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ISIS Report 08/12/10

Can GM Mosquitoes Eradicate Dengue Fever?

A risky strategy experimented on humans without informed consent, and based on data from unpublished studies Prof. Joe Cummins

A fully referenced version of this report is posted on ISIS members website and is available for download here

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What is dengue?

Dengue fever is an illness caused by an RNA flavivirus spread by the bites of Aedes aegypti mosquitoes. The symptoms include fever, headache, rash, severe pains in the muscles and joints, and pain behind the eyes. Dengue fever is rarely fatal, while the related dengue haemorrhagic   fever is a severe disease that leads to death in approximately 5 percent of cases. Dengue hemorrhagic fever is seen most often in children younger than 15 years old. It is also seen most often in individuals with fever and gastrointestinal symptoms followed by capillary haemorrhage [1] (Terminator Mosquitoes to Control Dengue? SiS 39). Incidence of dengue has grown dramatically around the world in recent decades. Some 2.5 billion people – two fifths of the world's population – are now at risk from dengue. The World Health Organization estimates there may be 50 million dengue infections worldwide every year [2]. For more details, see Box.

Terminator mosquitoes tested on mosquitoes and people

Genetically modified (GM) OX513A  Aedes aegypti male mosquitoes are being released in an area inhabited by people. The living modified organism (LMO) released contains a fluorescent molecular marker and a ‘self-limiting’ genetic construct. When female Aedes aegypti mosquitoes mate with OX513A male mosquitoes, all offspring will die, hence preventing the emergence of the next generation. OX513A male Aedes aegypti mosquitoes, developed by Oxitec Ltd. in Oxford, UK, carry a late-acting dominant-lethal gene [1]. The dominant-lethal gene requires only one copy to work, and is expressed conditionally. In the case of the Oxitec creation, the condition that acts to kill the mosquito larvae bearing a dominant gene is absence of the antibiotic tetracycline. The males reared for terminating their larvae offspring when mated with wild type females are maintained on antibiotic-containing media.  The males are homozygous diploids for OX513A so that all of their offspring are doomed in the absence of antibiotic. The lethal factors are active in late stages of larval development [3].

The field test is being done by Oxitec Limited, Oxford, United Kingdom in Grand Cayman. The Cayman Islands are a parliamentary democracy with judicial, executive and legislative branches. The present constitution, which came into effect on 6 November 2009, makes the Cayman Islands a British Overseas Territory.  Results of the Oxitech experiments claiming to prove the safety and benefits of the terminator mosquito have been reported at a press conference in London, UK [4], and at a meeting of the American Association of Tropical Hygiene in Atlanta, Georgia, USA [5], but have not yet appeared in a peer-reviewed journal. As the Cayman Island is under British jurisdiction, the human experimentation involving exposure to GM mosquito appeared to have been undertaken without informed consent as would be required by British law.

Construction of the GM mosquitoes

The OX513A mosquito was constructed using a transposon (jumping gene) piggyBac LA513 isolated from Baculovirus, a common soil dwelling DNA plant virus. LA513 is a non-autonomous piggyBac-based transposon of 8.4 kb. Transgenic mosquitoes are readily identified by red fluorescence due to expression of DsRed2, a red fluorescent protein from a marine microbe. The GM construct contains tTAV, coding for a tetracycline-repressible transcriptional activator, under the control of its binding site, tetO, a minimal promoter from Drosophila heat shock protein 70, and a 3' UTR (untranslated repeat) sequence from another Drosophila gene fs(1)K10. In the absence of tetracycline, tTAV binds to tetO and drives the expression of more tTAV, in a positive feedback loop. In the presence of tetracycline, tTAV binds tetracycline; the tetracycline-bound form does not bind tetO and so no more tTAV is expressed. Consequently, this construct gives very high levels of expression of tTAV in the absence of tetracycline, but only low, basal expression in the presence of tetracycline. High level expression of tTAV is toxic, possibly due to interactions of its VP16 domain in transcription activation of lethal genes, so this construct provides a tetracycline-repressible lethal system [3]. The VP16 domain is the transcriptional activation domain of the herpes virus protein VP16, and is a common feature of transcription activation proteins.  It seems clear that the absence of the antibiotic triggers enhanced transcription factors leading to lethality; however, the final product(s) causing late death at the larvae-pupae boundary have not been identified, which surely should have been done before people are exposed to the transgenic mosquitoes.

