Diagnostic and therapy of Viral and Fungal Infection

BAGIAN MIKROBIOLOGI FKUH BAGIAN MIKROBIOLOGI FKUH

DiagnosIs and Therapy of

Viral and Fungal Infection

Isolation and Identification of Viruses (1)

CELL and ORGAN CULTURE

Cells are derived from tissue source, allowed to grow in nutrient media until a confluent one cell layer is obtained.

Organ culture uses a tissue fragment with a specialized function (e.g. Fetal trachea with ciliated epithelial cells)

Isolation and Identification of Viruses(2)

Primary cell culture: cells derived from the initial growth of cells from a tissue source

Secondary cell culture: redispersed and regrowth of primary cell culture.

Cell lines: cells that have transformed spontaneously and become immortal, or cells are obtained from cancerous tissue

3

Isolation and Identification of Viruses(3)

Detection of Viral growth

1. Observing CPE; alteration or cytopathic effect in cells due to viral infection. Alteration may be change in morphology or death of cells

Tissue culture of epitheloid cells

Tissue culture of fibroblasts cells.

Enterovirus

Herpetovirus

Paramyxovirus

Isolation and Identification of Viruses(4)

2. Observing hemadsorption or interference

on cells that do not show CPE.

Interference: cells that do not show CPE after infected with a virus, but the cells may show CPE if infected with another virus (challenged). But the challenging virus cannot infect the cell culture in the presence of the first virus

Expressing infection on lymphocytes (EBV and HIV)

Isolation and Identification of Viruses(5)

Quantitation of Viruses

Hemagglutination Assay:

Viruses have attachment proteins (=hemagglutinins) that can be bound on red blood cells, thus causing hemagglutination.

Dilution of virus preparation reacted with a constant amount of red blood cells will show the decreasing amount of hemagglutination. Agglutinated RBCs settle dispersed on the bottom, but unagglutinated RBCs forms a tight button shaped sedimentation on the bottom of the well/tube. The titer is the reciprocal of the last dilution that still shows agglutination.

Plate HAI

Isolation and Identification of Viruses(6)

Plaque Assay

Virus at several different concentration is reacted into a one layer of semisolid culture cells. The growth of any one virus is therefore limited in its initial place, forming a round cleared area (plaque) due to the death of infected cells. The number of plaques is then counted and calibrated according to the dilution factor. Viral titer is the number of Plaque forming units per millimeter (Pfu/ml)

Plaque formed in an agar plate

http://www.virology.ws/2009/07/06/detecting-viruses-the-plaque-assay/

Isolation and Identification of Viruses(7)

In Vivo Isolation methods

Embryonated hens egg isolation and propagation of influenza A virus

Animal inoculation suckling mouse