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Laboratory Diagnosis of Viral Infection Professor Sudheer Kher 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 hen’s egg – Laboratory animal inoculation eg mice Detection of antobody in serum – IgG – Rising titre in paired sample – IgM – Indicates current / recent infection Microscopy Electron Microscope / Immune Electron Microscopy Light microscope – Inclusion bodies eg Negri Body in Rabies Fluorescent Microscope Fluorescent antibody technique Demonstration of Viral Antigens Precipitation on gel eg HBsAg Immunofluorescence Counter Immuno Electro Phoresis (CIEP) Enzyme Linkes Immuno Sorbant Assay (ELISA) Isolation of Virus Laboratory animals Fertilized Hen’s Egg – Chorioallantoic membrane – Allantoic cavity – Amniotic cavity – Yolk sac Organ/Tissue/Cell Culture Growth identified by serological method like neutralization. Virus Culture Embryonated Egg Chorioallantioc membrane (CAM) Allantoic cavity Amniotic cavity Yolk Sac Cell Lines/ Tissue cultures Primary Diploid/ Secondary Continuous Animal inoculation Suckling mice Embryonated Hen’s Egg 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 Yolk sac – Chlmyadia, Rickettsiae & some viruses Embryonated Hen’s Egg Inoculation Harvesting Cell Culture Routinely used for growing viruses Classified into 3 types: – Primary cell culture – normal cells freshly taken from body & cultured, limited growth 1. Rhesus monkey kidney 2. Chick embryo fibroblast 3. Human amnion cell culture – 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 – Continuous cell lines – malignant cells, indefinite subcultivtion 1. 2. 3. 4. HeLa: Human Ca of cervix cell line HEP-2: Human epithelioma of larynx Vero: Vervet monkey kidney McCoy, Detroit-6, BHK-21, Kb Cell Culture Tissues Individual cells trypsin & mechanical shaking Cells are washed, counted & suspended in a growth medium. Growth medium – Minimum Essential Medium (MEM): essential aminoacids, vitamins, salts, glucose & bicarbonate in 5% CO2 with 5% fetal calf or calf serum, antibiotics & phenol red indicator Cell Culture Bottles / Tubes Detection of virus growth in cell cultures 1. Cytopathic effects (CPE) – morphological changes in cultured cells, seen under microscope, characteristic CPE for different groups of viruses 2. Metabolic Inhibition – no acid production in presence of virus 3. Hemadsorption – influenza & parainfluenza viruses, by adding guinea pig erythrocytes to the culture Detection of virus growth in cell cultures 4. 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 5. Transformation – oncogenic viruses induce transformation & loss of contact inhibition – microtumors 6. Immunofluorescence – test for viral Ag in cells from viral infected cultures. 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 content of a specimen: Total no. of 1. Virus particles – EM, HA 2. Infectious virions only Assay of Infectivity: two types 1. Quantitative assays – actual no. of infectious particle in an inoculum 2. Quantal assays – indicate the presence or absence of infectious viruses, carried out in animals, eggs or tissue cultures Viral Assay Assay of Infectivity: Quantitative assays – – Plaque assay in monolayer cell cultures Pock assay on CAM *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 Rising titre of antibody in paired sample of sera is diagnostic – First sample – At the earliest – Second sample – After 2 weeks Single sample - IgM type of antibody detection. Indicates recent / current infection. Techniques – Neutralization, ELISA, CFT, Haemagglutination Inhibition (HAI)Test