The piggyBac transposon and horizontal gene transfer

The piggyBac transposon was discovered in cell cultures of the moth Trichopulsia, also known as the cabbage looper. The looper cells caused extensive mutation in an insect Baculovirus. The transposon is being used extensively as it acts as a gene transfer vector for a number of unrelated insects. Transposons are related to viruses but lack the ability to be packaged in a virion. The piggyBac transposon is flanked by short inverted repeats that initiate non-replicative insertion into the insect chromosome, mediated by an enzyme called transposase that targets the sequence TTAA on the chromosome. When the transposon inserts into a gene sequence, it may cause a mutation, or the transposon may act as a recombination site leading to duplication and deletion of sequences in the chromosomes.

The transposon is usually multiplied in the laboratory by being inserted in a bacterial plasmid. The transposon-bearing plasmids are injected into early insect embryos, where they may insert into the genome of germ cells and become maintained in a lineage arising from the germ cells. The plasmids injected into an embryo include the transposon carrying the gene(s) to be transferred and lacking a transposase, to ‘disable’ the transposon and prevent it from moving by itself. The transposase function is provided by a helper plasmid that has a transposon with the transposase gene, but is otherwise also disabled because it has one terminal short repeat missing, so it cannot insert into the chromosome [6] (piggyBac a name to remember, ISIS report).

The major hazard of horizontal transfer of the piggyBac transposon has been thoroughly addressed in a previous submission to the USDA objecting to the release of the pink bollworm in 2001 and again in our recent submission objecting to the release of the current transgenic .mosquito [1]. We provided evidence that the disabled vector carrying the transgene, even when stripped down to the bare minimum of the border repeats, was nevertheless able to replicate and spread, basically because the transposase function enabling the piggyBac inserts to move can be by transposons potentially present in all genomes, including that of the mosquito. The main reason for using transposons as vectors in insect control is precisely because they can spread the transgenes rapidly by ‘non-Mendelian' mean within a population, i.e., by replicating copies and jumping into genomes, including those of the mammalian hosts. Although each transposon has its own specific transposase enzyme that recognizes its terminal repeats, the enzyme can also interact with the terminal repeats of other transposons, and evidence suggest “extensive cross-talk among related but distinct transposon families” within a single eukaryotic genome.

The use of the piggyBac transposon has been plagued by problems of instability in transformed Aedes aegypti [7]; and large unstable tandem inserts of the piggyBac transposon were prevalent [8]. In spite of instability and resulting genotoxicity, the piggyBac transposon has been used extensively also in human gene therapy [9]. A number of human cell lines have been transformed, and even primary human T cells, using piggyBac [10]. The piggyBac transposon was found to induce genome wide insertion mutations disrupting gene functions when activated in mice [11].  Female Aedes aegypti mosquitoes mate as a rule before taking a first blood meal [12].  Thus living human blood will be exposed to the piggyBac carried by the mated female.  What would it take to activate the mosquito-borne transposon to infect human blood? No more than an encounter with Baculovirus that could enter through open cuts or sores, or with inhaled dust.  The piggyBac transposon GM construct could wreak havoc in the human genome, causing numerous insertion mutations and other untold, unpredictable damage.

Dengue virus replication cycle and infection

Infection with dengue virus begins when the mosquito takes a blood meal and the virus is introduced into the host. The virus binds to and enters a permissive host cell via receptor-mediated endocytosis. Fusion of viral and endosome-vesicle membranes allows the naked virus to enter the cytoplasm, where its and genome is further stripped of its protein coat. Translation of the protein-coding strand takes place; after which, the virus switches to the synthesis of a negative- (non-coding) strand intermediate that forms a circle and  serves as a template for the production of multiple copies of positive-strand viral RNA (vRNA). Successive rounds of translation produce high levels of viral proteins; the structural protein capsid or core (C), premembrane (prM), and envelope (E) proteins, along with vRNA, are assembled into progeny virions, which are transported through the Golgi compartment and secreted [13].

