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MICR 306 Advanced
Applications of Viruses
(Part 4.2)
Prof. J. Lin
University of KwaZulu-Natal
Westville campus
Microbiology Discipline
2014
School of Life Sciences
Biotechnology
• Organisms (bacteria, fungi, viruses) with
desirable properties
• Environments – Conditions
Enrichment process
Competitive exclusive principle
APPLICATIONS
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Molecular cloning (RDNA 220)
Vaccine Development
Gene Therapy
Cancer therapy
Phage Therapy (Drug Resistance Problem)
Nanoscience – where physics, chemistry and biology
collide
• Synthetic Biology
• Biological Control Agents
• Protein Engineering
Phage Therapy (Resistance problems)
• Narrow range: host (strain) specific
– Results in less harm to the normal body flora and ecology than
commonly used antibiotics
– As bacteria evolve resistance, the relevant phages naturally
evolve alongside.
– Staphylococcus, Streptococcus bacteriophages (√ )
– TB, E. coli 0157H7 etc….. (????)
• Disadvantages:
– The development of a large collection of well-characterized phage
for a broad range of pathogens
– Methods to rapidly determine which of the phage strains in the
collection will be effective for any given infection.
• Currently there are more than 10 phage bio-companies.
Comparison of the Prophylactic and/or Therapeutic Use of Phages and Antibiotics
Comments
Bacteriophages
Antibiotics
High specificity may be considered to be a disadvantage
Very specific
Target both pathogenic
phages  Disease-causing bacterium must be identified
Dysbiosis and chances of microorganisms and normal of
before applications. Antibiotics have a higher probability of
developing secondary
microflora which may lead to being effective than phages when the identity of the etiologic
infections are avoided.
serious secondary infections. agent has not been determined.
Replicate at the site of
infection where they are
most needed.
No serious side effects
Phage-resistant bacteria
remain susceptible to other
phages having a similar
target range.
Selecting new phages is a
relatively rapid process that
can frequently be
accomplished in days or
weeks.
They are metabolized and
eliminated from the body and
do not necessarily
concentrate at the site of
infection.
Multiple side effects, including
intestinal disorders, allergies,
and secondary infections
The "exponential growth" of phages at the site of infection
may require less frequent phage administration in order to
achieve the optimal therapeutic effect.
Resistance to antibiotics is
not limited to targeted
bacteria.
Because of their more broad-spectrum activity, antibiotics
select for many resistant bacterial species, not just for
resistant mutants of the targeted bacteria.
Developing a new antibiotic is
a time-consuming process
and may take several years.
Evolutionary arguments support the idea that active phages
can be selected against every antibiotic-resistant or phageresistant bacterium by the ever-ongoing process of natural
selection.
A few minor side effects reported for therapeutic phages
may have been due to the liberation of endotoxins from
bacteria lysed in vivo by the phages. Such effects also
may be observed when antibiotics are used.
Host immunity
Phage Therapy
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Advantages:
– Useful where multiple antibiotic resistance has developed
– host specific - won't kill off commensal bacteria
– Rapid action – exponential replication
– self-limiting infection once pathogenic bacteria are killed
– cheap - single dose - self propagates
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Disadvantage - strain specific
– need to generate, keep and archive large bank of phage serotypes
– need accurate diagnosis
– must give cocktail of phage types to prevent bacterial escape
Multi drug
resistant
Pseudomonas
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Also use for detecting pathogenic bacteria - phage infects bacterial lawn - assay
plaques by antibody or by phage-encoded marker gene expression
Breaking bacterial resistance
by designer phages
• Engineered bacteriophage T7 to constitutively
express DspB: an enzyme that hydrolyses
β-1,6-N-acetyl-d-glucosamine, which is an adhesin
that is required for biofilm formation and integrity in
Staphylococcus spp. and Escherichia coli
clinical isolates.
• Following lysis, T7DspB and DspB are released
into the biofilm, which leads to re-infection and
degradation of β-1,6-N-acetyl-d-glucosamine.
