Alarm Spreads in Brazil Over a Virus and a Surge in Malforme

Re: Alarm Spreads in Brazil Over a Virus and a Surge in Malf

Postby admin » Sun Feb 07, 2016 4:54 pm

GMOs, A Global Debate: Brazil, Second Largest GMO Producer in World
By Joana Ferreira
Epoch Times Staff
July 8, 2013

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Reports of neural defects and craniofacial malformations from regions where glyphosate-based herbicides (GBH) are used led us to undertake an embryological approach to explore the effects of low doses of glyphosate in development. Xenopus laevis embryos were incubated with 1/5000 dilutions of a commercial GBH. The treated embryos were highly abnormal with marked alterations in cephalic and neural crest development and shortening of the anterior-posterior (A-P) axis. Alterations on neural crest markers were later correlated with deformities in the cranial cartilages at tadpole stages. Embryos injected with pure glyphosate showed very similar phenotypes. Moreover, GBH produced similar effects in chicken embryos, showing a gradual loss of rhombomere domains, reduction of the optic vesicles, and microcephaly.

-- Glyphosate-Based Herbicides Produce Teratogenic Effects on Vertebrates by Impairing Retinoic Acid Signaling, by Alejandra Paganelli, Victoria Gnazzo, Helena Acosta, Silvia L. Lopez, and Andres E. Carrasco*


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RIO DE JANEIRO—Brazil is the second largest producer of genetically modified organisms (GMOs) in the world, behind only the United States. Brazil has about 37 million hectares (92 million acres) of land planted with GM crops, according to agribusiness consultancy agency Céleres (pdf).

That is more than half of the 67.7 million hectares (167.3 million acres) used for agriculture in Brazil overall,
according to the 2013 estimates of the Brazilian Institute of Geography and Statistics.

The use of transgenic products in Brazil began in the early 1990s, when farmers from the south began to cultivate genetically modified soybeans imported from Argentina. The government began regulating the product commercially in 1995.

The sale of GMOs was banned in 1998 due to a lawsuit by the Brazilian Institute for Consumer Defense. But in 2003, the government again permitted the marketing of GM products with regulations. In the same year, the Brazilian government issued the Labeling Decree (4680/2003), which requires producers and sellers to identify on food packaging products that contain more than 1 percent GM raw materials.

In March 2005, the Bio-Safety Act (11.105/05) went into effect, allowing the use of transgenic organisms without studies on the environmental impact. The act also outlined regulations for biotechnology research and created the organization that oversees this regulation, the Brazilian Technical Committee of National Biosafety (CTNBio).

Brazil has more varieties of GM soybeans than any other crop—a popular crop for export around the world. Céleres reports that 88.8 percent of soybean crops cultivated in Brazil in 2012–2013 were GM.

Is GMO necessary in Brazil?

Francisco Aragão, a researcher at the state-run Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA, Brazilian Enterprise for Agricultural Research), said Brazil needs to use more biotechnology, such as GM, to increase productivity, improve growing conditions, and reduce production costs. EMBRAPA is the research arm of the Brazilian Ministry of Agriculture, Livestock, and Food Supply.

“There are some problems that conventional methods cannot solve,” Aragão said. He noted the importance of agriculture as not only a source of food, but also of biofuel, textiles, and pharmaceuticals.

Sarah Agapito, agronomist and researcher at the Federal University of Santa Catarina, disputes the idea that GMOs increase productivity or reduce the use of pesticides.

She gave the example of Roundup-Ready (RR) soybeans. Roundup is a herbicide created by agriculture giant Monsanto. Its principal ingredient is glyphosate. The RR soybeans are genetically modified to be glyphosate resistant, so farmers can douse the crops with Roundup and only kill the weeds.

“There is already scientific evidence that the production of RR soybeans has contributed to the emergence of weeds resistant to glyphosate,” Agapito said. She said farmers end up with additional costs, needing to apply more Roundup or other herbicides.

Flavio Finardi Filho, president of CTNBio, said the use of GM seeds is essential, because Brazil’s tropical and humid climate makes it especially susceptible to pests. Plants can be genetically modified to make them resistant to pests and climate conditions.

He said in the last 15 years, agriculture has accounted for approximately 15 percent of the Brazilian GDP.

“Undoubtedly, the competitiveness of agribusiness [relies on] adoption of transgenic as an alternative to overcome limitations and to add new features to agricultural production,” Finardi Filho said.

Gabriel Bianconi, a researcher at ASPTA (Advisory Services for Projects in Alternative Agriculture), said researchers are worried GMO production could lead to the further dominance of a few companies in the global seed market, the contamination of non-GM crops, and the increasing difficulty of producing GM-free crops. ASPTA is a nonprofit that supports family agriculture and sustainable rural development.

Bianconi said Brazil is fully capable of returning to complete non-GM production. The smaller scale family farm production model and organic production already account for 70 percent of Brazil’s basic food items, according to the 2010 agricultural census.

Studies in Brazil on the Effects of GMO Food

Brazilian studies on the human health effects of GMOs are few.

Maria Clara Coelho, a doctoral student at the Brazilian National School of Public Health did a study on the studies.

She reviewed the portals of the Scientific Electronic Library Online (SciELO) and of the Coordination of Improvement of Higher Education Personnel (Capes). Of 716 articles on GMOs, only eight explore the issue of food safety.

The study concludes that the eight analyzed studies do not approve genetically modified foods as safe, but rather raise concerns about the safe consumption of such foods.

Coelho asked, “What studies provided the basis for the permission of CTNBio for commercial release of GMOs if in the sample [studies] analyzed by us all claim that such foods are not safe?”

According to a report published by human rights advocacy group Earth of Rights in 2011, the evaluations made by CTNBio to check the safety of GMOs in relation to the risks to the environment and human health are kept confidential.

The Epoch Times called CTNBio twice to request information on the studies it has performed. CTNBio asked the Epoch Times to inquire via email. It did not respond to the Epoch Times email within the three weeks preceding the publication of this article.

Professor Rubens Nodari of the Department of Plant Science and Laboratory of Physiology and Genetics of Development at the Federal University of Santa Catarina, said such information is lacking in the country because: “Obviously the companies want to keep the monopoly. But they can only achieve that when governments are subservient.”

The Epoch Times is exploring the issue of genetic modification, especially as it pertains to food products, with a series titled “GMOs, A Global Debate.” Each article in this series focuses on the role and reception of genetically modified organisms (GMOs) in a different country.
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Re: Alarm Spreads in Brazil Over a Virus and a Surge in Malf

Postby admin » Sun Feb 07, 2016 5:16 pm

Was Zika outbreak caused by release of genetically modified mosquitoes in Brazil? The genetically engineered insects were designed to stop the spread of dengue fever but critics now fear the programme may have had a deadly consequence
BY ELLE GRIFFITHS
31 JAN 2016

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Birth defect: Was the Zika outbreak caused by genetically-modified mosquitoes?
Getty
Dr. Angela Rocha examines Ludmilla Hadassa Dias de Vasconcelos
Birth defect: Was the Zika outbreak caused by genetically-modified mosquitoes?


The Zika virus outbreak currently gripping the Americas could have been sparked by the release of genetically modified mosquitoes in 2012, critics say.

The insects were engineered by biotechnology experts to combat the spread of dengue fever and other diseases and released into the general population of Brazil in 2012.

But with the World Health Organisation (WHO) now meeting in Geneva to desperately discuss cures for the Zika virus, speculation has mounted as to the cause of this sudden outbreak.

The Zika virus was first discovered in the 1950s but the recent outbreak has escalated alarmingly, causing birth defects and a range of health problems in South and central America.

The first cases were reported in Brazil last May with up to 1.5 million people now thought to be affected by the virus which is spread by mosquitoes endemic to Latin America.

FUMIGATIONS AND WARNINGS NOT TO HAVE BABIES UNTIL 2018


The Aedes aegypti mosquito sub-species that carries both the Zika virus and dengue was the type targeted with genetically modified mosquitoes.

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MIRROR MAN VISITS EPICENTRE OUTBREAK AND MEETS CHILD VICTIM


The aim was to release only male Aedes mosquitoes into the wild and they would in turn produce offspring with their virus carrying female counterparts.

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Getty
Aedes Aegypti mosquitos
Carriers: Aedes Aegypti mosquitoes spread the virus


This offspring would then die off before breeding again due to the GM coding in their genes.

But experts expressed concerns about the programme at the time of its inception, arguing that further studies were needed on the potential consequences.

On Saturday, senior health experts shockingly announced that the outbreak could pose a bigger threat to global health than the Ebola epidemic that killed more than 11,000 people in Africa.

WHAT IS THE ZIKA VIRUS?


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Outbreak: Experts have warned the Zika virus poses a greater threat to world health than Ebola

"In many ways the Zika outbreak is worse than the Ebola epidemic of 2014-15," Jeremy Farrar, head of the Wellcome trust told The Guardian.

"Most virus carriers are symptomless.

"It is a silent infection in a group of highly vulnerable individuals – pregnant women – that is associated with a horrible outcome for their babies.”

The UK is unlikely to be affected by the outbreak because the virus carrying mosquitoes could not survive the climate.

