How can we detect viruses?

How can we detect viruses? Identifying the etiology of a new disease

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.

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

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.

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.

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

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

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

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)

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

How can we detect viruses?