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Microimm 2500 > (V) 21: Viral Infectious Cycle > Flashcards

(V) 21: Viral Infectious Cycle Flashcards

1
Q

Why must viral genome be packed?

A

to protect it

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2
Q

Overview of viral infectious cycle

A

viruses are obligate INTRACELLULAR pathogens

  1. attachment
  2. entry
  3. uncoating (loses shell and expose nucleic acid)
  4. replication (use host’s ribosome machinery to make viral proteins)
  5. maturation
  6. release (cell lysis)
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3
Q

Coronavirus infection cycle

A
  1. binding + viral entry via membrane fusion or endocytosis
  2. uncoating - loses shell, exposes nucleic acid + makes viral proteins
  3. translation of polypeptide

eventually gets to formation of virion and exocytosis

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4
Q

What is needed for viral infectious cycle?

A

Host cell machinery (plasma memb, golgi, ER, nucleus)

needed for
- energy
- transport vesicles
- protein translation machinery

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5
Q

What type of cell takes up a virus and allow replication?

A

A susceptible AND permissive cell

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6
Q

Susceptible cell

A

FUNCTIONAL RECEPTOR for virus

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7
Q

Permissive cell

A

allows virus to replicate

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8
Q

Resistant cell

A

no receptor
- virus can’t get in

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9
Q

Hosts for viral replication

A
  • whole animal hosts
  • fertilized chicken eggs
  • cell culture
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10
Q

Fertilized chicken eggs

A
  • made of multiple cell types
  • used to replicate INFLUENZA virus (making flu vaccine)
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11
Q

Cell culture

A

Healthy cell population
- clear, stretched, flat
- NO growth on top of one another

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12
Q

Cytopathic effects

A

different changes that a virus induces inside a cell

ex.
- cell lysis
- syncytia
- transformation

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13
Q

Light microscopy

A

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

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14
Q

Syncytia

A

fusion of adjacent plasma cell membranes
- results in multinucleated array of cells

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15
Q

Transformation

A

cells are no longer flat but divide uncontrollably to become piles of round cells

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16
Q

How to tell what viruses are doing to cells

A
  1. Cytopathic effects (CPEs)
  2. Measuring infectivity
  3. Physical measurement
17
Q

Measuring infectivity

A
  • plaque assay
  • Particle to PFU ratio
  • transformation assay
18
Q

Physical measurement

A
  • hemagglutination assay
  • viral enzyme activity
  • immunostaining
  • immunoblotting
  • sequencing
  • fluorescent proteins
19
Q

Infectivity

A

understanding infection that has gone through ALL steps of viral infection

20
Q

Plaque assay

A

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

21
Q

Bacteriophage

A

viruses that infect bacteria
- bacteria is host

22
Q

How to perform plaque assay?

A
  • 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
23
Q

HOW TO MEASURE # OF INFECTIOUS PARTICLES IN PLAQUE ASSAY

A
  • 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

24
Q

Plaque assay with crystal violet

A

Crystal violet will stain cells that are alive

Alive = purple
Dead = white spots (NOT sticky) – infected cells

25
Q

Are all viral particles infectious?

A

Particle-to-PFU ratio: # of virus particles/number of infectious particles

small # ratio = more infectious
larger # = less infectious particles

26
Q

Transformation assay

A
  • 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

27
Q

What does measuring virus particles do?

A

measures the PRESENCE of viruses
NOT infectivity

28
Q

Hemaglutination assay

A

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

29
Q

Viral enzyme activity

A

retroviruses contain Reverse Transcriptase (modifies RNA to DNA)

30
Q

Immunostaining

A

tests if a viral protein is present

  • looking under microscope using specific antibodies
  • NOT measuring infectivity
31
Q

Sequencing

A

useful for low viral abundance genes

exponential growth of gene product

32
Q

Do physical assays measure infectivity?

A

NO

Microimm 2500 (29 decks)

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