But women returning from affected areas are warned to postpone trying for a baby for at least a month in case they have been infected.
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Re: Alarm Spreads in Brazil Over a Virus and a Surge in Malf

Postby admin » Sun Feb 07, 2016 5:36 pm

GMO mosquitoes could be cause of Zika outbreak, critics say
by RT.com
30 Jan, 2016

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The latest contagious virus freaking out the globe, particularly women worried about birth defects, may have been caused by the presence of genetically-modified mosquitoes (GMMs) in Brazil.
With international health experts convening in Geneva to discuss possible cures for the Zika virus, questions are being raised as to whether they are actually to blame.

In mid-2012, British biotech company Oxitec released the super bugs with the aim of reducing the overall mosquito population that spreads dengue fever, the Zika virus, and chikungunya in northeast Brazil.

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RT ✔ @RT_com
#ZikaVirus spreading explosively, 4mn may be infected, no vaccine expected for years - WHO http://on.rt.com/731b
7:50 PM - 28 Jan 2016


At the time, concerns were raised about the release of GMMs without further studies into possible side effects.

"It's a very experimental approach which has not yet been successful and may cause more harm than good," Dr Helen Wallace, director of GeneWatch, told the Guardian in 2012.

The first cases of Zika in humans were reported in the south American country last May with up to 1.5 million now thought people affected by the virus, which Oxitec’s critics note is the same area where the GMMs were released.

Image
The D.C. Clothesline @DCClothesline
Zika Outbreak Epicenter In Same Area Where GM Mosquitoes Were Released In 2015 http://www.dcclothesline.com/?p=56429
10:14 AM - 29 Jan 2016


Since the outbreak, there have been over 4,000 cases of babies born with microcephaly in Brazil, although various others causes can also be attributed to the rise.

The Aedes aegypti mosquito sub-species that carries both the Zika virus and dengue is the very type Oxitec targeted with its GMMs.

k. @kathydiim
"Zika Outbreak Epicenter in Same Area Where GM Mosquitoes Were Released in 2015" What a coincidence
5:24 AM - 30 Jan 2016


The aim of Oxitec's GM program was to release only male Aedes mosquitoes into the wild and they would in turn produce offspring with their virus carrying female counterparts.

This offspring would then die off before breeding age due to the GM coding in their genes, as long as the antibiotic tetracycline wasn't present, which would override the GM DNA.

Sounds like the plot of a Hollywood blockbuster.



There is already a known survival rate of up to five percent in these GMMs regardless of the presence of tetracycline.

This antibiotic, which is often used to treat teenage acne, can be found in nature too, showing up in soil, surface water, and food, with some research stating that the GMM survival rate could potentially increase to 15 percent.


Oxitec critics also suggest that in the absence of studies into the potential knock-on effects of this in these mutated mosquitoes, it’s possible they could in fact thrive in the wild with unknown mutations taking place in GMMs, which in turn could worsen the spread the Zika virus.

“Mosquitoes are food for lots of animals. We would still want to see studies of when birds and bats and amphibians eat these genetically modified animals,” Jaydee Hanson, a senior policy analyst at the US-based Center for Food Safety, told Bloomberg News. “They’re introducing into the ecosystem some genetic constructs that have never been there before.”

Hanson also raised the possibility that other mosquito species could still carry the Zika virus. “It doesn’t solve the problem.”


Calls for further studies into GMMs prior to their release by Oxitec to answer such questions went unanswered, however.

While this Jurassic Park-type scenario could have been accidental, the recent UK TV series "Utopia" suggests it could be a deliberate plan to reduce the global population.

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Re: Alarm Spreads in Brazil Over a Virus and a Surge in Malf

Postby admin » Sun Feb 07, 2016 5:49 pm

No, GM Mosquitoes Didn’t Start The Zika Outbreak.
By Christie Wilcox
Discover Magazine
January 31, 2016

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A new ridiculous rumor is spreading around the internets. According to conspiracy theorists, the recent outbreak of Zika can be blamed on the British biotech company Oxitec, which some are saying even intentionally caused the disease as a form of ethnic cleansing or population control. The articles all cite a lone Redditor who proposed the connection on January 25th to the Conspiracy subreddit. “There are no biological free lunches,” says one commenter on the idea. “Releasing genetically altered species into the environment could have disastrous consequences” another added. “Maybe that’s what some entities want to happen…?”

For some reason, it’s been one of those months where random nonsense suddenly hits mainstream. Here are the facts: there’s no evidence whatsoever to support this conspiracy theory, or any of the other bizarre, anti-science claims that have popped up in the past few weeks. So let’s stop all of this right here, right now: The Earth is round, not flat (and it’s definitely not hollow). Last year was the hottest year on record, and climate change is really happening (so please just stop, Mr. Cruz). And FFS, genetically modified mosquitoes didn’t start the Zika outbreak.

Background on Zika

The Zika virus is a flavivirus closely related to notorious pathogens including dengue, yellow fever, Japanese encephalitis, and West Nile virus. The virus is transmitted by mosquitoes in the genus Aedes, especially A. aegypti, which is a known vector for many of Zika’s relatives. Symptoms of the infection appear three to twelve days post bite. Most people are asymptomatic, which means they show no signs of infection. The vast majority of those who do show signs of infection report fever, rash, joint pain, and conjunctivitis (red eyes), according to the U.S. Centers for Disease Control. After a week or less, the symptoms tend to go away on their own. Serious complications have occurred, but they have been extremely rare.

The Zika virus isn’t new. It was first isolated in 1947 from a Rhesus monkey in the Zika Forest in Uganda, hence the pathogen’s name. The first human cases were confirmed in Uganda and Tanzania in 1952, and by 1968, the virus had spread to Nigeria. But since then, the virus has found its way out of Africa. The first major outbreak occurred on the island of Yap in Micronesia for 13 weeks 2007, during which 185 Zika cases were suspected (49 of those were confirmed, with another 59 considered probable). Then, in October 2013, an outbreak began in French Polynesia; around 10,000 cases were reported, less than 100 of which presented with severe neurological or autoimmune complications. One confirmed case of autochthonous transmission occurred in Chile in 2014, which means a person was infected while they were in Chile rather than somewhere else. Cases were also reported that year from several Pacific Islands. The virus was detected in Chile until June 2014, but then it seemed to disappear.

Fast forward to May 2015, when the Pan American Health Organization (PAHO) issued an alert regarding the first confirmed Zika virus infection in Brazil. Since then, several thousand suspected cases of the disease and a previously unknown complication—a kind of birth defect known as microcephaly where the baby’s brain is abnormally small—have been reported from Brazil. (It’s important to note that while the connection between the virus and microcephaly is strongly suspected, the link has yet to be conclusively demonstrated.)

Currently, there is no vaccine for Zika, though the recent rise in cases has spurred research efforts. Thus, preventing mosquito bites is the only prophylactic measure available.

The recent spread of the virus has been described as “explosive”; Zika has now been detected in 25 countries and territories. The rising concern over both the number of cases and reports of serious complications has led the most affected areas in Brazil to declare a state of emergency, and on Monday, The World Health Organization’s Director-General will convene an International Health Regulations Emergency Committee on Zika virus and the observed increase in neurological disorders and neonatal malformations. At this emergency meeting, the committee will discuss mitigation strategies and decide whether the organization will officially declare the virus a “Public Health Emergency of International Concern.”

GM to the Rescue

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Aedes aegypti: the invasive mosquito in Brazil that carries Zika virus and other awful diseases.
Photo by James Gathany, c/o the CDC


The mosquito to blame for the outbreak—Aedes aegypti—doesn’t belong in the Americas. It’s native to Africa, and was only introduced in the new world when Europeans began to explore the globe. In the 20th century, mosquito control programs nearly eradicated the unwelcome menace from the Americas (largely thanks to the use of the controversial pesticide DDT); as late as the mid 1970s, Brazil and 15 other nations were Aedes aegypti-free. But despite the successes, eradication efforts were halted, allowing the mosquito to regain its lost territory.

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The distribution of Aedes aegypti in the Americas in 1970 and 2002, from the Centers for Disease Control

Effective control measures are expensive and difficult to maintain, so at the tail end of the 20th century and into the 21st, scientists began to explore creative means of controlling mosquito populations, including the use of genetic modification. Oxitec’s mosquitoes are one of the most exciting technologies to have emerged from this period. Here’s how they work, as I described in a post almost exactly a year ago:

While these mosquitoes are genetically modified, they aren’t “cross-bred with the herpes simplex virus and E. coli bacteria” (that would be an interkingdom ménage à trois!)—and no, they cannot be “used to bite people and essentially make them immune to dengue fever and chikungunya” (they aren’t carrying a vaccine!). The mosquitoes that Oxitec have designed are what scientists call “autocidal” or possess a “dominant lethal genetic system,” which is mostly fancy wording for “they die all by themselves”. The males carry inserted DNA which causes the mosquitoes to depend upon a dietary supplement that is easy to provide in the lab, but not available in nature. When the so-called mutants breed with normal females, all of the offspring require the missing dietary supplement because the suicide genes passed on from the males are genetically dominant. Thus, the offspring die before they can become adults. The idea is, if you release enough such males in an area, then the females won’t have a choice but to mate with them. That will mean there will be few to no successful offspring in the next generation, and the population is effectively controlled.


