(V) 21: Viral Infectious Cycle Flashcards
Why must viral genome be packed?
to protect it
Overview of viral infectious cycle
viruses are obligate INTRACELLULAR pathogens
- attachment
- entry
- uncoating (loses shell and expose nucleic acid)
- replication (use host’s ribosome machinery to make viral proteins)
- maturation
- release (cell lysis)
Coronavirus infection cycle
- binding + viral entry via membrane fusion or endocytosis
- uncoating - loses shell, exposes nucleic acid + makes viral proteins
- translation of polypeptide
eventually gets to formation of virion and exocytosis
What is needed for viral infectious cycle?
Host cell machinery (plasma memb, golgi, ER, nucleus)
needed for
- energy
- transport vesicles
- protein translation machinery
What type of cell takes up a virus and allow replication?
A susceptible AND permissive cell
Susceptible cell
FUNCTIONAL RECEPTOR for virus
Permissive cell
allows virus to replicate
Resistant cell
no receptor
- virus can’t get in
Hosts for viral replication
- whole animal hosts
- fertilized chicken eggs
- cell culture
Fertilized chicken eggs
- made of multiple cell types
- used to replicate INFLUENZA virus (making flu vaccine)
Cell culture
Healthy cell population
- clear, stretched, flat
- NO growth on top of one another
Cytopathic effects
different changes that a virus induces inside a cell
ex.
- cell lysis
- syncytia
- transformation
Light microscopy
shortly after infection
- cells are very round = necrosis/apoptosis
- cells unstick from monolayer on surface of plate = dark spots
after more time
- tight, round nuclei
- dark spots btwn
- cells are not sticky to surface
after 24h
- everything is bunched up
Syncytia
fusion of adjacent plasma cell membranes
- results in multinucleated array of cells
Transformation
cells are no longer flat but divide uncontrollably to become piles of round cells
How to tell what viruses are doing to cells
- Cytopathic effects (CPEs)
- Measuring infectivity
- Physical measurement
Measuring infectivity
- plaque assay
- Particle to PFU ratio
- transformation assay
Physical measurement
- hemagglutination assay
- viral enzyme activity
- immunostaining
- immunoblotting
- sequencing
- fluorescent proteins
Infectivity
understanding infection that has gone through ALL steps of viral infection
Plaque assay
first used for bacteriophage
- count # of plaques to establish plaque forming units (PFU)/mL
- shows POTENCY of virus
agar plate: bacterial lawn
plaques: area where bacteria have been infected w/ virus
Bacteriophage
viruses that infect bacteria
- bacteria is host
How to perform plaque assay?
- add virus to cells
- overlay cells w/ a gel-like substance (agar) = stops continuous budding = virus won’t get to next cell = lmites virus to a SINGLE replication
- when infected cells release progeny, spread is halted by gel
HOW TO MEASURE # OF INFECTIOUS PARTICLES IN PLAQUE ASSAY
- 1 mL of concentrated virus is diluted in 9 mL of buffer = 10 fold dilution
Step is repeated 8 times = dilution of final tube is 10^8
One mL of 10^8 virus is diluted in 9 mL of buffer in an agar plate and added to susceptible and permissive cells
- since we add ONE mL, we can measure PFU/mL
Average the # of plaques on 4 different plates and calculate
Plaque assay with crystal violet
Crystal violet will stain cells that are alive
Alive = purple
Dead = white spots (NOT sticky) – infected cells
Are all viral particles infectious?
Particle-to-PFU ratio: # of virus particles/number of infectious particles
small # ratio = more infectious
larger # = less infectious particles
Transformation assay
- certain viruses do not form plaques but form FOCI
- can count foci + get foci forming units per mL (more foci = more infectivity)
ex. Rous Sarcoma Virus (RSV) form foci
What does measuring virus particles do?
measures the PRESENCE of viruses
NOT infectivity
Hemaglutination assay
does NOT measure infectivity
- susceptible host is RBC
- certain viruses have proteins that bind to RBCs
- if a sample contains viruses, they will bind RBC and form a LATTICE that coats the tube
LACK OF VIRUS = RBCs FORM A DOT
- no hemaglutinin = RBCs make a dot b/c no virus to interact with
Viral enzyme activity
retroviruses contain Reverse Transcriptase (modifies RNA to DNA)
Immunostaining
tests if a viral protein is present
- looking under microscope using specific antibodies
- NOT measuring infectivity
Sequencing
useful for low viral abundance genes
exponential growth of gene product
Do physical assays measure infectivity?
NO
