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Viral Growth

Viral Growth

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Viral Growth

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  1. Viral Growth Dr Paul Brown paul.brown@uwimona.edu.jm BC10M: Introductory Biochemistry Lecture 7

  2. Characteristics of Viruses • Non-living agents that infect all life forms (Bacteriophages, plant viruses, and animal viruses) • One virus particle = “virion” (size: 10 – 500 nm) • Virus architecture • Nucleic acid (DNA or RNA – not both!) • Protein coat = capsid of various shapes (isometric, helical, complex) • Naked virions vs. enveloped viruses • Genome: ss-/ds-RNA, ss-/dsDNA; linear or circular

  3. Methods of Study • Much more expensive and difficult to study animal viruses than bacteriophages • Cultivation in host cells • Living animal • Embryonated chicken eggs • Cell or tissue culture (= in vitro)

  4. Methods of Study cont: Quantitation • Plaque assay (useful for infective and lytic viruses) • Virion counting with EM • Quantal assay (ID50 or LD50) • Haemagglutination (e.g.influenza virus)

  5. Culturing viruses requires an appropriate host cell. In this example a bacteriophage is grown using bacterial cells as host cells. Animal or plant viruses can be grown in tissue culture.

  6. Plaques in a lawn of bacterial cells caused by viruses lysing bacterial cells.

  7. Replication Cycle - Overview • Obligate intracellular parasites using host cell machinery • Very limited number of genes encode proteins for • Capsid formation • Viral nucleic acid replication • Movement of virus into and out of cell • Kill or live in harmony within the host cell – Outside the cell, viruses are inert Fig 13.3 Size comparison(see also Table 13.1)

  8. Bacteriophage (Phage) • Obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery • Significance • Models for animal cell viruses • Gene transfer in bacteria (transduction) • Medical applications • Identification of bacteria - phage typing • Treatment and prophylaxis??? • Examples: T4 and Lambda ()

  9. Head/Capsid Contractile Sheath Tail Tail Fibers Base Plate Composition and Structure • Composition • Nucleic acid • Genome size • Modified bases • Protein • Protection • Infection • Structure (T4) • Size • Head or capsid • Tail

  10. Infection of Host Cells • Irreversible attachment • Adsorption • LPS for T4 • Sheath Contraction • Nucleic acid injection

  11. Lytic Bacteriophage • Lytic or virulent phage: Phage that can only multiply within bacteria and kill the cell by lysis. (e.g., T4)

  12. One-step growth curve of a lyticbacteriophage

  13. Total Phage Extracellular Phage Number of Infectious Particles Lysis Intracellular accumulation phase Eclipse Time after Infection Lytic Phage Multiplication Cycle • Eclipse • Early genes • Phage DNA synthesis • Late genes • Intracellular accumulation • Lysis and Release

  14. Time course of events occurring during one-step growth curve of bacteriophage T4.

  15. Lysogenic Bacteriophage • Lysogenic or temperate phage: Phage that can either multiply via the lytic cycle or enter a quiescent state in the bacterial cell. (e.g.,) • Expression of most phage genes repressed • Prophage • Lysogen

  16. Lysogenic Phage • DNA integrates into host chromosome • Phage DNA = Prophage • Infected bacterial cell = lysogenic cell or lysogen • Prophage state can be indefinite • Lysogenic conversion confers new properties onto host cells (e.g.: toxin production of S. pyogenes – scarlet fever) • Phage induction converts lysogenic to lytic state

  17. Replication cycle in a lysogenic bacteriophage

  18. Cohesive Ends Ligase Linear Double Stranded Closed Circle Opened Circle Events Leading to Lysogeny • Circularization of the phage chromosome • Cohesive ends

  19. bio gal gal bio bio gal Events Leading to Lysogeny • Site-specific recombination • Phage coded enzyme • Repression of the phage genome • Repressor protein • Specific • Immunity to superinfection

  20. gal bio bio bio gal gal bio gal Termination of Lysogeny • Induction • Adverse conditions • Role of proteases • recA protein • Destruction of repressor • Gene expression • Excision • Lytic growth

  21. Lytic vs Lysogenic Cycle? • Role of repressor • Role of cro gene product • Role of proteases

  22. Significance of Lysogeny • Model for animal virus transformation • Lysogenic or phage conversion • Definition: A change in the phenotype of a bacterial cell as a consequence of lysogeny • Modification of Salmonella O antigen • Toxin production by Corynebacterium diphtheriae

  23. Host Range of Phages Phage host cell interaction usually very specific Limiting factors for host range • Phage has to bind to bacterial surface receptors • Bacterial surface receptors mutate  resistant cell • Lysogenic conversion changes surface receptors and protects host • Restriction modification system of host cell

  24. Medical Applications of Phage • “I strongly believe phage could become an effective antibacterial tool” - Carl Merril, Chief of the Laboratory of Biochemical Genetics, National Institute of Mental Health, NIH. • “It might be another string on the bow, such that when (conventional antibiotics) fail, here’s something that has a chance of working. But it’s not going to be a panacea” - Joshua Lederberg, Sackler Foundation Scholar at The Rockefeller University Reassessment of Medicinal Phage Spurs Companies to Study Therapeutic Uses American Society for Microbiology News 64:620-623, 1998

  25. Medical Applications of Phage • Exponential Biotherapies (Rockville, MD) • Vancomycin resistant Enterococcus faecium and Streptococcus pneumoniae • Phage Therapeutics (Bothell, WA) • Staphylococcus aureus and Staphylococcus epidermidis • Intralytix, Inc. (Baltimore, MD) • Salmonella in meat and poultry • Biopharm Ltd. (Tblisi, Georgia) • Infections associated with burns • University of Idaho • Escherichia coli O157:H7 in cattle Reassessment of Medicinal Phage Spurs Companies to Study Therapeutic Uses. American Society for Microbiology News 64:620-623, 1998. Phages eyed as agents to protect against harmful E. coli. American Society for Microbiology News 65:666-667, 1999. The End