Dengue infection with any of the four serotypes of the dengue virus can produce the full continuum of illness, ranging from mild, nonspecific symptoms, to classic dengue fever, to the most serious, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Up to half of all infections are asymptomatic; and a person can become infected from the bite from one infected mosquito. DHF is characterized by the acute onset of flu-like symptoms 3 to 14 days after a bite. The febrile phase includes a high temperature (> 38.3°C) and possibly frontal headache, facial flushing, joint and muscle pains, pain behind the orbit of the eye, rash, nausea, anorexia, and bone pain. A positive tourniquet test (for fragile capillaries) increases the likelihood of dengue. Definitive diagnosis requires the detection of dengue-specific antibodies or isolation of the virus. Haemorrhagic manifestations include petechiae (red or purple spots on the body caused by broken capillaries), hematomas (blood-filled swelling), nose-bleeding, and gum bleeding. Weakness and malaise may persist for several weeks. DHF and DSS are potential life-threatening. DHF critical phase begins 24 to 48 hours after fever subsides and is defined by four criteria: (A) fever or recent fever lasting 2 to 7 days; (B)  any haemorrhagic sign or symptom; (C) thrombocytopenia (abnormally low count of platelets in blood); and (D) evidence of increased blood vessel permeability. Persistent vomiting, severe abdominal pain, and difficulty breathing may develop. Swings in body temperature (hypo- to hyperthermia) and mental status changes may occur. Severe haemorrhagic symptoms may progress to vaginal or intracranial bleeding. In mild to moderate DHF cases, symptoms will begin to dissipate once the fever subsides [14].

To conclude

Dengue virus is a growing threat to human health. The problem is compounded by the absence of effective vaccines. However, as we pointed out [1], there are recent safe, effective, and affordable alternatives to controlling the insect vector. In contrast, the current release of terminator male mosquitoes on Cayman Island is a risky strategy. To add insult on injury, there has been no warnings issued to tourists, and most residents on the Island do not appear to have given informed consent to be exposed to the GM male mosquitoes, in blatant violation of human rights with regard to human experimentation.  If the strategy has succeeded, as claimed, the islanders may have been granted temporary respite from the insect vector for Dengue; though replacement mosquito vectors are likely to be blown in from neighbouring islands almost immediately while GM mosquitoes are spreading to them from Cayman Island. The UK government appears not to have exercised appropriate jurisdiction over the human experimentation in its territory; while the scientific community should condemn the use of data to justify the experiment from studies that had not passed peer review.

