Presentation on theme: "How can we detect viruses?"— Presentation transcript:
1How can we detect viruses? Identifying the etiology of a new disease
2Learning 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.
3Isolation/purification of virions CentrifugationDifferential centrifugation - high vs low speed to separate cells from virusesGradient centrifugation - separate by size or densityFiltration can be used
4BCBL-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.
5Archael 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 filtersFiltrate 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 dialyzedFurther concentration with filter if needed.
6Identifying Viruses in Cell Culture/specimen Plaques: Plaque purification - assumes “clone” from single plaqueMicroscopy - cytopathic effects (CPE)SyncytiaCell roundingMembrane proliferationsVacuolizationInclusion bodiesFocus (foci)Hemadsorption
7Identifying Viruses in Cell Culture/specimen Fluorescent abNA hybridization(HPV)PCR/RT-PCR
8Quantification Plaques/cpe Electron Microscopy Virus=arrowhead Latex bead = arrow
9Hantavirus Tried EM, cell culture and no success Did serological survey and got a positive with a hantavirusNot previously known as respiratory pathogenDid RT-PCR and amplified a region that they sequencedFound a new HantavirusFound evidence of virus in local deer mice (urine)
10Kaposi’s sarcoma Tried culture, serology, EM and failed Representational difference analysis (RDA) - amplifies difference in NA between tumor and normal tissueYielded a partial sequence that showed similarity to a herpesvirusVerifying the results