• Bacteriophage M13 was engineered to
express LexA3, which suppresses the
SOS DNA repair system that bacteria
require to counteract antibiotic-induced
oxidative stress. Infection by this designer
phage sensitizes E. coli to quinolone
antibiotics.
Synthetic Biology
Scientists have assembled the first synthetic virus and
bacteria (2010). The US researchers built the infectious agent
from scratch using the genome sequence for polio. To
construct the virus, the researchers say they followed a recipe
they downloaded from the internet and used gene sequences
from a mail-order supplier.
Having constructed the virus, which appears to be identical to its
natural counterpart  injected it into mice to demonstrate that
it was active. The animals were paralysed and then died.
A series of synthetic virus-like particles useful in the
characterization of human papillomavirus type 16 (HPV16)
infection and assays employing the synthetic particles have
been developed.
Idea
Synthetic Biologists who are interested in
testing the idea that since natural
biological systems are so complicated, we
would be better off re-building the natural
systems that we care about, from the
ground up, in order to provide engineered
surrogates that are easier to understand
and interact with.
A correlation-based gene network
key enabling technologies
• DNA sequencing
– Functional level
• Fabrication
– de novo DNA synthesis and assembly of fragments of
DNA  gene synthesis.
• Modeling
• Measurement
– Precise and accurate quantitative measurements of
biological systems
– Differences between predicted and measured system
behavior can identify gaps in understanding and
explain why synthetic systems don't always behave as
intended
Potential applications
Six categories:
• bioenergy,
• agriculture and food production,
• environmental protection and remediation
• consumer products,
• chemical production,
• human health.
Current developing products
• The Sleeping Beauty transposon system
– extinct Tc1/mariner-type transposons
• Biosensor technology
– to detect viruses, bacteria, hormones, drugs,
toxics, pollutants and DNA sequences
• Biological controls
• Nanotechnology
Virus/Virus Like Particle
Structural stability and tolerance
towards manipulation
(nm ranges, self assembly)
 serve as building blocks for
novel nano-materials
Viral genetic
Viruses are highly efficient
replicators & viral gene
expression is adapted to host
systems  large production
Biomolecules (DNA/RNA)
 manipulation
(Molecular biology)
Nanoscience – where physics,
chemistry and biology collide
Biological systems emerge from evolved sophisticated processes
developed by Nature. Understanding of how the biological
materials function can help us to synthesize, control and
manipulate materials at an atomic scale. Researchers also begin
this biological manufacturing process by assembling nucleic acids of
a selected bacterium, then essentially infecting them with a virus.
When the virus infects the bacterium, the bacterium can't distinguish
between its own genome and the viral genome, and it starts making
the protein the virus is telling it to make. The result is the production
of novel proteins, which have commercial uses.
Advantages
• They represent very stable and self-assembled
architectures at the nanometer level with sizes ranging
from 10 nm to 200 nm, which are otherwise very difficult
to make by standard synthetic methods in the laboratory.
• Three dimensional structures can be characterized at
near atomic resolution
• They can be purified inexpensively on a large scale, a
crucial advantage when considered for materials
development.
• For each type of virus and virus-like protein assembly, all
the particles are identical. We can therefore envision
them as truly mono-disperse nanoparticles.
