Laboratory Diagnosis of Viral Infection

VIRAL LIFE CYCLE ATTACHMENT PENETRATION HOST FUNCTIONS UNCOATING Transcription Translation REPLICATION VIRAL LIFE CYCLE ASSEMBLY (MATURATION) RELEASE MULTIPLICATION

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 Screening for Blood transfusion Treatment available

Microscopy Electron Microscope Light microscope – Inclusion bodies 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.

Serological Reactions Rising titre of antibody in paired sample of sera First sample – At the earliest Second sample – After 2 weeks Single sample IgM type of antibody detection Techniques – Neutralization, ELISA, CFT, Haemagglutination Inhibition (HAI)Test

Specimens According to the disease Respiratory – Throat swab Eyes- Conjunctival scrapings Liver – Blood PUO – Blood Skin - Scrapings

Virus Culture Embryonated Egg Cell Lines/ Tissue cultures 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

Cell Culture Routinely used for growing viruses Classified into 3 types: Primary cell culture – normal cells freshly taken from body & cultured, limited growth Rhesus monkey kidney Chick embryo fibroblast Human amnion cell culture Diploid cell strains – cells of single type (fibroblast cells) that can be subcultivated for limited number of times, mostly 50 WI-38: human embryonic lung cell HL-8: Rhesus embryo cell Continuous cell lines – malignant cells, indefinite subcultivtion 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 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

Detection of virus growth in cell cultures 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.