MGY378 25: EM, cell cultures

EM

• Does not require viral specific reagents

  • Useful if you suspect growth of a virus in cells but CPE may not be evident

• Detectable virus in EM indicates high viral load can suggest it is the causative
  agent rather than virus that is just present

  • Ex. Polyomavirus in urine

• Virus can be directly detected from sample

• Useful when cell culture systems are not developed

• Can provide insight into viral attachment

• Metagenomics has taken the place of EM

• Although EM is useful as a confirmatory assay

viral morphologies of:

• variola

• varicella

• variola

  • brick-shape, like vaccinia

• varicella

  • rectangular shape

  • some coated with “mucus”

EM process

• Centrifuge samples to pellet cellular debris and bacteria

• Sample placed onto a grid or undergoes ultracentrifugtion
  followed by negative staining with 2% uranyl acetate

• Negative stain allows for viewing small details (spikes, envelope)

• Thin sectioning of tissues can be done to look for viruses in present in biopsies

• Immunolocalization

  • Viral antibodies and gold-labeled secondary antibodies can show the location inside cells of various viral protein

types of cell culture

1. primary

2. semicontinuous

3. continuous/ transformed

4. hematopoietic

primary cell culture

directly from source

semicontinous cell culture

allow for limited passages before senescing

ex: MRC-5 fibroblast good for RSV

continuous/ transformed cell culture

immortalized cells from cancer that continue to grow and habe no limit to number of passages

• LLC-MK2 for coronaV NL63

• HeLa cells

• Vero cells: don’t have IFNs to kill virus so we can study virus in them

CPE

= cytopathic effects

• evidence of cell death/ destruction = virus there

• rounding, plaques, detached, floating cells

• characteristic CPEs of viruses:

  • inclusion bodies (of virus and cells)

  • multinucleated cells

  • syncytia (cytoplasmic mass with many nuclei)

HSV-1 CPE

cell ballooning

CMV CPE

owl’s eyes inclusions

rapid cell culture

• centrifugation of virus and cells in a shell vial

  • forces virus to contact cell and get inside it

  • shortens virus contact time from weeks to 24-72h

• part of research labs

• pre-determined mixes can detect: CMV, HSV1, HSV2, VSV, resp viruses

• direct immunofluorescent antibody staining

direct immunofluorescent antibody staining

• to visualise

• virus-specific antibody conjugated with fluorochrome

• use fluor microscope

• can detect virus even w/o evident CPE

TCID₅₀

= 50% tissue culture infectious dose

Quantify the amount of virus that is present in a cell culture assay

• If u wanna know if patient is infectious

• How well a vaccine lowers a viral load

 

Steps:

• Do a series of 10 fold dilutions

• Add them to different wells that have a near-confluent (?) layer of cells

• After incubation period, look for CPE

  • Directly - light microscopy

  • Indirect - dyes, amino-histochemical methods

•  Look for the dilution where 50% of the cells show CPE = TCID_50

• can be visualized using color-changing media (pink → yellow)

  • cell dies (CPE) → more acidic → more yellow

plaque assay

• Similar to TCID50

• Series of 10-fold dilutions

• Add to a larger plate of a cell monolayer

• remove dilutions and overlay w agarose + media

  • semi-solid media that prevents virus from spreading to other cells

• Virus sets and adheres

 

Whenever you see a plaque (tiny dot) = 1 single virus that has killed cells around it, but could not spread any further

• You can go in with a pipette to take out that 1 virus

• Larger plaque = more virulent? Killing more cells around it at a faster rate before it dies out

• Gold standard for isolating 1 virus.

 

Advantage over TCID50 - u can find 1 single virus

1-step growth curve

= tells us how the virus grows (studying pathogenesis

• Testing for inhibitors requires that u show how ur virus stops growing in its presence

• 1 million cells and 1 million viruses = MOI of 1

• Add your drug

• Collect supernatant of the cells at various timepoints (shoukd have some virus)

  • Do TCID_50 or plaque assay - viral quantification

• At what time point does your virus reach a plateau?

PRNT₅₀

= plaque reduction neut test

• detect abs that are specific to virus OI and prevent virus entry

• dilute ab/serum and add to virus for ~1h

• add virus/ab mixture to a cell layer

• overlay with agarose and media

• monitor for plaques

• determine the dilution where you see 50% less plaques than you ab-free control (should have many plaques)

BSL3 labs

high individual risk, low community risk

BSL3 resp viruses (6)

• MERS

• SARS2

• SARS1

• Hantavirus

• H5N1

• H7N9

BSL3 systemic viruses (3)

• Mpox

• chikungaya

• dengue

BSL3 encephalitis-causing viruses (3)

• west nile

• powassan

• paramyxoviruses

BSL4 labs

both high individual and high community risk

BSL4 hemorrhagic fever viruses

• ebola

• marburg

• lassa

• crimean-congo hem fev virus

BSL4 resp viruses

1918 flu

BSL4 encephalitis viruses

• herpes B

• hendra

• nipah