Professional Documents
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Learning Objectives
Describe the principles, techniques, standards and recording of results and interpretation of different methods used in diagnosis of viral infections
Difficulties
Can not be seen under light microscope Can not be cultivated easily Do not grow on culture media Treatment was not available Changed situation Rapid techniques have emerged Screening for Blood transfusion Treatment available
Techniques used
Microscopy Detection of Viral Antigen Growing and detecting viruses in
Tissue / Organ / Cell culture Fertilized hens egg Laboratory animal inoculation eg mice
Microscopy
Electron Microscope / Immune Electron Microscopy Light microscope Inclusion bodies eg Negri Body in Rabies Fluorescent Microscope Fluorescent antibody technique
Precipitation
on gel eg HBsAg Immunofluorescence Counter Immuno Electro Phoresis (CIEP) Enzyme Linkes Immuno Sorbant Assay (ELISA)
Isolation of Virus
Laboratory animals Fertilized Hens Egg
Virus Culture
Embryonated Egg
Chorioallantioc membrane (CAM) Allantoic cavity Amniotic cavity Yolk Sac
Animal inoculation
Suckling mice
Chorioallantoic membrane (CAM) visible lesions called pocks. Each infectious virus particle forms one pock. e.g. Variola, Vaccinia virus Allantoic cavity Influenza virus (vaccine production) & paramyxoviruses Amniotic cavity primary isolation of Influenza virus
Inoculation
Harvesting
Cell Culture
Primary cell culture normal cells freshly taken from body & cultured, limited growth
Diploid cell strains cells of single type (fibroblast cells) that can be subcultivated for limited number of times, mostly 50
1. WI-38: human embryonic lung cell 2. HL-8: Rhesus embryo cell
Cell Culture
Tissues
trypsin & mechanical shaking
Individual cells
Cytopathic effects (CPE) morphological changes in cultured cells, seen under microscope, characteristic CPE for different groups of viruses Metabolic Inhibition no acid production in presence of virus Hemadsorption influenza & parainfluenza viruses, by adding guinea pig erythrocytes to the culture
2.
3.
Interference growth of a non cytopathogenic virus can be tested by inoculating a known cytopathogenic virus: growth of first virus will inhibit the infection by second Transformation oncogenic viruses induce transformation & loss of contact inhibition microtumors Immunofluorescence test for viral Ag in cells from viral infected cultures.
5.
6.
Viral Hemagglutination
Hemagglutination
Originally seen with the Influenza virus by Hirst in 1941. A convenient method of detection & assay of Influenza virus. Due to the presence of Hemagglutinin spikes on the surface.
Viral Assay
Viral Assay
*Each plaque/ pock represents one infectious virus. Plaques are clear zones that develop on lawns of host cells. The virus plaque is analogous to the bacterial colony.
Specimens
According
to the disease
Respiratory Throat swab CNS CSF Eyes- Conjunctival scrapings Liver Blood PUO Blood Skin - Scrapings
Serological Reactions
Single sample - IgM type of antibody detection. Indicates recent / current infection. Techniques Neutralization, ELISA, CFT, Haemagglutination Inhibition (HAI)Test