Most commonly used viruses
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Cowpea mosaic virus (CPMV)
Cowpea chlorotic mottle virus (CCMV)
Bacteriophage MS2
Bacteriophage M13
Tobacco mosaic virus (TMV)
Turnip yellow mosaic virus (TYMV)
Conventional protein conjugation
strategies on bionanoparticles (BNP)
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Drug delivery
Bio-imaging
Bio-mineralization
Tissue engineering
Applications
Table Genetic modifications in BNPs
BNP
Type of insert
Expression host
TMV
Antigenic peptides, RGD peptides cysteine,lysine,His6 tag
(Arginylglycylaspartic acid – a cell adhesion motif)
Tobacco plants and bacterial
expression
CPMV
Peptide antigens, cysteine, lysine, His6 tag
Cowpea plants
CCMV
subE mutant, cysteine
Cowpea plants and bacterial
expression
Ferritin
AG4, Co2 peptide,
Native host
P22
FHV
Cysteine
Antigenic insert
Bacteria host
Native (insect cells) and
recombinant (baculovirus)
MjHsp
Metal binding peptides, cysteine, RGD peptide
Bacteria host
MS2
HBVLP
Cysteine, unnatural amino acid
Unnatural amino acid, His6peptide
Bacteria host
Recombinant
M13
Qβ
Metal binding peptides, proteins
Unnatural amino acids
Bacteria host
Bacteria host and cell free
Viruses for Peptide display: M13 Phage or
plant virus (TMV) Coat Protein Fusions
Need :
non-enveloped virus
many repeat capsid subunits
ordered capsid array - amplified display
external loops or termini available for
peptide addition via gene fusion
Mass peptide display
on outer surface of
TMV particle
N
C
60S
loop
Tobacco mosaic virus
TMV
VIRION
Assembly of mixed TMV capsids
carrying epitope variants = useful
vaccine vs highly variable pathogen
Cysteine-mutant TMV
Bacteriophages
+ metal binding
peptide
 Semiconductor
Bacteriophage M13
+ amine rich peptide
CoPt-FePt; ZnS-CdS
 Magetic
 Liquid Cystal
 Bioconjugational
techniques
M13 -semiconductor
The genetically engineered hepatitis B virus particle (green) binds
to nickel and antibodies (dark blue) specific for troponin I, a marker
associated with heart attacks
Developing molecular-scale technologies include Zettacore, which is
examining ways to design and use molecules in modern electronic
applications, and NanoMagnetics, which makes magnetic particles
grown inside uniform hollow protein spheres 12 nanometers in
diameter. The technology has applications in water purification,
data storage and medical imaging.
Virus-Assembled Batteries
More than half the weight and size of today's batteries comes from
supporting materials that contribute nothing to storing energy.
Genetically engineered viruses can assemble active battery
materials into a compact, regular structure, to make an ultra-thin,
transparent battery electrode that stores nearly three times as much
energy as those in today's lithium-ion batteries. The scientists used M13
viruses to make the positive electrode of a lithium-ion battery, which they
tested with a conventional negative electrode. By adding sequences of
nucleotides to the virus' DNA, the researchers directed these proteins
to form with an additional amino acid that binds to cobalt ions. The
viruses with these new proteins then coat themselves with cobalt
ions in a solution, which eventually leads, after reactions with water,
to cobalt oxide, an advanced battery material with much higher storage
capacity than the carbon-based materials now used in lithium-ion
batteries.
Applications for Electrical Devices
Virus battery
Virus-Assembled
Batteries
Recipe for using viruses
to make an electrode:
1Dip a polymer electrolyte
in a solution of genetically
engineered viruses.
2These form a uniform
coating on the electrolyte.
3Dip the coated polymer
into a solution of battery
materials.
4The viruses coat
themselves with the
battery material,
transforming into
nanowires with a regular
crystal structure good for
high-energy batteries.
New virus-built battery could
power cars, electronic devices
2nd April, Science 2011
Electronic devices
MIT researchers use genetically modified virus to produce structures
that improve solar-cell efficiency by nearly one-third. --2011
Viruses as Energy converting
Materials or Catalysts
• High reactivity and large surface area
• Conjugation of chromophores to viral
template  photocatalyst  solar energy
cell
• Virus template as catalyst supports 
enhance the activity
Nanotechnology
Questions
• Safety and Security
• Social and Ethical
• Scientists are very intelligent people, very
dedicated to their work and their fields of
endeavor. But many times they fail to
communicate to the public policymakers
the importance of the things that they’re
involved in