Male mosquitoes don’t bite people, so they cannot serve as transmission vectors for Zika or any other disease. As for fears that GM females will take over: less than 5% of all offspring survive in the laboratory without tetracycline, and as Glen Slade, director of Oxitec’s Brazilian branch notes, those are the best possible conditions for survival. “It is considered unlikely that the survival rate is anywhere near that high in the harsher field conditions since offspring reaching adulthood will have been weakened by the self-limiting gene,” he told me. And contrary to what the conspiracy theorists claim, scientists have shown that tetracycline in the environment doesn’t increase that survival rate. The proponents of this conspiracy theory say that an internal Oxitec memo showed they could survive as much as 15% if fed cat food containing tetracycline, as if it was some secret information being covered up by the company. But that number was reported in their paper on the mosquitoes in 2013, while it was also noted that such a level in was unlikely outside of the lab. Oxitec further went out and tested to see if the environmental levels in Brazil were high enough to raise survival rates, and they weren’t. As Simon Warner, Chief Scientific Officer for Oxitec, explains:

Researchers from the University of Campinas, Brazil, Imperial College London and outside CROs worked together with Oxitec to assess whether exposure to tetracycline in the environment could affect survival levels of OX513A. The highest levels of tetracycline found in mosquito breeding sites or nearby locations were below the minimal level of tetracycline needed to produce any change in survival rates.

What if a bowl of pet food and water was available for OX513A in Brazil? Firstly the bowl would need to be left uninterrupted for a couple of weeks for the OX513A to develop. Most pet owners clean their pet’s food more frequently and health officials advise that no standing containers of water be left for Aedes aegypti to breed in. If the OX513A did develop then as soon as they flew away from the dog bowl they would no longer have access to tetracycline and the self-limiting gene would become active and they would die. It would be impossible for them to continue to breed and become self-sustaining without access to doses of tetracycline.


Brazil, a hotspot for dengue and other such diseases, is one of the countries where Oxitec is testing their mozzies—so far, everywhere that Oxitec’s mosquitoes have been released, the local populations have been suppressed by about 90%.

Genetics Schmenetics (added Feb 2-4)

Oliver Tickell, journalist and editor of the Ecologist, came out with a point by point “hypothesis” seeking to legitimize the conspiracy theory after my initial post. It hinges on his claim that “The promiscuous piggyBac transposon now present in the local Aedes aegypti population takes the opportunity to jump into the Zika virus, probably on numerous occasions.” But we know for a fact this isn’t the case.

First off, it’s not possible. Tickell attempts to make a convoluted connection between the gene insertion system used to add the genetic modification—PiggyBac—and the an increase in virulence in Zika. He cites a dodgy anti-GM website (calling it a “review article”, when it is not a scientific journal article of any kind and has not passed peer review) which claims that PiggyBac moves around all the time. But for the risk assessment to get the permits and approval of the local Brazilian government, Oxitec had to demonstrate that their insertion doesn’t move. They had to demonstrate that the insertion is stable and follows “Mendelian inheritance” (which means it stays in the same place on the chromosome). And they did, to the satisfaction of the regulators—you can read the risk assessment yourself. As Simon Warner, Chief Scientific Officer for Oxitec, explains:

The OX513A mosquito has now been through more than 100 generations and there has never been an instance where the self-limiting gene has behaved as if it were a jumping gene. In human terms, 100 generations is equivalent to a period of time from the early AD years to the present day. Additionally, the self-limiting gene confers a strong selective disadvantage to any insect that carries it, so in the impossible event that the gene did move, there would be no selection that would mean that gene could persist and spread in any population.


But most importantly, it’s not possible for a PiggyBac transposon to move into the Zika genome because PiggyBac is a double-stranded DNA element which only inserts into double-stranded DNA at specific sites (TTAA elements). Zika has no DNA. It’s a single strand RNA virus roughly 10.8kb in size which never goes through a DNA phase when replicating, nor does it enter the cellular nucleus where the mosquito genome is located.

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How flaviviruses like Zika replicate, courtesy of the National Institutes of Health’s National Institute of Allergy and Infectious Diseases

Virologist Kenneth Stedman, who recently published a review on the interaction between viral genes and genomes, explained:

There are a quite small, but not insignificant, number of examples of partial genomes of RNA and single-stranded DNA viruses that have been incorporated into cellular DNA genomes and even fewer examples of a gene from a purely RNA virus which appears to have been acquired by an ssDNA virus (which we originally discovered, see Diemer and Stedman, 2012). However, there are NO examples of the inverse, that is to say purely RNA viruses or ssDNA viruses picking up genes from host genomes. This is not from want of trying, there have been extensive sequencing efforts on purely RNA viruses, including Zika, and none of these viruses have been found to contain cellular genes.

There are 2 reasons for the unidirectionality, purely RNA viruses, such as the flaviviruses, of which Zika is a member, have RNA genomes that make copies of their genome from RNA, there is no DNA intermediate and they use a special virus-specific RNA polymerase to do this. The second is that most ssRNA viruses (and flaviviruses in particular) are very constrained in terms of how much RNA they can hold, so they are extremely unlikely to pick up any “extra” genes without causing the virus to be non-viable. On the other hand, cellular genomes are not under such constraints and can acquire RNA and ssDNA virus genes by fascinating mechanisms (some of which are outlined in my Annual Reviews of Virology paper).


To Stedman’s last point: the transposon used by Oxitec is 8.4kb – so almost the same size as the entire Zika virus genome!

But perhaps most to the point, mosquito genes, from genetic modification or otherwise, are not present in the Zika virus in Brazil. The whole genome of the Zika virus is tiny, and it’s easily sequenced—which is exactly what scientists in Brazil have done. That means there was no “jumping DNA” responsible, period. Given the importance of this outbreak, scientists published their sequencing results as openly and as quickly as possible. I’ll say it again: They did not find any transposons or mosquito genes of any kind. They did, however, find some interesting mutational changes which may explain why the outbreak in Brazil seems to be worse than previous outbreaks; the mutational changes may have led to an increase in viral titers.

The family of viruses that Zika belongs to are known to cause birth defects in rare cases, including microcephaly, so an increase in viral titers could be sufficient to explain the sudden uptick in cases, especially when you consider the other confounding factors—factors which, as Oliviera Melo and his colleagues explain, include an increase in reporting (the more we look for a disease, the more we tend to find cases of it), a lack of childhood exposure to the virus in the outbreak areas (if you are infected by Zika as a kid, your body has some resistance or even immunity, so even if someone is exposed when pregnant, they don’t have the same complications), or the rarity of the disease until now (it takes a very large number of cases to detect an increase in very rare complications).

Wrong Place, Wrong Time

Now that we’ve covered that, let’s dig into the conspiracy theory as it was originally posted. We’ll start with the main argument laid out as evidence: that the Zika outbreak began in the same location at the same time as the first Oxitec release:

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The D.C. Clothesline @DCClothesline
Zika Outbreak Epicenter In Same Area Where GM Mosquitoes Were Released In 2015 http://www.dcclothesline.com/?p=56429
10:14 AM - 29 Jan 2016


Though it’s often said, it’s worth repeating: correlation doesn’t equal causation. If it did, then Nicholas Cage is to blame for people drowning (Why, Nick? WHY?). But even beyond that, there are bigger problems with this supposed correlation: even by those maps, the site of release is on the fringe of the Zika hotspot, not the center of it. Just look at the two overlaid:

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The epicenter of the Zika outbreak is clearly on the coast, hundreds of kilometers from the noted location.

The epicenter of the outbreak and the release clearly don’t line up—the epicenter is on the coast rather than inland where the map points. Epidemiologists have tracked the outbreak back to where it started, and now say that the Zika outbreak “almost certainly” began in Recife, Brazil, a city almost 400 miles from the nearest Oxitec release location. There are no early cases, however few, from 400 miles inland; the viral outbreak started on the coast and then, as the disease spread, moved inland. If the Oxitec conspiracy theory is correct: how did a virus mutated in Juazeiro get 400 miles away before causing any microcephaly cases?

I see a lot of people gloss over this, saying “well, what if the person infected by the mutant virus immediately drove to Recife” or hypotheses along those lines. Let’s take a moment to think about how unlikely a scenario that is. The theory argues that there was a group of people in Juazeiro with Zika virus and that all of them had no signs whatsoever, because if they had gone to the doctor and had signs, those cases would be reported and we would know that Zika was there. Then one of those people had to be bitten by GM mosquitoes, even though the vast majority of female mosquitoes would be non-GM, while their viremia (the number of viruses in their blood stream) was high enough (but yet not high enough to cause symptoms). Then the virus has to mutate in that mosquito (see above on why that didn’t happen), and then that mosquito has to bite another person and infect them. And THEN that person has to high tail it out of Juazeiro 400 miles to the coast before coming down with the illness, meanwhile, the virus in the Juazeiro population stays hidden with no one getting symptoms until after the outbreak everywhere else spreads. Does that sound like a plausible scenario?

To claim that anything done in Juazeiro caused an outbreak to occur 400 miles away is the same as claiming that whatever is done in Phoenix, AZ is directly responsible for disease outbreaks in Los Angeles, CA. In fact, those two cities are nearly 50 miles closer together than Juazeiro, BA and Recife, PE:

Dr. Christie Wilcox @NerdyChristie
Epidemiologists say #Zika outbreak "almost certainly" began in Recife, almost 396 miles from Juazeiro, the site of the #Oxitec releases.

