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

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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 302/Browser/kSHVID/step2.ht mlhttp://biology.fullerton.edu//biol 302/Browser/kSHVID/step2.ht ml


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