Intracellular Parasite, Cytopathic Effect, Cell Culture


Department
Microbiology and Immunology
Course Code
MICROIMM 2500A/B
Professor
Kelly Summers
Lecture
2

Page:
of 2
Lecture 2:
Infectious cycle overview
1. Attachment = virus surface recognizes specific cell surface and attaches
2. Endocytosis = become obligate intracellular parasite
3. Genome exposure=remove particle to expose viral genome
- this can either lead to protein synthesis or make more viruses
4. Protein Synthesis = using host machinery to make viral proteins
5. Assemble = fully assembled virus is called virion
6. Release = virions are vehicles to transmit to a new host (and start new infectious cycle)
Basic viral replication kinetics
- activity requires Energy => mitochondria =>virus dont have mitochondria
- Transport Vesicles=>need to get to certain locations inside host cells to serve specific functions
- Protein Translation Machinery => need to use host machinery to translate mRNA ->proteins
Hosts utilized to study infectious cycle in labs
- whole animal hosts=>costly=>fertilized chicken eggs
- contains many cell types, used to grow influenza virus to make flu vaccine
- cell culture (flask+plastic coated surface for cell attachment and formation of a monolayer)
-light microspectropy cant be used to see these viruses
How to asses infectious cycle=>What viruses are doing to cells
- Cytopathic Effects= different changes that a virus induces inside a cell
- only certain types result in CPE display in hosts, cannot be seen using light microscope
-infected monolayer: less cells, nuclei become rounded+ bulking up(=initial sign of death)
- dead cells can no longer attach to surface=>less and less cells to be seen
- specific example of CPEs:
- Cell Lysis= Breaking down the membrane of the cell
-Syncytia(ie HIV)=fusion of adjacent cell membranes =>multinucleated array of cells
-Transformation=instead of having flat cells, they divide uncontrollably=>piles of round, bulky cells
- virus inhibit normal cell functioning from attaching to plastic surface=>cells move upwards
and form “mountains”=foci=dense and dark matters
-Infectivity=when cells fail to display CPE (some virus dont cause CPE)
-Plaque Assay: first used for bacteriophage (virus that infects bacteria, ie Ebola)
- agar plate = bacterial lawn for hosts
-phage plaques= spots =area where bacteria have been infected (bacteria are replicating virus),
bacteria no longer forming a lawn bcz they’re dead
- measures Plaque Forming Units (pfu/ml) by counting # of plaques=measures infectivity
-add virus to cells(must be susceptible and permissive)=>overlay cells with gel-like substance, agar
(cells on bottom, virus on top)=>infection occurs via transmission there4 spreads cell to cell, overlaying
infected cell layer can block virion from transmission and end up w/ plaques(=one single round of
infection), NOT all virus form plaques
- 1mL in 9mL of buffer=10 fold dilution => repeat dilution 8 times and end up with 108 dilution (too
many virus if w/out dilution)
- average # of plaques/mL x 108=PFU/mL
-a plaque represents the infectivity from a SINGLE viral clone (homogenous virus, no mutated virus)
-Particle to PFU Ratio= #of virus particles/ number of infectious particles
-many step from viral infectious cycle will fail and not all products are virions
-certain viruses dont form plaques but form foci (ie RSV, rous sarcoma virus)=>foci forming
units/mL
-Physical Measurements of viral infectious cycle
- measure virus particles (not infectivity but #s), fail to display CPE =>cant measure infectivity using
plaque/transformation assays
-Hemaglutination Assay= certain virus have proteins that binds to red blood cells=>infected sample
will bind RBC and form a Lattice=+ve hemaglutination (lack of virus will result in RBC forming a
“dot”=-ve)
-measures the first step of the cycle: recognition
-Viral Enzyme Activity = retroviruses(HIV) contain active enzymes, Reverse Transcriptase (makes
DNA=>RNA) => set up radioactive assay to measure the ability of DNA to become RNA
-Immunostaining(HIV)= viral particle will generate anitibodies=>link antibodies to diff fluorescent
proteins to see if ells are infected, use microscopy
-Immunoblotting
-Sequencing= for low abundance viral genes=>use specific enzyme to amplify genome
-Fluorescent Proteins= insert fluorescent proteins sequence into viral genome=>makes f luorescent viral
proteins=>montor viral transmission and see specific areas inside cell thats infected using fluorescent
miroscopy
Notes:
- virus replicates very differently from bacteria (=binary fission=splitting of a single cell into 2)
- suitable host cells must be susceptible and permissive
- susceptible = functional receptor for cells to recognize (and attach to)
- permissive = have all intracelluar pieces to allow virus to replicate (go thru its cycle)
- a resistant cell: has no receptors but may still support replication
-Primary Cell Lines= extracted from live animals and can be cultured for a week or two
-Continuous Cell Lines=immortal cells that can live forever thru replicating in cell culture, attractive to
tumours
-Viral Burst = usually happens when CPE is observed
- cytopathic viruses bursts cell membrane after infection (kills it)=>cells have a finite ability to produce
virus
- does NOT apply to viruses that are NOT cytopathic
Adsorption=attachment
Eclipse=time when infectivity disappears due to uncoating
Latent=replication of genome + protein synthesis
Maturation=assembly of geome and viral proteins
-lag phase where initial steps of the viral infectious cycle takes
place/gets going and virus is not infectious
-time is 8-40hr in animal hosts, assembly varies as well