Dr. Christie Wilcox @NerdyChristie
If Recife and Juazeiro are "the same area"... Juazeiro-Recife: 395.9 mi. LA-Phoenix: 357.2 mi. Boston-DC: 394.2 mi pic.twitter.com/MtEJGVa2bw
2:34 AM - 2 Feb 2016

Image


That’s not even mentioning that the location on the map isn’t where the mosquitoes were released. That map points to Juazeiro de Norte, Ceará, which is a solid 300 km away from Juazeiro, Bahia—the actual site of the mosquito trial. That location is even more on the edge of the Zika-affected area:

Image
1: Juazeiro de Norte, the identified location in by conspiracy theorists. 2: Juazeiro, the actual location of Oxitec’s release trial, about 300 km away and even further from the outbreak epicenter

The mistake was made initially by the Redditor who proposed the conspiracy theory and has been propagated through lazy journalistic practices by every proponent since. Here’s a quick tip: if you’re basing your conspiracy theory on location coincidence, it’s probably a good idea to actually get the location right.

They’re also wrong about the date. According to the D.C. Clothesline:

By July 2015, shortly after the GM mosquitoes were first released into the wild in Juazeiro, Brazil, Oxitec proudly announced they had “successfully controlled the Aedes aegypti mosquito that spreads dengue fever, chikungunya and zika virus, by reducing the target population by more than 90%.”


However, GM mosquitoes weren’t first released in Juazeiro, Bahia (let alone Juazeiro de Norte, Ceará) in 2015. Instead, the announcement by Oxitec was of the published results of a trial that occurred in Juazeiro between May 2011 and Sept 2012—a fact which is clearly stated in the methods and results of the paper that Oxitec was so excited to share.

A new control effort employing Oxitec mosquitoes did begin in April 2015, but not in Juazeiro, or any of the northeastern states of Brazil where the disease outbreak is occurring. As another press release from Oxitec states, the 2015 releases of their GM mosquitoes were in Piracicaba, São Paulo, Brazil:

Following approval by Brazil’s National Biosafety Committee (CTNBio) for releases throughout the country, Piracicaba’s CECAP/Eldorado district became the world’s first municipality to partner directly with Oxitec and in April 2015 started releasing its self-limiting mosquitoes whose offspring do not survive. By the end of the calendar year, results had already indicated a reduction in wild mosquito larvae by 82%. Oxitec’s efficacy trials across Brazil, Panama and the Cayman Islands all resulted in a greater than 90% suppression of the wild Ae. aegypti mosquito population–an unprecedented level of control.

Based on the positive results achieved to date, the ‘Friendly Aedes aegypti Project’ in CECAP/Eldorado district covering 5,000 people has been extended for another year. Additionally, Oxitec and Piracicaba have signed a letter of intent to expand the project to an area of 35,000-60,000 residents. This geographic region includes the city’s center and was chosen due to the large flow of people commuting between it and surrounding neighborhoods which may contribute to the spread of infestations and infections.

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Piracicaba mosquito control results


Piracicaba, for the record, is more than 1300 miles away from the Zika epicenter:

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1: Juazeiro de Norte, the identified location in by conspiracy theorists. 2: Juazeiro, the actual location of Oxitec’s 2011-2012 trial, and 3: Piracicaba, the location where mosquitoes began to be released starting in April 2015, more than 2,000 km from the disease epicenter.

So not only did the conspiracy theorists get the location of the first Brazil release wrong, they either got the date wrong, too, or got the location of the 2015 releases really, really off. Either way, the central argument that the release of GM mosquitoes by Oxitec coincides with the first cases of Zika virus simply doesn’t hold up.

And, if the nails aren’t already in the coffin, then there’s this: when Zika hit French Polynesia in 2014, they also saw a trend of increasing microcephaly. There are no GM mosquitoes in French Polynesia, then or now: so how did they end up with the supposed “mutant” virus that caused birth defects?

And just for those who aren’t convinced Zika has anything to do with this: if the virus isn’t involved, and the mosquitoes are “mutating” people directly (also not possible, as I explained in my post last year on why they are not damaging), then why are there more cases from areas where there aren’t GM mosquitoes than areas where they were released? And where are there microcephaly cases in the Cayman Islands, or other places where these mosquitoes have been tested?

Scientists Speak Out

As this ludicrous conspiracy theory has spread, so, too, has the scientific opposition to it. “Frankly, I’m a little sick of this kind of anti-science platform,” said vector ecologist Tanjim Hossain from the University of Miami, when I asked him what he thought. “This kind of fear mongering is not only irresponsible, but may very well be downright harmful to vulnerable populations from a global health perspective.”

Despite the specious allusions made by proponents of the conspiracy, this is still not Jurassic Park, says Hossain.

“We have a problem where ZIKV is spreading rapidly and is widely suspected of causing serious health issues,” he continued. “How do we solve this problem? An Integrated Vector Management (IVM) approach is key. We need to use all available tools, old and new, to combat the problem. GM mosquitoes are a fairly new tool in our arsenal. The way I see it, they have the potential to quickly reduce a local population of vector mosquitoes to near zero, and thereby can also reduce the risk of disease transmission. This kind of strategy could be particularly useful in a disease outbreak ‘hotspot’ because you could hypothetically stop the disease in its tracks so to speak.”

Other scientists have shared similar sentiments. Alex Perkins, a biological science professor at Notre Dame, told Business Insider that rather than causing the outbreak, GM mosquitoes might be our best chance to fight it. “It could very well be the case that genetically modified mosquitos could end up being one of the most important tools that we have to combat Zika,” Perkins said. “If anything, we should potentially be looking into using these more.”

Brazilian authorities couldn’t be happier with the results so far, and are eager to continue to fight these deadly mosquitoes by any means they can. “The initial project in CECAP/Eldorado district clearly showed that the ‘friendly Aedes aegypti solution’ made a big difference for the inhabitants of the area, helping to protect them from the mosquito that transmits dengue, Zika and chikungunya,” said Pedro Mello, secretary of health in Piracicaba. He notes that during the 2014/2015 dengue season, before the trial there began, there were 133 cases of dengue. “In 2015/2016, after the beginning of the Friendly Aedes aegypti Project, we had only one case.”

It’s long past time to stop villainizing Oxitec’s mosquitoes for crimes they didn’t commit. Claire Bernish, The Daily MFail, Mirror and everyone else who has spread these baseless accusations: I’m talking to you. The original post was in the Conspiracy subreddit—what more of a red flag for “this is wildly inaccurate bullsh*t” do you need? (After all, if this is a legit source, where are your reports on the new hidden messages in the $100 bill? or why the Illuminati wants people to believe in aliens?). It’s well known that large-scale conspiracy theories are mathematically challenged. Don’t just post whatever crap is spewed on the internet because you know it’ll get you a few clicks. It’s dishonest, dangerous, and, frankly, deplorable to treat nonsense as possible truth just to prey upon your audience’s very real fears of an emerging disease. You, with your complete lack of integrity, are maggots feeding on the decay of modern journalism, and I mean that with no disrespect to maggots.
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Re: Alarm Spreads in Brazil Over a Virus and a Surge in Malf

Postby admin » Sun Feb 07, 2016 6:02 pm

Pandora's box: how GM mosquitos could have caused Brazil's microcephaly disaster
Oliver Tickell
1st February 2016

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Aedes Aegypti mosquito feeding on human blood. This is the species that transmits Zika, and that was genetically engineered by Oxitec using the piggyBac transposon. Photo: James Gathany via jentavery on Flickr (CC BY).

In Brazil's microcephaly epidemic, one vital question remains unanswered: how did the Zika virus suddenly learn how to disrupt the development of human embryos? The answer may lie in a sequence of 'jumping DNA' used to engineer the virus's mosquito vector -- and released into the wild four years ago in the precise area of Brazil where the microcephaly crisis is most acute.

These 'promiscuous' transposons have found special favour with genetic engineers, whose goal is to create 'universal' systems for transferring genes into any and every species on earth. Almost none of the geneticists has considered the hazards involved.


Since August 2015, a large number of babies in Northeast Brazil have been born with very small heads, a condition known as microcephaly, and with other serious malformations. 4,180 suspected cases have been reported.

Epidemiologists have found a convincing correlation between the incidence of the natal deformities and maternal infections with the Zika virus, first discovered in Uganda's Zika Valley in 1947, which normally produces non-serious illness.

The correlation has been evidenced through the geographical distrubution of Zika infections and the wave of deformities. Zika virus has also been detected in the amniotic fluids and other tissues of the affected babies and their mothers.


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Map showing the concentration of suspected Zika-related cases of microcephaly in Brazil. Image: Claire Bernish / AntiMedia.

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Map showing the location of Juazeiro, Bahia, Brazil. Image: thanks to John Fedor-Cunningham.

This latter finding was recently reported by AS Oliveira Melo et al in a scientific paper published in the journal Ultrasound in Obstetrics & Gynecology, which noted evidence of intra-uterine infection. They also warn:

"As with other intrauterine infections, it is possible that the reported cases of microcephaly represent only the more severely affected children and that newborns with less severe disease, affecting not only the brain but also other organs, have not yet been diagnosed."

