1. ｱﾊﾞｵﾗﾘｰﾈﾙﾋﾞﾙﾊﾞｵ January 26, 2010
Applied Molecular Biology of Livestock にしかわ先生
Controlling New Influenza Virus Using Genetic Engineering Techniques
According to Wikipedia, genetic engineering (which includes recombinant DNA
technology, genetic modification/manipulation and gene splicing) is a term that
applies to the direct manipulation of an organism's genes. Genetic engineering is
different from traditional breeding, where the organism's genes are manipulated
indirectly. Genetic engineering uses the techniques of molecular cloning and
transformation to alter the structure and characteristics of genes directly. The
techniques include the use of hybridomas (hybrids of rapidly multiplying cancer cells
and of cells that make a desired antibody) to make monoclonal antibodies; gene
splicing or recombinant DNA, in which the DNA of a desired gene is inserted into the
DNA of a bacterium, which then reproduces itself, yielding more of the desired gene;
and polymerase chain reaction, which makes perfect copies of DNA fragments and is
used in DNA fingerprinting.
The question now is how can we control new influenza viruses using genetic
engineering techniques? By studying the characteristics of new influenza viruses
through the manipulation of the DNA, recombinant virus vector vaccine can be
developed. Avian influenza viruses are major contributors to viral diseases in poultry
as well as humans.1 Outbreaks of high-pathogenicity avian influenza viruses cause
high mortality in poultry, resulting in significant economic losses. The potential of
avian influenza viruses to re-assort with human stains resulted in global pandemics in
1957 and 1968, while the introduction of an entirely avian virus into humans claimed
several lives in Hong Kong in 1997. Despite considerable research, the mechanisms
that determine the pathogenic potential of a virus or its ability to cross the species
barrier are poorly understood.
Fortunately, reverse genetics methods, i.e., methods that allow the generation of an
influenza virus entirely from cloned cDNAs, have provided scientist with the means to
address these issues. In addition, reverse genetics is an excellent tool for vaccine
production and development. This technology should increase preparedness for
future influenza virus outbreaks. In particular, the virus responsible for the 1918
Spanish flu pandemic (which killed an estimated 50 million people worldwide) has
been reconstructed by genetic engineering in a high-security US laboratory.2
Preliminary studies show that it is an avian flu virus that mutated to spread quickly
between people (just as many experts fear will happen soon with the current H5N1
strain of bird flu in Asia). The US National Institutes of Health approved the research,
despite its apparent risk, because it will help scientists find new treatments for the
most dangerous types of flu.
1
Neumann G, Hatta M, Kawaoka Y. Reverse genetics for the control of avian influenza. Department of Pathobiological
Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive West, Madison, WI 53706, USA.
2
Clive Cookson, Science Editor、Financial Times, 5 October, 2005.
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2. The Centers for Disease Control laboratory in Atlanta made a live virus with the full
genetic sequence of Spanish flu, using the reverse genetics techniques developed at
Mount Sinai Hospital in New York. The CDC team felt that they had to recreate the
virus and run these experiments to understand the biological properties that made
the 1918 virus so exceptionally deadly. They wanted to identify the specific genes
responsible for its virulence, with the hope of designing anti-virals or other
interventions that would work against virulent influenza viruses.
The key genetic data for the experiment came from the Armed Forces Institute of
Pathology in Washington DC. Over the past eight (8) years, scientists there have
pieced together the entire Spanish flu genome, from viral fragments isolated from
preserved lung samples of patients who died in 1918 and from a female victim whose
body was fortuitously frozen in Alaskan permafrost. Many of the flu viruses
circulating today were descendants of the H1N1 strain that swept the world in 1918.
So the population still had some protective immunity against it. Because of this, it is
unlikely that a1918-like virus would be able to cause a pandemic today.
The research suggested that Spanish flu arose in a different way to the viruses that
caused the two (2) other 20th century pandemics. In 1957 and 1968 an existing
human virus underwent genetic mixing with a bird virus to produce a new
"reassorted" strain in one step. Ominously, the H5N1 strain currently circulating in
Asia is undergoing similar humanizing mutations though it has not yet accumulated
as many changes as Spanish flu.
Recombinant vaccines can be created based on the results of genetic engineering on
new influenza viruses. They are created by utilizing bacteria or yeast to produce
large quantities of a single viral or bacterial protein. This protein is then purified and
injected into the patient, and the patient's immune system makes antibodies to the
disease agent's protein (protecting the patient from the viruses).
The advantage of the recombinant vaccine technology is that there is virtually no
chance of the host becoming ill from the agent. This is because it is just a single
protein, not the organism itself. Traditional vaccine risks come from the organism not
being totally weakened (attenuated) or a reversion to a virulent (disease-causing)
form. Another advantage of a recombinant vaccine is that it does not need an
adjuvant. An adjuvant is an agent that stimulates (irritates) the immune system to
find and react to the vaccine agent. Some adjuvants have been implicated in
causing cancer in some animals over time.
In reverse genetics, a flu virus contains eight gene segments.3 One of the gene
segments codes for the surface antigen hemagglutinin (HA) and another codes for
the surface antigen neuraminidase (NA). Scientists can custom-make a flu strain by
assembling genes that code for the desired features. Two genes representing the
HA and NA antigens are selected from the target strain (flu strain 1) while the
remaining six genes come from a virus that's time-tested for its ability to grow inside
an egg (flu strain 2). Although the influenza virus actually uses RNA as its genetic
material, the researchers make complementary pieces of DNA because DNA is
easier to work with.
3
http://www3.niaid.nih.gov/topics/Flu/Research/basic/ReverseGeneticsIllustration.htm
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3. The illustration details the following steps in creating the vaccine (please see Figure
1):
• After removing the dangerous part of the HA gene, scientists splice the HA
and NA genes from flu strain 1 into circular pieces of DNA called plasmids;
• Additional plasmids are created using the remaining six genes found in flu
strain 2;
• Scientists insert the HA and NA plasmids from flu strain 1 and the six
plasmids carrying genes from flu strain 2 into animal cells growing in the
laboratory; and
• The genes in the plasmids instruct the animal cells to make the desired new
flu strain.
Figure 1. Steps in Vaccine Production to Combat a Flu Virus
Source: US National Institute of Allergy and Infectious Diseases
In conclusion, using genetic engineering techniques will lead to control of new
influenza viruses. Based on the knowledge gained from genetic engineering,
genetically recombinant vaccines can be created.
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