MCB 308 Part II
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  • 2. 1.Observe the situation 2. Ask a question 3. Turn that question into a testable hypothesis 4. Predict the outcome of your experiment 5. Perform your experiment 6. Analyze the results Redeemer ’s
  • 3. Viruses are?: 1. 1. 2 2. 3. 4. 5 Redeemer ’s
  • 4. Viruses .…….1 • 1. Infectious agent found in virtually all life forms, (humans, animals, plants, fungi, and bacteria) • 2. Not considered free-living, since they cannot reproduce outside of a living cell • 3. Replicates by transmitting their genetic information from one cell to another Redeemer ’s
  • 5. Viruses…..2 • 4. Consist of genetic material—either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) • 5. Surrounded by a protective coating of protein, called a capsid, + or - an outer lipid envelope. • 6. Vary in size from poxvirus (450nm - 0.000014 in) to poliovirus (30 nm - 0.000001 Redeemer in) in length, so too small to be seen by light ’s
  • 6. Redeemer ’s
  • 7. Identification 3 main methods: i. Microscopy ii.Isolation iii.Serology Redeemer ’s
  • 8. Isolation & Identification Microscopy • Light Microscopy • Virus size precludes use of LM • Pathogenic effect of virus on infected cells • inclusion bodies:e.g. Negri bodies in Rabies and Councilman bodies in YF, Guanieri bodies, Smallpox. • Fluorescence Microscopy • Electron Microscopy Redeemer ’s
  • 10. ELECTRONMICROSCOPY Arenaviridae Haemorrhagic Fever Viruses (Lassa, Junin, Machupo, Guanarito) Filoviridae Flaviviridae (Ebola, Marburg) (dengue, yellow fever, TBE group) Bunyaviridae (CCHF, RVF, Hantaviruses) Redeemer ’s
  • 11. Viruses Structure – Small pox virus A colored-transmission electron micrograph shows a group of Orthopoxvirus variola, the virus that causes smallpox. Once greatly feared for its ability to kill or disable its victims, smallpox was eradicated by 1979 through a worldwide vaccination campaign Note the unique brick shaped structure. Redeemer ’s
  • 12. Viruses Structure - Adenovirus Non enveloped icosahedral virus The adenovirus is one of the 100 virus types that causes the common cold. The adenovirus can enter a human host through the respiratory and GITs or the eye Redeemer ’s
  • 13. Techniques for Virus isolation Two major techniques - Classical and Molecular  Classical Cell Culture Virus isolation and quantitation Preparation of Antigen Virus Purification Electron Microscopy Traditional serological tests Immunoassys Cell mediated immunity Redeemer ’s
  • 14. Techniques for Virus isolation Two major techniques - Classical and Molecular  Molecular RNA transcription, transfection & quantitation DNA viruses: DNA extraction, purification & characterization Virus mutants Polypeptides Viral enzyme assays Redeemer ’s
  • 15. Techniques for Virus isolation: Classical  Cell Culture  Primary- fresh organ of lab animal human embryo (kidney)  Continuous- from primary grown continuously limited subcultures generic variation, often die out 15-25 passages  Euploid- Normal human diploid cells with exact multiples of normal chromosomal numbers, up to 50 passages, stop dividing  Aneuploid – Mouse fibroblasts, tumour cells not an exact multiple of normal chromosomal numbers  Insect – Aedes aegypti and A. albopictus Redeemer ’s
  • 16. Virus Isolation Techniques - Classical Methods  Cell Culture   Art of growing cells in vitro Over 3,200 chracterized cell lines available  Derived from 75 species (including hybridomas and plant cultures)  Banked in cell culture banks: ATCC, ECACC  Select cell type with unique characteristics for  Confirm life expectancy for study (15-20 passages or subcultures from seed stock to ensure retention of characteristics)  Types of Cell cultures  Primary cells  Continuous cells  Insect cells  Propagation of cell cultures  Media and buffers  Endotoxin free water  Sterilization  Propagation of cells by scraping and enzyme treatment  Subculture protocol  Suspension cultures  Lymphocyte cultures Redeemer  Maintenance of adherent and suspension cells ’s
  • 17. Entry by fusing with plasma membrane Fusion of a virus with t he membrane of an endosome Redeemer ’s
  • 18. Cytopathic Effect of Viruses Virus replication leads to cell damage Cell Lysis Redeemer ’s
  • 19. FIGURE 44-1 Development and progression of viral cytopathology. Human embryo skin muscle cells were infected with human cytomegalovirus and stained at selected times to demonstrate (A) uninfected cells, (B) late virus cytopathic effects (nuclear inclusions, cell enlargement), (C) cell degeneration, and (D) a focus of infected cells in a cell monolayer Redeemer (i.e., a plaque), hematoxylin and eosin stain. ’s