The Brazilian Health Minister, Marcelo Castro, says he has "100% certainty" that there is a link between Zika and microcephaly. His view is supported by the medical community worldwide, including by the US Center for Disease Control.

Oliveira Melo et al draw attention to a mystery that lies at the heart of the affair: "It is difficult to explain why there have been no fetal cases of Zika virus infection reported until now but this may be due to the underreporting of cases, possible early acquisition of immunity in endemic areas or due to the rarity of the disease until now.

"As genomic changes in the virus have been reported, the possibility of a new, more virulent, strain needs to be considered. Until more cases are diagnosed and histopathological proof is obtained, the possibility of other etiologies cannot be ruled out."

And this is the key question: how -- if indeed Zika really is the problem, as appears likely -- did this relatively innocuous virus acquire the ability to produce these terrible malformations in unborn human babies?

Oxitec's GM mosquitoes

An excellent article by Claire Bernish published last week on AntiMedia draws attention to an interesting aspect of the matter which has escaped mainstream media attention: the correlation between the incidence of Zika and the area of release of genetically modified Aedes aegypti mosquitos engineered for male insterility (see maps, above right).

The purpose of the release was to see if it controlled population of the mosquitos, which are the vector of Dengue fever, a potentially lethal disease. The same species also transmits the Zika virus.

The releases took [place] in 2011 and 2012 in the Itaberaba suburb of the city of Juazeiro, Bahia, Northeast Brazil, about 500 km west of the coastal city of Recife. The experiment was written up in July 2015 in the journal PLOS Neglected Tropical Diseases in a paper titled 'Suppression of a Field Population of Aedes aegypti in Brazil by Sustained Release of Transgenic Male Mosquitoes' by Danilo O. Carvalho et al.

An initial 'rangefinder of 30,000 GM mosquitos per week took place between 19th May and 29th June 2011, followed by a much larger release of 540,000 per week in early 2012, ending on 11th February.

At the end of it the scientists claimed "effective control of a wild population of Ae. aegypti by sustained releases of OX513A male Ae. aegypti. We diminished Ae. aegypti population by 95% (95% CI: 92.2%-97.5%) based on adult trap data and 78% (95% CI: 70.5%-84.8%) based on ovitrap indices compared to the adjacent no-release control area."

So what's to worry about?

The idea of the Oxitec mosquitoes is simple enough: the males produce non-viable offspring which all die. So the GM mosqitoes are 'self-extinguishing' and the altered genes cannot survive in the wild population. All very clever, and nothing to worry about!

But in fact, it's not so simple. In 2010 geneticist Ricarda Steinbrecher wrote to the biosafety regulator in Malaysia -- also considering a release of the Oxitec mosquitoes -- with a number of safety concerns, pointing out the 2007 finding by Phuc et al that 3-4% of the first generation mosquitos actually survive.

The genetic engineering method employed by Oxitec allows the popular antibiotic tetracycline to be used to repress the lethality during breeding. But as a side-effect, the lethality is also reduced by the presence of tetracycline in the environment; and as Bernish points out, Brazil is among the world's biggest users of anti-microbials including tetracycline in its commercial farming sector:

"As a study by the American Society of Agronomy, et. al., explained, 'It is estimated that approximately 75% of antibiotics are not absorbed by animals and are excreted in waste.' One of the antibiotics (or antimicrobials) specifically named in that report for its environmental persistence is tetracycline.

In fact, as a confidential internal Oxitec document divulged in 2012, that survival rate could be as high as 15% -- even with low levels of tetracycline present. 'Even small amounts of tetracycline can repress' the engineered lethality. Indeed, that 15% survival rate was described by Oxitec."

She then quotes the leaked Oxitec paper: "After a lot of testing and comparing experimental design, it was found that [researchers] had used a cat food to feed the [OX513A] larvae and this cat food contained chicken. It is known that tetracycline is routinely used to prevent infections in chickens, especially in the cheap, mass produced, chicken used for animal food. The chicken is heat-treated before being used, but this does not remove all the tetracycline. This meant that a small amount of tetracycline was being added from the food to the larvae and repressing the [designed] lethal system."

So in other words, there is every possibility for Oxitec's modified genes to persist in wild populations of Aedes aegypti mosquitos, especially in the environmental presence of tetracycline which is widely present in sewage, septic tanks, contaminated water sources and farm runoff.

'Promiscuous' jumping genes

On the face of it, there is no obvious way in which the spread of Oxitec's GM mosquitos into the wild could have anything to do with Brazil's wave of microcephaly. Is there?

Actually, yes. The problem may arise from the use of the 'transposon' ('jumping' sequence of DNA used in the genetic engineering process to introduce the new genes into the target organism). There are several such DNA sequences in use, and one of the most popular is known as known as piggyBac.

As a 2001 review article by Dr Mae Wan Ho shows, piggyBac is notoriously active, inserting itself into genes way beyond its intended target: "These 'promiscuous' transposons have found special favour with genetic engineers, whose goal is to create 'universal' systems for transferring genes into any and every species on earth. Almost none of the geneticists has considered the hazards involved ...

"It would seem obvious that integrated transposon vectors may easily jump out again, to another site in the same genome, or to the genome of unrelated species. There are already signs of that in the transposon, piggyBac, used in the GM bollworms to be released by the USDA this summer.

The piggyBac transposon was discovered in cell cultures of the moth Trichopulsia, the cabbage looper, where it caused high rates of mutations in the baculovirus infecting the cells by jumping into its genes ... This transposon was later found to be active in a wide range of species, including the fruitfly Drosophila, the mosquito transmitting yellow fever, Aedes aegypti, the medfly, Ceratitis capitata, and the original host, the cabbage looper.

"The piggyBac vector gave high frequencies of transpositions, 37 times higher than mariner and nearly four times higher than Hirmar."

In a later 2014 report Dr Mae Wan Ho returned to the theme with additional detail and fresh scientific evidence (please refer to her original article for references): "The piggyBac transposon was discovered in cell cultures of the moth Trichopulsia, the cabbage looper, where it caused high rates of mutations in the baculovirus infecting the cells by jumping into its genes ...

"There is also evidence that the disabled piggyBac vector carrying the transgene, even when stripped down to the bare minimum of the border repeats, was nevertheless able to replicate and spread, because the transposase enzyme enabling the piggyBac inserts to move can be provided by transposons present in all genomes.

"The main reason initially for using transposons as vectors in insect control was precisely because they can spread the transgenes rapidly by 'non-Mendelian' means within a population, i.e., by replicating copies and jumping into genomes, thereby 'driving' the trait through the insect population. However, the scientists involved neglected the fact that the transposons could also jump into the genomes of the mammalian hosts including human beings ...

"In spite of instability and resulting genotoxicity, the piggyBac transposon has been used extensively also in human gene therapy. Several human cell lines have been transformed, even primary human T cells using piggyBac. These findings leave us little doubt that the transposon-borne transgenes in the transgenic mosquito can transfer horizontally to human cells. The piggyBac transposon was found to induce genome wide insertion mutations disrupting many gene functions."


Has the GM nightmare finally come true?

So down to the key question: was the Oxitec's GM Aedes aegypti male-sterile mosquito released in Juazeiro engineered with the piggyBac transposon? Yes, it was. And that creates a highly significant possibility: that Oxitec's release of its GM mosquitos led directly to the development of Brazil's microcephaly epidemic through the following mechanism:

1. Many of the millions of Oxitec GM mosquitos released in Juazeiro in 2011/2012 survive, assisted, but not dependent on, the presence of tetracycline in the environment.

2. These mosquitos interbreed with with the wild population and their novel genes become widespread.

3. The promiscuous piggyBac transposon now present in the local Aedes aegypti population takes the opportunity to jump into the Zika virus, probably on numerous occasions.

4. In the process certain mutated strains of Zika acquire a selective advantage, making them more virulent and giving them an enhanced ability to enter and disrupt human DNA.

5. One way in which this manifests is by disrupting a key stage in the development of human embryos in the womb, causing microcephaly and the other reported deformations.
Note that as Melo Oliveira et al warn, there are almost certainly other manifestations that have not yet been detected.

6. It may be that the piggyBac transposon has itself entered the DNA of babies exposed in utero to the modified Zika virus. Indeed, this may form part of the mechanism by which embryonic development is disrupted.

In the latter case, one implication is that the action of the gene could be blocked by giving pregnant women tetracycline in order to block its activity. The chances of success are probably low, but it has to be worth trying.

No further releases of GM insects!

While I am certainly not claiming that this is what actually took place, it is at least a credible hypothesis, and moreover a highly testable one. Nothing would be easier for genetic engineers than to test amniotic fluids, babies' blood, wild Aedes mosquitos and the Zika virus itself for the presence of the piggyBac transposon, using well established and highly sensitive PCR (polymerase chain reaction) techniques.

[See Author's notes 1 & 2, below. Recent Zika genome snapshots examined as a result of this article are, in fact, free of the piggyBac transposon.]

If this proves to be the case, those urging caution on the release of GMOs generally, and transgenic insects bearing promiscuous transposons in particular, will have been proved right on all counts.

