viral isolation methods

1. Cultivationofviruses
Virology 3rd Lab

2. Cultivationofviruses
1. ANIMALS
2. CELLCULTURE
3. EMBRYONATED EGGS

3. Animals
For animal viruses, samples from affected animals
were collected and used to infect other animals, initially of
the same species to determine the susceptibility of other
species (host range).

4. ANIMALS
Advantages
Testingnewviralvaccinesornewantiviraldrugs
Studiesonpathogenesistomodelhumandisease
Sourceofprimarycellcultures
Productionofantibodiesfordiagnosticandresearchpurposes
Disadvantages
Expensive
Havetheirownindigenousviruses,mayinterferewiththeexperimentorin
somecasesare dangeroustohumanse.g.herpes virus.

5. Animal models
Symptoms andsigns
increased temperature
circling
sneezing
rash
pustules/pocks
Death

6. Tissues
Post-infection/post-mortemhistology
inclusion bodies
inflammatory response
cell death/necrosis
Cytoplasmic
inclusions
e.g.rabies,
smallpox.
Nuclear
inclusion
e.g.herpes
Nucleus
Necrosis
Inflammation

7. Inclusion Bodies
Rabbit cornea infectedwith vacciniavirus.
Arrows =intracytoplasmic inclusions (Guarnieribodies)
Hippocampus of dogwith Rabies showing large round
inclusion bodies (Negribodies)
Herpes encephalitis in brain showing large intranuclear inclusions and
margination ofchromatin.

8. CELLCULTURE
• Cell culture is the process by which cells are grown under controlled
conditions, generally outside their natural environment. After the cells of
interest have been isolated from living tissue, they can subsequently be
maintained under carefully controlled conditions.
• Cell culture has largely replaced the egg system, which is labor-intensive
and expensive

9. Media
Balanced salt solution+glucose
Amino acidsandvitamins
Supplement(usuallycalfserum) 10% =growthmedium
0.5-2%=maintenancemedium
Antibiotics
Penicillin/streptomycin(crystamycin)
Antifungalagent e.g.fungizone
pHregulation
Sodiumbicarbonate/CO2
Cell culture media
also known as growth media, is an umbrella term that encompasses any
gel or liquid created to support cellular growth in an artificial environment.

10. Monolayer: Cells grown on an
inert surface as a sheet one cell
thick. When the sheet of cellsfill
the surface area available, the
culture issaidto be “confluent.”
Suspension: Separate cells that
multiply without becomingattached
to an inertsurface
Confluent
No Confluency

11. Subculture
Confluent cellmonolayer
Cells attached to solid
surface
Detach cells
Trypsin(proteasedigestion)
EDTA(chelating agent)
“split”cells
Seed according to celltype
Continuous cell line - 1:4(or
more)
Semicontinuous/primary –1:2

12. Primary cultures
A culture taken directly from an organism,often
embryonic tissue e.g.chick embryofibroblasts
Remove embryo/organ
Wash
Manual disruption oftissue
Wash
Enzymatic disruption
Filtration
Enumeration
Culture

13. Primarycultures
Advantages:
Verysusceptibletoviruses,particularlyforisolationdirectlyfrom patientsina
diagnosticlaboratory
Diploidandcanthereforebeusedforviralvaccineproduction (e.g.
measles,polio)
Disadvantages:
Composedofdifferentcelltypeswithdiffer ssusceptibilityto
viruses
Maycontaincontaminatingvirusesfromtheoriginalhost
Difficulttosubcultureandusuallynotattempted
Tissuesourcesrarelyimmediatelyavailable

14. cell culture

15. Diploid celllines
Derived from human or animal fetal tissue. At least 75% of
the cells have the same karyotype as the organism from
which they were originally obtained e.g.human embryo
lung (HEL)cells

16. Diploid celllines
Advantages:
Very susceptible to viruses, particularly for isolation
directly from patients in a diagnosticlaboratory
More convenient for diagnostic laboratories as they can
be sub-cultured(passaged)
Diploid karyotype retained
Can be screened for extraneous viruses
cell cultures of choice for viral vaccineproduction
(e.g. rabies, hepatitisA)
Disadvantage:
Can only be sub-cultured for 50-60generationsbefore
they degenerate and die

17. Continuous celllines
Derived from tumours
Normal cells that have acquired tumour-like properties
(“transformed”cells)
Aneuploid
have an abnormal number of chromosomes. (e.g. H
e
L
a
cells)

18. Continuous celllines
George Gey.
First cultured HeLacells
HenriettaLacks.
Died from the cervicalcancerfrom
which HeLacells werederived

19. Continuous celllines
Advantages:
Canbesub-culturedindefinitelymakingthemvery convenientforboth
diagnosticandresearchlaboratories
Usuallyofonecelltypeonlyandthereforegiveconsistency toviralgrowth
experiments
Disadvantages:
Lesssusceptibletosomevirusescommonlyfoundbydiagnostic
laboratories
Theirtumour-likepropertiesmakethemunsuitablefortheproductionofviral
vaccinesforhumans

20. CELLTYPES
Epithelial-likecells:
Form sheets of closelyadherent
polygonal-shapedcells
Fibroblast-likecells:
Form sheets ofthin
elongatedcells

21. Cellculture
As virus grows, produces a characteristic effect on
the cells
CYTOPATHICEFFECT(CPE)
Effectis specific to virus family but NOT species

22. Cell rounding: e.g. HSV1
Rounded
cells
Uninfected
cells
Infection of Vero cells withHSV-1
showing discrete foci of roundedcells

23. Enterovirus CPE at various stages of development

24. Cell rounding & stranding
e.g.vaccinia virus

25. Syncytium formation
e.g. respiratory syncytial virus, measles virus,HSV.
Multiple
nuclei
Syncytium
Uninfected
cells

26. An additional distinguishing feature of measles is the presenceof
distinct eosinophilic inclusions (arrowed) in the nuclei of infectedcells.

27. HSV-infected Vero cells showing syncytialformation

28. Vero cells infected with respiratory syncytialvirus
(RSV) showing largesyncytia.

29. RSV-infected cells showing pale eosinophilic inclusionsin
the cytoplasm (seearrows).

30. Cytoplasmic vacuolation
e.g.measles virus
Cytoplasmic
vacuoles in
syncytium
Advanced measles virusCPE
showing cytoplasmic vacuoles insyncytia

31. Transformation
e.g.Rous sarcomavirus
Transformed cells
Uninfected cells

32. What about viruses which donot produce a
CPE?
Not all viruses producea CPE need to detect viralantigens
e.g.haemadsorption