1. How can we detect viruses?
Identifying the etiology of a new disease
2. Learning objectives
• Describe how viruses are isolated.
• Explain the theory and procedures of various
virus identification methods.
• Apply the appropriate method to the
identification of a virus under different
circumstances.
• Explain how virus titer is enumerated.
3. Isolation/purification of virions
• Centrifugation
– Differential centrifugation
- high vs low speed to
separate cells from viruses
– Gradient centrifugation -
separate by size or density
• Filtration can be used
4. • BCBL-1, a latently KSHV-infected primary lymphoma cell line, was
maintained in RPMI 1640 medium supplemented with 10% fetal
bovine serum. For virus production, BCBL-1 cells (0.5 x 106/ml) were
induced with 20 ng of phorbol-12-tetradecanoate-13-acetate (TPA)
per ml and 0.5 µM ionomycin for 5 to 7 days.
• The medium was cleared by centrifugation at 4,000 x g for 30 min and
then at 8,000 x g for 15 min to remove cells and cell debris.
• The supernatant was filtered through 0.45-µm-pore-size filters.
• Virions were pelleted at 27,000 rpm for 1 h through a 5% sucrose
cushion (5 ml) and resuspended in 1x phosphate-buffered saline (PBS)
plus 0.1% bacitracin in 1/100 of the original volume.
• The concentrated virus particles were centrifuged through a 20 to 35%
sucrose step gradient at 24,000 rpm for 2 h. The virus band at the
gradient junction was collected.
• The virions were then diluted with 1x PBS and pelleted at 27,000 rpm
for 1 h.
• The pellets were resuspended in 1x PBS and further purified through a
20 to 35% continuous sucrose gradient.
5. Archael virus purification
• Cells were removed by centrifugation (6000 x g for 10
min) and the supernatant filtered through a 0.8 and then
0.2 um filters
• Filtrate was concentrated by passage through filter
membranes (100,000 mw)to a volume of 8 ml.
• Retentate was loaded onto Cs sulfate and centrifuged at
247,000 x g for 20 h.
• Virus bands were removed, placed in 14,000 mw cutoff
dialysis tubing and dialyzed
• Further concentration with filter if needed.
6. Identifying Viruses in Cell
Culture/specimen
Plaques: Plaque purification -
assumes “clone” from
single plaque
Microscopy - cytopathic
effects (CPE)
Syncytia
Cell rounding
Membrane proliferations
Vacuolization
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Focus (foci)
Hemadsorption
7. Identifying Viruses in Cell
Culture/specimen
• Fluorescent ab
• NA
hybridization(HPV)
• PCR/RT-PCR
9. Hantavirus
• Tried EM, cell culture and no
success
• Did serological survey and got
a positive with a hantavirus
• Not previously known as
respiratory pathogen
• Did RT-PCR and amplified a
region that they sequenced
• Found a new Hantavirus
• Found evidence of virus in
local deer mice (urine)
10. Kaposi’s sarcoma
• Tried culture, serology, EM and
failed
• Representational difference
analysis (RDA) - amplifies
difference in NA between
tumor and normal tissue
• Yielded a partial sequence that
showed similarity to a
herpesvirus
• Verifying the results
• http://biology.fullerton.edu
//biol302/Browser/kSHVID
/step2.html