  • 20. Redeemer ’s
  • 21. Transmission electron micrograph of HIV-1, budding and free Redeemer ’s
  • 22. Redeemer ’s
  • 23. Cytopathic Effect of Viruses Virus replication leads to cell damage Enlarged round refractile cells Adenovirus Redeemer ’s
  • 24. Parainfluenza Bovine adeno Bovine herpes Enterovirus V.stomatitis Vaccinia Paramyxovirus Herpes Redeemer ’s
  • 25. Virus isolation and quantitation  Most commonly used in viral diagnosis  Collection of specimens – sterile, timely & preservation  Virus isolation and quantitation  Cell culture/Organ culture CPE, Hemadsorption, Interference  Laboratory animals Mouse, rabbits, birds, sheep goats cows etc  Fertile hen eggs Redeemer ’s
  • 26. Diagnosis Laboratory Test Specimen needed Serology  neutralization - (Neut)  hemagglutination inhibition (HI) blood  complement fixation (CFT)  ELISA - IgM & IgG Virus Detection blood or organs  culture, PCR, Ag detection Histopathology liver tissue Redeemer ’s
  • 27. Techniques for Virus isolation: Classical  Quantitation  Infectivity assay(  Plaque Assay  Pock Assay  Haemagglutinin Assay  TCID50, EID50, LD50,  Dilution of a virus required to infect 50% of experimental animal  Dilution of a virus required to kill or cause the death 50% of experimental animal  To calculate we do serial 10/5/2 -fold dilution and check number of animals sick or dead at end of experiment Redeemer ’s
  • 28. A plaque assay. Serial dilutions of virus have been plated on confluent monolayer cultures of cells. The cells are stained after a period of time in which a single virus infects a cell, produces new virus particles and infects surrounding cells. The white areas show areas of the culture in which the cells have been killed. Each "plaque" Redeemer is the result of the presence of one original infectious virus particle. ’s
  • 29. Calculation of TCID50 or LD50 Reed & Muench method (% positive above 50%) - 50% (% positive above 50%) – (%positive below 50%) = proportionate distance Spearman Karber method Highest dilution giving 100% CPE + 0.5 – Total number of test units showing CPE Number of test units per dilution = TCID50 Redeemer ’s
  • 30. Preparation of Antigen & Purification  Whole cell antigen  Freezing and Thawing  Sonication  Alkaline glycine treatment  Fractionation of antigens Tween 80 Beta-propiolactone extraction - Non-ionic detergent – separates envelope proteins from capsid  Virus Purification  Adsorption/elution from erythrocytes based on hemagglutinanility of virus  Adsorption/elution from CaHPO4.2H20 brushite (DNA from RNA Toga from Flavi) Redeemer ’s
  • 31. Preparation of Antigen & Purification  Chromatographic procedures  Gel filtration  Ion exchange or affinity  Ultracentrifugation  Precipitationntechnique using PEG 600 in a cold high –salt environment gentler than other techs  Sucrose gradient produces bands of pure virus  CsCL gradient  Filtration  Electron Microscopy  Negative staining virus particle do not scatter ions under EM, so carbon coating in plastic films Redeemer ’s
  • 32. Traditional serological tests  Neutralization Test  Complement foxation Test  Haemagglutination-inhibition test  Single radial hemolysis  Particle agglutination test with latex or labelled erythrocytes  Immunodiffusion test  Countercurrent Immunoelectrophoresis CIEOP Redeemer ’s
  • 33. Immunoassays ELISA FIA RIA Immunoassys Cell mediated immunity Redeemer ’s
  • 34. Medical Virology •Initial in vitro screening of drug candidates for potential antiviral activities •Detection of virus and viral antigen Redeemer ’s
  • 35. Virus Structure ……1 • Individual virus particle is called a virion • Contains genetic material, or genomes, in one of several forms • Viral genes may consist of either DNA or RNA. (cellular organisms genes only DNA) • Almost all viral DNA is double-stranded, with either a circular or a linear arrangement Redeemer ’s
  • 36. Redeemer ’s
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  • 39. Redeemer ’s
  • 41. Acknowledgement Information contained in this slide were obtained from various sources, including CELLS ALIVE, WIKIPEDIA, IMMUNOLOGY TUTORIALS, PERSONAL LECTURE NOTES AND VIROLOGY METHODS MANUAL (Mahy & Kangro) Redeemer ’s
  • 42. Acknowledgement Information contained in this presentation was obtained from various sources, including: CELLS ALIVE, WIKIPEDIA, IMMUNOLOGY TUTORIALS, PERSONAL LECTURE NOTES AND VIROLOGY METHODS MANUAL (Mahy & Kangro) Redeemer ’s