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 Theiler – 1903
• Suckling mice – coxsackie, arbovirus
• Guinea pigs, rabbits and ferrets are also used

5. 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)

9. POCK FORMATION

10. 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)

12. TISSUE CULTURE
CLASSIFICATION

15. ORGAN CULTURE

16. EXPLANT CULTURE

18. CELL CULTURE

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

33. CULTIVATION OF VIRUSESTissue 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

35. SOME CELL CULTURES IN COMMON USE
VIRAL CELL CULTURES

36. • 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

37. 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)

38. 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

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

40. THANK YOU