Topic 5 - Viruses Flashcards
when did virology begin as a science?
late 1800s
what did Dimitri Ivanovski discover?
infectious tobacco mosaic virus isolated in filtered, bacteria-free fluid;
means causative agent was smaller than bacteria (virus)!!!
What did Walter Reed discover?
yellow fever is a virus transmitted by mosquitoes (jaundice, yellow-looking eyes)
Felix D’Herelle discovered?
bacteriophages (coined term “plaque”)
virus (size, genome size, how they survive)
- intracellular obligate parasites
- typically btw 10-100nm
- genome typically a few thousand to 200,000 nucleotides long (very few genes!)
what are exceptions to the small size of viruses?
all amoeba viruses!
viral genome types & physical structure
- single/double-stranded, DNA/RNA, linear/circular
- protein shell (capsid) around genome
– composed of many capsomere proteins
– capsid and genome together = nucleocapsid - possible envelope (cell-derived membrane around capsid)
virus - what is a capsid?
protein shell (around genome)
virus - what is a nucleocapsid?
capsid and genome together
capsids shape
- often exhibit either helical or icosahedral (20 sides of triangles, 3 capsid proteins per triangle on corners)
- can take on irregular or complex shapes
bacteriophage structure (4)
top to bottom:
genome (inside capsid)
capsid (shell)
tail (pillar looking “body”)
tail fibers (like little legs)
what is common for bacteriophages?
to inject genomic information into host
viral envelopes
enveloped virus
- a plasma membrane surrounds the nucleocapsid
naked virus (nonenveloped virus)
- no plasma membrane
viral replication steps (6)
- adhere (receptor in host membrane recognized by viruses)
- stick to a host cell - enter
- uncoat
- release genome - synthesis
- express and replicate genome - assembly
- create new virus particles (viral DNA + viral proteins) - exit
- new particles leave host cell
what is arguably the most important part in viral replication cycle?
entering the host cell
what receptors do HIV attach to on host cells?
CD4 receptors
T/F: Animal viruses need to contend with a cell wall during entry; elaborate (2)
FALSE!
entering animal cells:
- endocytosis
- membrane fusion
which one is outside of the other structure part? viral envelope and nucleocapsid
the envelope is outside the nucleocapsid
steps of endocytosis of a non-enveloped virus
- virus attaches to cell receptor
- endocytosis initiated
- endosome forms with virus inside
- nucleocapsid escapes to cytoplasm and uncoats to release genome
steps of membrane fusion of an enveloped virus
- virus attaches to cell receptor
- a CONFORMATIONAL change in attachment protein and bound receptor initiates membrane fusion
- viral envelope fuses with plasma membrane (NO endosome!)
- nucleocapsid enters cytoplasm and uncoats to release genome
steps of endocytosis of enveloped virus
- virus attaches to cell receptor
- endocytosis initiated
- endosome forms with virus inside
- low pH(!!!) of endosome initiates fusion of viral envelope with endosome membrane. nucleocapsids are released
methods of entry for enveloped vs non-enveloped viruses
non-enveloped:
- endocytosis
enveloped:
- membrane fusion
- endocytosis
how do viruses enter plant cells? why?
- often depends on damage to plant tissues to OPEN a spot in cell wall (no receptors on cellulose!)
how do viruses enter bacterial cells?
- like a hypodermic syringe, DNA is injected directly into cell
- tail fibers attach to receptors
- conformational change in tail fibers bring base of tail in contact with host cell surface
- rearrangement of tail proteins let inner core tube proteins extend down into cell wall
- contact with the plasma membrane initiates DNA transfer through a pore, formed in the lipid bilayer
types of cycles for bacteriophage replication
1) lytic cycle
2) lysogenic cycle (temperate phage)
what is lytic cycle?
viruses enter, replicate, lyse host cell
lysogenic cycle?