But most important, such experiments, and any deployment of similar GM insects, must be immediately halted until the possibilities outlined above can be safely ruled out. There are plans, for example, to release similarly modified Anopheles mosquitos as an anti-malarial measure.

There are also calls for even more of the Oxitec Aedes aegypti mosquitos to be released in order to halt the transmission of the Zika virus. If that were to take place, it could give rise to numerous new mutations of the virus with the potential to cause even more damage to the human genome, that we can, at this stage, only guess at.

Author's note 1: A reader of this article, David Murphy, reports on Facebook (see thread here) as follows:

Zika, 4 strains, 2016 from the Center for Technological Innovation, Brazil

http://www.ncbi.nlm.nih.gov/nuccore/KU365777.1
http://www.ncbi.nlm.nih.gov/nuccore/KU365778.1
http://www.ncbi.nlm.nih.gov/nuccore/KU365779.1
http://www.ncbi.nlm.nih.gov/nuccore/KU365780.1

The releases of GM mosquitos took place in 2011 and 2012. Fortunately people sequenced zika before that.

Zika 01-AUG-2006

http://www.ncbi.nlm.nih.gov/nuccore/GU937109.1
id:AY632535.2

So lets compare. This isn't anything amazing, the viruses are tiny, this is rare in biology but we can actually eyeball the data since you could fit a whole viral genome on a single A4 sheet of paper.

View the alignment online:
http://web.archive.org/web/201602041137 ... 5505577-pg

http://toolkit.tuebingen.mpg.de/alnviz/results/8144137

If you take a peek at the last 2 links you can see that while they're not identical the differences are almost all small changes with no big new chunks of code added.

From a quick scan through eyeballing it, the only chunk of new bases was a 15 base sequence that doesn't show up in any piggyBac.

Zika is small, only 10000 bases long and it's hard to hide anything big in it.

piggyBac is not tiny, ( http://www.ncbi.nlm.nih.gov/nuccore/?term=piggybac ), you wouldn't have to do any amazing analysis to see that it's not been added in there. Zika virus strain BeH818995 polyprotein gene, complete cds - Nucleotide - NCBI National Center for Biotechnology Information, U.S. National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USANCBI.NLM.NIH.GOV

Author's note 2: I'm grateful to David Murphy for carrying out this work, and to James Babcock for drawing it to my attention. It appears that the hypothesis set out above is probably incorrect, and this must be a matter of considerable relief to all concerned. However it remains my opinion that considerable caution should be exercised with releases of GE insects containing 'promiscuous' DNA sequences such as piggyBac.

The Zika virus, as a pure RNA virus, is actually highly resistant to the uptake of DNA of any kind, and being very small, any such uptake would be rapidly detectable. However there are many other viruses, bacteria, disease vectors, etc, for which this remains a possiblity.

Another check that should also be made is to test for the presence of piggyBac in wild Aedes mosquitos around the release sites to see if, in fact, these 'programmed to die out' sequences are indeed as evanescent as claimed.

Oliver Tickell edits The Ecologist.
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Re: Alarm Spreads in Brazil Over a Virus and a Surge in Malf

Postby admin » Sun Feb 07, 2016 7:09 pm

Terminator insects give wings to genome invaders
by Dr. Mae-Wan Ho
March 19, 2001

NOTICE: THIS WORK MAY BE PROTECTED BY COPYRIGHT

YOU ARE REQUIRED TO READ THE COPYRIGHT NOTICE AT THIS LINK BEFORE YOU READ THE FOLLOWING WORK, THAT IS AVAILABLE SOLELY FOR PRIVATE STUDY, SCHOLARSHIP OR RESEARCH PURSUANT TO 17 U.S.C. SECTION 107 AND 108. IN THE EVENT THAT THE LIBRARY DETERMINES THAT UNLAWFUL COPYING OF THIS WORK HAS OCCURRED, THE LIBRARY HAS THE RIGHT TO BLOCK THE I.P. ADDRESS AT WHICH THE UNLAWFUL COPYING APPEARED TO HAVE OCCURRED. THANK YOU FOR RESPECTING THE RIGHTS OF COPYRIGHT OWNERS.


The United States Department of Agriculture has approved field release of GM pink bollworm this summer, which are made with a mobile genetic element that can jump many species. This is tantamount to giving wings to the most aggressive genome invaders. Dr. Mae-Wan Ho exposes evidence of instability in these GM insects, and warns of rampant horizontal gene transfer and recombination, should such GM insects become released.

Geneticists have created a particularly hazardous class of gene transfer vectors for engineering insects. These are transposons, or mobile genetic elements, which, as the name implies, are genetic units that can move from one site to another in the same genome or move between genomes belonging to unrelated species. Transposons are related to viruses and proviral sequences that, like transposons, are found in the genomes of all species.

A transposon consists of several genes flanked by terminal repeat sequences. One of the genes will code for the enzyme transposase, which is necessary for moving the element. However, elements that have lost the transposase gene can nevertheless get help from the enzyme coded in other transposons. Transposons come in groups, or superfamilies, many of which have members distributed widely across species belonging to different phyla of both animals and plants. These ‘promiscuous’ transposons have found special favour with genetic engineers, whose goal is to create ‘universal’ systems for transferring genes into any and every species on earth. Almost none of the geneticists has considered the hazards involved.

A group in Boston created a vector from mariner, a superfamily of transposons found across genomes of diverse species from insects to plants and vertebrates, including human beings. One element belonging to this superfamily, Hirmar1, isolated from the horn fly, was used to make ‘minitransposons’ consisting of the short inverted terminal repeats, between which any gene expression cassette(s) can be inserted. The researchers constructed a minitransposon with a kanamycin antibiotic resistance marker gene driven by a bacterial promoter. This minitransposon was found to jump easily into the E. coli and the Mycobacterium chromosome. It is known to recognize the dinucleotide TA. The probability of this dinucleotide occurring in any stretch of DNA is 0.252 or 6.25%. Within the 500 base pairs of the bacterial chromosome analysed, 21 of the 23 possible TA dinucleotide insertion sites were occupied (1).

The experiment shows that the transposon can be stripped down to the bare minimum of the flanking repeats, and it can still jump into genomes. The reason, as mentioned earlier, is that the transposase function can be supplied by a ‘helper’ transposon. Such helper transposons are ubiquitous. So, it would seem obvious that integrated transposon vectors may easily jump out again, to another site in the same genome, or to the genome of unrelated species. There are already signs of that in the transposon, piggyBac, used in the GM bollworms to be released by the USDA this summer.

The piggyBac transposon was discovered in cell cultures of the moth Trichopulsia, the cabbage looper, where it caused high rates of mutations in the baculovirus infecting the cells by jumping into its genes (2). The piggyBac is 2.5kb long with 13 bp inverted terminal repeats. It has a specificity for sites with the base sequence TTAA. (The probability of this sequence occurring is 0.254 or 0.4%.) This transposon was later found to be active in a wide range of species, including the fruitfly Drosophila, the mosquito transmitting yellow fever, Aedes aegypti, the medfly, Ceratitis capitata, and the original host, the cabbage looper (3). The piggyBac vector gave high frequencies of transpositions, 37 times higher than mariner and nearly four times higher than Hirmar.

In another experiment, the integrative piggyBac vector, with its transposase gene disabled and carrying the green fluorescent protein gene cassette, was used to transform the silkworm, Bombyx mori L (4). Transposase function was provided by a helper-plasmid containing a piggyBac transposon also disabled by having one of its terminal repeats removed. The integrative vector and helper plasmids were both injected into silkworm embryos. The adult fertile moths (G0) resulting from the injected embryos were mated in single pairs among themselves or backcrossed to the unmodified parent, and the resultant broods (G1) were analysed.

A total of 2498 embryos from two strains of silk worms were injected, 1164 (46.6%) of the embryos hatched resulting in 654 (26.7%) fertile adults, single-pair matings among which 12 broods (0.5%) expressing green fluorescent protein were found.

The genomic DNA of the broods were analysed with Southern blot (a technique that gives information on the inserts). Here is how the authors reported their results.

"Southern blot analyses of the DNA of transformed G1 insects showed that one to three different inserts were present in a single animal and that larvae from the same progeny [ie, brood] had different insertions. These insertions were inherited independently at the G2 generation…

"The presence of multiple independent inserts in many G1 larvae indicates that a single gamete from the G0 parent can harbor several insertions and that different gametes can have different insertions. Eighteen insertions were observed in 12 G1 individuals issued from three transformed parents. It is likely that this result underestimates the total number of insertion events that occurred in the G0 moths." (p.82)

What was the explanation for such a large number of different inserts? There were two possible explanations.

"Either the integration events [of the piggyBac vector] in the germ line occurred late during development [of the injected embryo]", so that the same adult carries a population of germ cells each with different insertions, "or successive rounds of transposition took place after an initial insertion event". The latter hypothesis, considered more likely, "would explain why –- despite the low frequency of insertion in the parental population [0.5%] -– the number of inserts is high in the transformed insects….. A similar situation was also observed in transgenic C. capitata, and it was also attributed to secondary mobilizations of an initial single insert." (p.82)

In other words, there is evidence that the inserts had moved between the G0 and the G1 generations, and possibly, again between the G1 and G2 generations. The "stable germ line transformation" claimed (p.83) is based on a dangerous instability of the insert, which is prone to secondary mobilization.