There are 9 comments on this article so far. Add your comment
Di Robinson Comment left 9th December 2010 09:09:06
A similar experiment is being conducted in Cairns?
joe cummins Comment left 10th December 2010 18:06:59
Di Robinson asks 'A similar experiment is being conducted in Cairns?' Thank you Di Robison for an excellent question. Cairns , Queensland, Australia is the home of a n important Mosquito research facility. I have searched the net to find out whether or not Cairns has done field releases with Oxitech genetically modified terminator mosquitoes but do not find such experiments. However, the Cairns laboratory is doing important work on the use of Wolbachia mosquitoes to control infection of mosquitoes by dengue virus. The Cairns research team led by Scott O’Neill plans to release female Aedes aegypti mosquitoes bearing the Wolbachia pipientis bacterium . The bacterium is an an intracellular endosymbiont which is transmitted through the egg (maternal inheritance). Wolbachia has been found to inhibit replication of the degue virus thus preventing spread of dengue virus to humans. Furthermore, the Wolbachia infections interferes with human blood feeding by the infected mosquitoes. The wolbachia bacteria used in the experiments is not genetically modified but is selected from natural bacterial colonies. The trial releases of the Wolbachia infected mosquitoes will not be done in Cairns but in nearby Yorkeys Knob and Gordonvale communities. I understand that the communities have been fully informed of the conduct and any risk associated either experimental release of the mosquitoes. The Cairns experiment seems to have covered the concerns related to human experiment. Presumable the results of the experiments’ will be published in full in a timely manner, The natural solution may save the world. References for further study 1. Enserink,M. Australia to test mosquito vaccine against human disease Science 2010, 330, 1460-61 2. Frentiu FD, Robinson J, Young PR, McGraw EA, O'Neill SL Wolbachia-mediated resistance to dengue virus infection and death at the cellular level.PLoS One. 2010 Oct 15;5(10):e13398. 3. Eliminate dengue, our challenge An alternate strategy to eliminate dengue fever 2010 http://www.mosquitoage.org/en/HOME.aspx 4. Moreira LA, Saig E, Turley AP, Ribeiro JM, O'Neill SL, McGraw EA. Human probing behavior of Aedes aegypti when infected with a life-shortening strain of Wolbachia. PLoS Negl Trop Dis. 2009 Dec 15;3(12):e568.
Todd Millions Comment left 22nd December 2010 15:03:19
Prof Cummins-Thanx for this and your exellent overveiw of contamination threats to vaccines. On the fail safe tetracycline requirement for the larva-Correct me if I'm wrong but,I seem to remembre that tetracycline was developed from cultures gathered from the mud of stagnant ponds. The story as I remembre reading it was a biologist noticed wounded racoons sniffing the mud and coating open wounds from one patch in particular.He later observed wounded muskcrats,using the same patch.But of course mosquities are never found breeding in stagnant ponds,so there is of course no danger of naturally occuring background levels of tetracycline.
joe cummins Comment left 6th January 2011 11:11:08
Replying o Todd Millions comment on the presence of tetracycline in the soil and surface water that will cause the offspring’s of mating between terminator male mosquitoes and wild females to survive and spread the dengue virus . That is an important point and one that should be investigated before further releases such as those in Malaysia are undertaken. Science literature provides information on the presence of tetracycline in soil and surface water samples. Low levels of the antibiotic have ben observed in soil, surface and ground water in locations with temperate climates but no studies have been reported for tropical or semi tropical climates. Todd made an important observation and one that should be studied.
lye Comment left 3rd January 2011 00:12:53
I'm very worry as the Malaysia government has recently approved the release of Genetic Modified OX513A in 2 city in Malaysia. We're lacking of scientific information on the risk of these release and I highly hope this confirmed release can raise the alert alarm of the international press. Here is the confirmed news about the release: http://www.thesundaily.com/article.cfm?id=46067
joe cummins Comment left 6th January 2011 11:11:58
In reply to lye I agree with you, the planned release of OX513A Aedes aegypti male mosquitoes in Malaysia is a grave concern. A report of Jan.5,2011 indicates that The rainy season in December prompted the postponement of the release of the genetically-modified (GM) Aedes aegypti mosquito in Bentong and Alor Gajah. In spite of the heroic efforts of Lim Li Ching from Third World Network and other NGO members it seem likely that the release will go ahead as soon as the weather permits. Should the release happen it will be wise to ensure that the release is monitored to protect residents and that the any adverse effects of the release should be reported to the public fully and immediately. Efforts to continue to seek the use of safer control measures such as the use of Wolbachia bacteria which have not been genetically modified should continue. Reference: http://thestar.com.my/news/story.asp?file=/2011/1/5/nation/20110105152553&sec=nation
Katheryn Kerr Comment left 7th February 2016 07:07:53
Does anybody have anything to say about the possible connection between the latest outbreak of the Zika virus and the release of the gm mosquitoes by Oxitec? Gotta love this day and age of science knows best, to kill a few (the poor and weak)but know in the end you have done humanity a good sacrifice.
mae-wan ho Comment left 7th February 2016 07:07:43
Hi Katheryn, Oliver Tickell of the Ecologist has made this connection. There is no sequence of piggyback found in the zika virus. maewan
Amber Comment left 11th February 2016 14:02:00
So they haven't linked the piggybac to zika, but what of tTav, has anyone made that connection, also I know they haven't connected all birth defects yet to zika, in fact we don't know yet if all have zika in thier cerebral fluids. I know they are also checking the strain of zika, are they checking it for changes, nothing hopped a ride and hitched itself to zika? How did most of Yap island get zika outbreak a few years ago, and we heard of no such birth defects there?

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