Viral Growth

1
Viral Growth

• Dr Paul Brown
• paul.brown_at_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
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
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
Events Leading to Lysogeny

• Circularization of the phage chromosome
• Cohesive ends

19
Events Leading to Lysogeny

• Site-specific recombination
• Phage coded enzyme
• Repression of the phage genome
• Repressor protein
• Specific
• Immunity to superinfection

20
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, heres
  something that has a chance of working. But its
  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 64620-623, 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 O157H7 in cattle

Reassessment of Medicinal Phage Spurs Companies
to Study Therapeutic Uses. American Society for
Microbiology News 64620-623, 1998. Phages eyed
as agents to protect against harmful E. coli.
American Society for Microbiology News
65666-667, 1999.
The End