These artificial transposons are already aggressive genome invaders, and putting them into insects is to give them wings, as well as sharp mouthparts for efficient delivery to all plants and animals and their viruses. The predictable result is rampant horizontal gene transfer and recombination across species barriers. The unpredictable unknown is what kinds of new deadly viruses might be generated (5), and how many new cases of insertion mutagenesis and carcinogenesis they may bring (6). It is the height of folly and irresponsibility to release such GM insects, let alone GM insects carrying female-killing genes (7).

_______________

Notes:

1. Rubin EJ, Akerley BJ, Novik VN, Lampe DJ, Husson RN, and Mekalanos JJ. In vivo transpostion of mariner-based elements in enteric bacteria and mycobacteria. Proc. Natl. Acad.Sci USA 1999: 96: 164-1650. See also "Can such rampant gene shuffling be safe?" Mae-Wan Ho and Angela Ryan, ISIS News 4, March 2000
2. See "Terminator insects – a primer" by Joe Cummins, ISIS Report, March 2001
3. Lobo N, Li X and Fraser Jr. MJ. Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti andTrichoplusia ni. Mol Gen Genet 1999: 261: 803-10.
4. Toshiki T, Chantal T, Corinne R, Toshio K, et al. Germline transformation of the silkworm Bombyx mori L. using a piggyBactransposon-derived vector. Nature Biotechnology 2000: 18: 81-84.
5. See "Genetic engineering superviruses" by Mae-Wan Ho, ISIS Report, March 2001
6. See "Unregulated hazards, ‘naked’ and ‘free’ nucleic acids" ISIS Report, Jan. 2000
7. Thomas DD, Donnelly CA, Wood RJ and Alphey LS. Insect population control using a dominant, repressible, lethal genetic system.Science 2000: 287: 2474-6.
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Re: Alarm Spreads in Brazil Over a Virus and a Surge in Malf

Postby admin » Sun Feb 07, 2016 7:13 pm

Beware the new 'Breakthrough' Transgenic Mosquitoes
by Dr Mae Wan Ho
6/24/14

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Mosquitoes engineered with a jumping gene vector to express a DNA-cutting enzyme produce >95 % male offspring; unfortunately both enzyme and vector target genomes of diverse species from slime moulds to humans. Dr Mae Wan Ho

This commentary has been sent to the editors of the journal Nature Communications, inviting them and the researchers who have reported the creation of the new transgenic mosquitoes in the journal to reply.

Please circulate widely and repost, but you must give the URL of the original and preserve all the links back to articles on our website. If you find this report useful, please support ISIS by subscribing to our magazine Science in Society, and encourage your friends to do so. Or have a look at the ISIS bookstore for other publications


A good trick but no consideration of risks

A team led by Andrea Crisanti at Imperial College London in the UK was widely reported to have made a breakthrough or even a ‘quantum leap’ in creating transgenic mosquitoes that could eradicate malaria [1]. Unfortunately, it is potentially the most hazardous genetically modified organism (GMO) to have been created, and should go no further from the laboratory. The researchers have not considered the risks involved, which would have been obvious from a casual review of existing literature.

Their fast-tracked online report in Nature Communications stated [2]: “Here we generate a synthetic sex distortion system by exploiting the specificity of the homing endonuclease I-PpoI, which is able to selectively cleave ribosomal gene sequences of the malaria vector Anopheles gambiae that are located exclusively on the mosquito’s X-chromosome. We combine structure-based protein engineering and molecular genetics to restrict the activity of the potentially toxic endonuclease to spermatogenesis. Shredding of the paternal X-chromosome prevents it from being transmitted to the next generation resulting in fully fertile mosquito strains that produce >95 % male offspring.”

Simply considered as a genetic trick, it is ingenious. Shredding the X chromosome of the male will make all of its offspring males. That is because female mosquitoes (like female humans) have two X chromosomes, one from the male parent and the other from the female parent, so without the contribution of the X chromosome from the male parent, only male offspring will result. A completely sterile male mosquito is useless, as it just dies out without affecting the population. But a fully fertile one that breeds exclusively males and pass on the sex-distorter trait would be ideal, as it would indeed wipe out the natural population, provided the trait is stably inherited. It would have been the perfect solution to destroying the natural populations of mosquitoes that transmit malaria; except that the DNA-cutting enzyme is by no means “specific” to “ribosomal gene sequences located exclusively on the mosquito’s X-chromosome” as stated. On the contrary, it cuts at a target sequence in ribosomal RNA (rRNA) genes -- numerous copies of which are present in all eukaryote genomes -– plus other sites as well, and the transgenic mosquitoes have been created using a jumping gene (transposon) vector that promiscuously invades all genomes. It is the female mosquitoes that bite people and transmit disease; so any transgenic female mosquitoes among the offspring would inject GM DNA containing the vector and I-PpoI transgene for horizontal transfer into people’s cells to shred their genomes.

The creation of transgene mosquitoes with heritable sex distortion

The wild-type I-PpoI has been engineered into mosquitoes. The expression of the wild-type enzyme during spermatogenesis in transgenic mosquitoes causes cleavage of the paternal X chromosome, but also results in complete male sterility because the protein is stable and persists in mature sperm cells, leading to subsequent cleavage of the maternal X chromosome in the zygote after fertilization, thereby killing the zygote. Thus, the sterile males would just die out without leaving any offspring, and not make any difference to the natural population, unless they are continually released into the wild.

To overcome that, the team mutated amino acid residues in the hydrophobic core of the endonuclease to obtain recombinant proteins with reduced stability. The melting temperature of the protein was decreased from 54.4 °C in the wild type to 49.4 °C in mutant L111A and 35.1 °c in a double mutant L111A/W124L. The thermal half-life at 37 °C ranged from 73.5h for the wild type enzyme to 2 h for H106A. The L111A/W 124L double mutant had a half-life of about 4 min. The specific activity of the wild type was ~7 pmol/min/mg compared to about a third of that in mutant H106A.

Germ line transformation constructs were created to express the I-PpoI variants placed under the control of the male spermatogenesis-specific b2-tubulin promoter. The constructs were designed to express both enhanced green fluorescent protein (EGFP) as in frame fusion protein or, using a 2A ribosomal stuttering signal, as distinct protein chains. The constructs were put into the promiscuous piggyBac vectors for transgenesis. Constructs that had integrated on the X chromosome failed to show significant levels of I-PpoI expression in the testes, probably because the X chromosome is transcriptionally silent during male meiosis.

Reduced in vitro thermal stability translated into significantly lower protein levels of EGFPI-PpoI protein in vivo. The destabilized I-PpoI distorts the sex ratio towards males as measured in emerging adults, fertility was measured by larval hatching rate and number of eggs in crosses of transgenic male mosquitoes to wild type females. As a control transgenic female mosquitoes of each strain were crossed to wild type males. Male mosquitoes expressing the wild-type enzyme were sterile as previously found. No effect on fertility or sex ratio was observed in all strains expressing the double mutant L111A/W124L, which had the lowest in vitro stability; or in strains carrying X-linked transgenes. Significantly, male biased sex ratios ranging from 70.2 to 97.4 % were found in the progeny of males carrying the remaining I-PpoI variants; of those, only three lines, 111A-2 and 124L-2, 124L-3 had comparable hatching rates to the wild type. These strains also showed the lowest mRNA levels. The sex distortion phenotype was stably inherited from male mosquitoes to their transgenic sons. For four subsequent generations, 111A-2 fathers showed comparable levels of fertility and male-biased sex ratios in their offspring. Homozygous males in strains 111A-2 and 124L-3 caused a reduction in the hatching rate, but had no effect on fertility in 124L-2.

It is important to note that the sex distortion is not complete, which means that a variable number of transgenic females will be left to bite people and transmit the potentially lethal transgenes into people’s cells. Such transgenic females were found to have more female offspring when mated to wild type males (see below). In the wild, the proportion of surviving transgenic females, and indeed, I-PpoI-resistant transgenic females could also arise (see below). All that would make it more likely to spread the transgenes into human and other mammals.

Female offspring of transgenic male mosquitoes showed evidence of misrepair and copy number variation in the ribosomal gene cluster in their genome, suggesting chromosomal damage. There was also a significant female-biased sex ratio in the progeny of female survivors crossed to wild type males suggesting that loss of vitality occurred in individuals that had inherited only a damaged X chromosome from the transgenic female.

But have the transgenic female offspring inherited integrated I-PpoI endonuclease genes in the X-chromosome, which would make the chromosome resistant to further endonuclease damage (as integration destroys the target site, see below)? To test for this possibility, the transgenic females were crossed to wild type males, and the transgenic male progeny, which have a 50 % chance of carrying the potentially resistant X-chromosome was then crossed to wild type females, and the sex ratio of individual crosses was recorded. The analysis showed a male bias in the progeny of all males that did not differ significantly from the 111A-2 stock, suggesting that the X chromosomes of female survivors are damaged but still susceptible to further cleavage.

