Laboratory Diagnosis of Viral Infection

1. Laboratory Diagnosis of Viral Infection Professor Sudheer Kher

2. Learning Objectives • Describe the principles, techniques, standards and recording of results and interpretation of different methods used in diagnosis of viral infections

3. 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

4. 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

5. Microscopy • Electron Microscope / Immune Electron Microscopy • Light microscope – Inclusion bodies eg Negri Body in Rabies • Fluorescent Microscope -Fluorescent antibody technique

6. Demonstration of Viral Antigens • Precipitation on gel eg HBsAg • Immunofluorescence • Counter Immuno Electro Phoresis (CIEP) • Enzyme Linkes Immuno Sorbant Assay (ELISA)

7. 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.

8. Virus Culture Embryonated Egg Chorioallantioc membrane (CAM) Allantoic cavity Amniotic cavity Yolk Sac Primary Cell Lines/ Tissue cultures Diploid/ Secondary Continuous Animal inoculation Suckling mice

9. 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

10. Embryonated Hen’s Egg Inoculation Harvesting

11. 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

12. 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

13. Cell Culture Bottles / Tubes

14. 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

15. 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.

16. 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.

18. Viral Assay • Viral content of a specimen: Total no. of • Virus particles – EM, HA • Infectious virions only • Assay of Infectivity: two types • Quantitative assays – actual no. of infectious particle in an inoculum • Quantal assays – indicate the presence or absence of infectious viruses, carried out in animals, eggs or tissue cultures

19. Viral Assay • Assay of Infectivity: Quantitative assays • Plaque assay in monolayer cell cultures • Pock assay on CAM *Each plaque/ pock represents one infectious virus. • Plaquesare clear zones that develop on lawns of host cells. • The virus plaque is analogous to the bacterial colony.

20. Specimens • According to the disease • Respiratory – Throat swab • CNS – CSF • Eyes- Conjunctival scrapings • Liver – Blood • PUO – Blood • Skin - Scrapings

21. 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