Lecture 3 and 7 part I: virus culture and lab safety

Viruses can be grown in

Tissue (cell) culture
Embryonated eggs
Animals

Laminar flow hoods

They provider an ultra clean work area, air is passed though a HEPA filter, removes 99.97% of particles of 0.3 microM of higher from the air

One-step growth experiment

1. Infect cells, incubate in CO2 incubator

2. Monitor the experiment via inverted microscope

3. Collect the lysate at various points of infection

4. Do serial dilutions, do plaque assays

5. Stain and analyze, record results

HeLa cells

human epithelial cells of a strain maintained in tissue culture since 1951 and used in research, especially in virology.
Derived from Henrietta Lacks

plaque

a visible area of cell lysis on a confluent monolayer of cells caused by the infection of those cells with a virus

Cytophatic effects (CPEs)

Structural changes in host cells that are caused by viral invasion
Can be observed with inverted light microscopes

CPEs visible indicators

Rounding/detachment from the flask
Shrinkage
Cell lysis /death
Increased refractility
Syncytia/fusion
Aggregation

Syncytia

fusion of multiple damaged host cells into single large cells containing multiple nuclei (giant cells)

Refractility

The degree to which a cell or other structure is capable of refracting light.

chorioallantoic membrane inoculation

Into the shell membrane of an egg
Herpes simplex virus
Pox virus
Rous sarcoma virus

Inoculation

The introduction of a pathogen or antigen into a living organism to stimulate the production of antibodies.
Eggs used for creating the influenza vaccine (not anymore)

Amniotic inoculation

Into amniotic cavity
Influenza virus
Mumps virus

Yolk sac inoculation

Into the yolk sac
herpes simplex virus

Allantoic inoculation

Into the allantoic cavity (white part)
influenza, mumps, newcastle disease, avian adenovirus

Laboratory safety levels

BSL-1 = minimum containment
BSL-2
BSL-3
BSL-4 = suit lab-maximum containment

BSL-1

Microorganisms not known to cause disease in healthy adults
Present minimal `potential hazard to lab personnel and the environment

BSL-2

Handling moderately hazardous agents
Procedures conducted in biosafety cabinets (BSCs)

BSL-3

Indigenous or exotic microorganisms that cause serious or potentially lethal disease through respiratory transmission
Protective gear
Closed air ventilation (HEPA), sealed windows, seams, floors, walls, ceiling surfaces

BSL-4

Microorganisms that are dangerous and exotic with high risk of aerosol transmitted infections. Rarely are there treatments or vaccines for these microorganisms, and the diseases they cause are frequently fatal.
Entry through an airlock and airtight doors, showers required, negative pressure inside is maintained