- also possible for human viruses (like coldsores), not just bacteriophages
- phage integrate their genome into host (i.e. LYSOGEN) cell’s genome, becoming a PROPHAGE
- prophage genome is replicated along with host cell’s until STRESS! can then enter lytic phase
temperate phage vs lytic phage
- temperate phage can be both lytic or exist as a prophage (lysogeny)
- lytic phage are lytic
3 hypotheses for origin of viruses (names + explanation)
- not necessarily mutually exclusive
Coevolution hypothesis
- viruses originate about the same time as other microbes and have been coevolving with them
Regressive hypothesis
- viruses are previously alive organisms that have evolutionarily regressed into host-dependent particles
Progressive hypothesis
- viruses originated from genetic material that gained the ability to replicate and be transmitted semi-autonomously
cultivating bacteriophage steps
- small volume of susceptible bacterial host cells are added to phage sample
- mixture added to molten agar, mixed
- pour onto nutrient agar base, let solidify
- plaques appear after sufficient incubation
what do plaques show/mean on bacteriophage cultures?
plaques show where the viruses are (like little clearings)
cultivating animal viruses steps
- tissue culture of host cells used to grow viruses (or in embryonated chicken/duck eggs)
- cultures must be sterile and bacteria-free
viral purification
- usually starts with simple filtration to remove large cells and cellular debris
- viruses then purified and concentrated with centrifugation (differential centrifugation & gradient centrifugation)
differential centrifugation
start with: cells and virus suspension
- start with low speed
- transfer supernatant and centrifuge med speed (pellet of whole and broken cells at bottom)
- transfer supernatant and centrifuge high speed (ultracentrifugation) (pellet of nuclei and other large organelles at bottom)
end with: pellet of virus
gradient centrifugation
- depends on diff densities of viral components and particles
- one step, faster than differential centrifugation
- tube is successively filled with layers of decreasing conc of sucrose
- suspension containing virus is layered on top
- centrifuge
end with: band of cell debris + band of intact virus particles
viral quantification (measured as? 4 methods?)
- usually measured as a titer or concentration of a virus prep
- methods incl: direct count, hemagglutination assay, plaque assay, endpoint assay
direct count
- electron microscope used to visualize a known volume of material
- viruses within it counted and scaled up to determine titer
– expensive, specialized microscope
– doesn’t differentiate between infectious and non-infectious viral particles
hemagglutination assay
- exploits trait of some viruses to stick to RBCs!, causing them to form a GEL MAT
– cheap, fast, easy (no microscope)
– some viruses don’t do this
– doesn’t differentiate viable/non-viable viruses
– doesn’t provide a virus number - uses dilutions of viral samples
- button well = small dot in middle (less virus)
- shield well = full dot (a lot of viruses)
plaque assay
- virus diluted and placed on target cells
- plaques are counted to determine plaque-forming units (PFU) titer of original suspension (not counting individual viruses! counting PFU!)
- useful for phages and plant viruses
endpoint assays
- endpoint - cytopathic (infected cell)
- tissue culture infectious dose 50 (TCID50) (preferable) -> amount of virus needed to induce a CPE in 50% of cultured cells
- lethal dose 50 (LD50) -> amount of virus needed to kill 50% of test animal subjects
virus naming (3)
historically varied
- simple letter/number combo
- infected organism
- discovery location
- appearance
- disease caused
ICTV = international committee on taxonomy of viruses
- classify viruses based on Order, Family, Subfamily, Genus, Species
- morphology, genome structure, biological features, disease caused, envelope, and genomes
Baltimore Classification System
- based on mRNA production methods
- separates viruses into 7 classes
- made by David Baltimore
Virus identification (2 methods)
electron microscopy
- visual observation of viral morphology (not infallible)
nucleic acid analysis
- PCR and reverse-transcriptase PCR
– used to identify viruses by genome sequence
– used to study viral evolution patterns
virus-like particles (2, details)
viroids
- consist only of naked circular RNA
- very small! (<400 nucleotides)
- high degree of internal complementarity
- resistant to ribonucleases
- only causes diseases in plants (so far observed)
prions
- proteinaceous infectious particles (“prions”)
- no nucleic acid, no genes, only protein
- different “infectious” agent
- responsible for transmissible spongiform encephalopathies (TSEs) like mad cow disease
what does prions stand for?
proteinaceous infectious particles
how are prions thought to replicate?
unclear, thought to revolve around conversion of protein conformations from normal to abnormal over time
- proteins that misfold lead to more misfolding (domino effect), leading to disease
how is virology used today?
- CRISPR
- cancer-causing oncoviruses (e.g., HPV)
- cancer-destroying oncolytic viruses
- gene therapy
- phage therapy
