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
Inclusion bodies
Focus (foci)
Hemadsorption

7. Identifying Viruses in Cell Culture/specimen
Fluorescent ab
NA hybridization(HPV)
PCR/RT-PCR

8. Quantification Plaques/cpe Electron Microscopy Virus=arrowhead
Latex bead = arrow

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