Cultivation of Virus

The above power point presentation describes the various methods of cultivating viruses and their cytopathic effects on cell culture

1. CULTIVATION OF VIRUSES

2. INTRODUCTION Obligate Intracellular parasites Cannot be grown on artificial media 3 methods are employed for growth – Animal Inoculation Embryonated eggs Tissue cultures

3. ANIMAL INOCULATION Reed and colleagues – Yellow fever in human Landsteiner and Popper – Monkey – Polio Mice are widely used by T heiler – 1903 Suckling mice – coxsackie , arbovirus Guinea pigs, rabbits and ferrets are also use d

4. EMBRYONATED EGG CULTIVATION Good Pasteur - 1931 Duck eggs better Chorioallantoic membrane – vaccinia / variola forms pocks Allantoic cavity – influenza, paramyxovirus isolation and vaccine production Amniotic cavity – influenza virus Yolk sac – chlamydiae , rickettsiae , vaccine production yellow fever (17D strain), rabies (flurry strain)

5. POCK FORMATION

6. CELL CULTURE Steinhardt 1913 – maintained vaccinia virus in cornea of Rabbit Major Problem in tissue culture – Bacterial contamination After antibiotics – Routinely used Enders, Weller, Robbins – grew Polio in non neural tissue – 1949 (MOST IMP)

7. TISSUE CULTURE CLASSIFICATION

8. ORGAN CULTURE

9. EXPLANT CULTURE

10. CELL CULTURE

11. DIPLOID CELL STRAINS Single type Retain the original diploid chromosome Subcultivation for about 50 times Human Fibroblasts – isolation of Fastidious organism Viral vaccine production

12. CULTIVATION OF VIRUSES Tissue culture Organ culture – tracheal ring, coronavirus Explant culture – adenoid tissues, adenovirus Cell culture Primary cell culture – normal cells from body and cultured, used for isolation, vaccine production Diploid cell lines – human fibroblast Continuous cell lines – cells of single type from cancer cells, HeLa , HEp2

13. SOME CELL CULTURES IN COMMON USE VIRAL CELL CULTURES

14. Cytopathic effects Enterovirus – rapid degeneration of cells Measles virus – syncytium formation Herpes virus – discrete focal degeneration Adenovirus – grape-like large granular clumps SV 40 – cytoplasmic vacuolation DETECTION OF VIRUS GROWTH IN CELL CULTURES

15. Virus-specific CPE in simian kidney cell line LLC-MK2. Non-inoculated cells (A) cells inoculated with plasma specimen 1225/2014, 8 days post-inoculation (B)

16. DETECTION OF VIRUS GROWTH IN CELL CULTURES Metabolic inhibition – virus growth inhibits cell metabolism Hemadsorption – red blood cells adsorb to virus multiplying cells – influenza, parainfluenza Interference – growth of non-cytopathogenic virus in cell culture inhibits subsequent cytopathogenic virus – viral interference

17. Transformation – oncogenic viruses causing tumours Immunofluorescence – viral antigens are detected by fluorescent conjugated antibody (antiserum)

18. THANK YOU