In five independent cage experiments, the release of hemizygous 111A-2 males at a ratio of 3X controls was effective in suppressing caged wild type populations (achieving elimination within 6 generations in four out of five cages. As expected the release of sterile males expressing the wild-type enzyme had no measurable effect in three populations. Sex distorter is much more effective than sterile males by 2 orders of magnitude, and 2-70 times more effective than equivalent releases of female-killing alleles.

This sex-distorter system is clearly an improvement on the Oxytec transgenic mosquitoes engineered with an ill-characterized, ineffective, as well as hazardous system [3, 4] (Regulation of Transgenic Insects Highly Inadequate and Unsafe, Transgenic Mosquitoes Not a Solution, SiS 54). There is an alternative non-transgenic system based on a common symbiotic bacterium that can stop the dengue virus multiplying in the mosquito host, which effectively makes those transgenic mosquitoes obsolete [5] (Non-transgenic Mosquitoes to Combat Dengue, SiS 54). But the ill-advised Brazilian government has approved Oxytec’s transgenic mosquitoes for commercial release in April 2014 [6].

In terms of safety, the new sex distortion transgenic mosquito is no better and possibly worse.

Homing endonuclease originally from slime mould

The homing endonucleases are a collection of enzymes encoded either as freestanding genes located within introns (intervening noncoding sequence in protein-coding genes), as fusions with host proteins, or as self-splicing inteins (sequences within proteins that splice themselves out after translation). They cut genomic DNA within the cells that synthesize them at target sites. Repair of the cut DNA by the host cell frequently results in the gene encoding the homing endonuclease being copied into the cleavage site, hence the term 'homing' to describe the movement of these genes. Homing endonucleases can thereby transmit their genes horizontally within a host population, increasing their frequency more rapidly than classical Mendelian genes [7, 8].

Homing endonuclease (HEN) recognize target sequences of 15 to 40 bp long. Generally, owing to the homing mechanism, the gene encoding the endonuclease is located within the recognition sequence which the enzyme cuts, thus interrupting the homing endonuclease recognition sequence and limiting DNA cutting only to sites that do not (yet) carry the HEN. However, target recognition is not completely specific, and off target cleavage and integration has been known at least since the 1990s.

The I-Ppol homing endonuclease used in the construction of the sex distorter transgenic mosquitoes [1, 2] was originally isolated from the slime mould Physarum polycephalum. It is a member of the His-Cys box family found in myxomycetes and amoebae. The His-Cys box protein motif consists of two conserved histidine and three conserved cysteine residues within a 30 residue region of protein. These conserved His and Cys residues appear to contribute to a Zn2+-binding motif and to the endonuclease active site. The I-Ppol target site of 15 bp and cleavage position is as follows [9]. The cleavage at TTAA leaves a 4 nucleotide overhang at the cut ends.

Image

However, a substantial degree of degeneracy of the target sequence appeared to be tolerated, as demonstrated in a study in which partially randomized cleavage sites were created by mutagenesis [9]. Not only that, multiple target sites exist in all eukaryote genomes.

Target sites ubiquitous in all eukaryote species including humans

The native I-PpoI target site is by no means exclusive to the mosquito X-chromosome, it is actually present in multiples copies in the highly conserved 28S ribosome RNA (rRNA) genes of all eukaryotes species including humans, and currently being deployed in gene therapy experiments [10]. Each diploid human cell has about 600 copies of the rRNA genes in five clusters located to the short arms of chromosomes 13, 14, 15, 21, and 22. On account of the multitude of rRNA genes and the presence of possibly inert spacers between the gene repeats, the rDNA is considered an appealing safe haven for transgene integration. A team of researchers in Finland have created an HIV-1 integrase-1 PpoI fusion protein in order to guide the integration of an HIV viral vector into the rDNA [10].

The team also carried out a study on cytotoxicity of the construct. Although a genome-wide interaction study found that the IN-fusion PpoI proteins bind to their target sequence containing 28 S rRNA genes with 100-fold enrichment compared to controls, there was significant off-target binding to non-rRNA gene sites, and considerable cytotoxicity for all human cell lines tested from the double-stranded breaks in DNA produced by the I-PpoI endonuclease [11]. The researchers put a positive gloss on the findings by noting that the construct was more toxic to cancer than normal cells, and suggesting that the endonuclease could be used for cancer therapy.

PiggBac a promiscuous transposon now used in gene therapy experiments

I have long warned against using promiscuous transposons as gene transfer vectors, especially for insects that bite people [12] (Terminator insects give wings to genome invaders, ISIS report). The piggyBac transposon was discovered in cell cultures of the moth Trichopulsia, the cabbage looper, where it caused high rates of mutations in the baculovirus infecting the cells by jumping into its genes [13] (Terminator insects –- a primer, ISIS Report). The piggyBac is 2.5kb long with 13 bp inverted terminal repeats. It has specificity for sites with the base sequence TTAA (same as the I-PpoI endonuclease cleavage site, see above). The probability of this sequence occurring at random in any genome is 0.254 or 0.4%. There is also evidence that the disabled piggyBac vector carrying the transgene, even when stripped down to the bare minimum of the border repeats, was nevertheless able to replicate and spread, because the transposase enzyme enabling the piggyBac inserts to move can be provided by transposons present in all genomes. The main reason initially for using transposons as vectors in insect control was precisely because they can spread the transgenes rapidly by ‘non-Mendelian' means within a population, i.e., by replicating copies and jumping into genomes, thereby ‘driving’ the trait through the insect population. However, the scientists involved neglected the fact that the transposons could also jump into the genomes of the mammalian hosts including human beings. Although each transposon has its own specific transposase enzyme that recognizes its terminal repeats, the same enzyme can also interact with the terminal repeats of other transposons, and evidence suggests extensive cross-talk among related but distinct transposon families within a single eukaryotic genome (reviewed in [3]).

The use of the piggyBac transposon has been plagued by problems of instability in transformed Aedes aegypti [14]; and large unstable tandem inserts of the piggyBac transposon were prevalent [ 15]. In spite of instability and resulting genotoxicity, the piggyBac transposon has been used extensively also in human gene therapy [16]. Several human cell lines have been transformed, even primary human T cells using piggyBac [17]. These findings leave us little doubt that the transposon-borne transgenes in the transgenic mosquito can transfer horizontally to human cells. The piggyBac transposon was found to induce genome wide insertion mutations disrupting many gene functions.

To conclude

Transgenic mosquitoes are not the solution to eradicating dengue or malaria. On the contrary, they are among the most hazardous GMOs created, and should never be released into the wild on any commercial basis.

Researchers should consider the risks involved before embarking on a project, and science journal editors and commentators should also question whether works for publications carry risks to health and the environment.

_______________

References

1. “GM mosquitoes a ‘quantum leap’ towards tackling malaria”, Adam Vaughan, The Guardian, 10 June 2014,http://www.theguardian.com/environment/2014/jun/10/gm-mosquitos-malaria-genetic-modification
2. Galizi R, Doyle LA, Menishelli M, Bernardini F, Deredec A, Burt A, Stoddard BL, Windbichler N and Crisanti A. A synthetic sex-ratio distortion system for the control of the human malaria mosquito. Nature Communications 2014, published 10 June, doi:10.1038/ncommuns4977
3. Ho MW. Regulation of transgenic insects highly inadequate. Science in Society 54, 20-21, 2012.
4. Ho MW. Transgenic mosquitoes not a solution. Science in Society 54, 22-23, 2012.
5. Ho MW. Non-transgenic mosquitoes to control dengue. Science in Society 54, 24-25, 2012.
6. “Brazil approves use of genetically modified mosquitoes”, Hal Hodson, New Scientist, 23 April 2014, http://www.newscientist.com/article/dn2 ... 6h_dHnjjIU
7. Homing endonuclease, Wikipedia, 12 May 2014, http://en.wikipedia.org/wiki/Homing_end ... d8928227-2
8. Belfort M and Perlman PS. Mechanisms of intron mobility. J Biol Chem 1995, 270, 30237-40.
9. Argast GM, Stephens KM, Emonds MJ and Monnat Jr RJ. I-PpoI and I-CreI homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment. J Mol Biol 1998, 280, 345-53.
10. Schenkwein D, Turkki V, Ahlroth MK, Timonen O, Airenne KJ and Ylä-Herttuaia S. rDNA-directed integration by an HIV-1 integrase-I-Ppol fusion protein. Nucleic Acids Research 2012, 1-10, doi:10.1093/nar/gks1438,http://nar.oxfordjournals.org/content/early/2012/12/25/nar.gks1438.full.pdf+html
11. Turkki V, Schenkwein D, Timonen O, Husso T, Lesch HP and Ylä-Herttuaia S. Lentiviral protein transduction with genome-modifying HIV-1 integrase-I-PpoI fusion proteins: studies on specificity and cytotoxicity. BioMed Research Interna 2014, article ID 370340, 11 pp, http://www.hindawi.com/journals/bmri/2014/379340/
12. Ho MW. Terminator insects give wings to genome invaders. ISIS Report, 19 March 2001, http://www.i-sis.org.uk/terminsects-pr.php
13. Cummins J. Terminator insects – a primer. ISIS Report, 15 March 2001, http://www.i-sis.org.uk/piggybac-pr.php
14. Adelman ZN1, Jasinskiene N1, Peek C1, Travanty EA2, Olson KE2, James AA1. Instability of the piggyBac element in transformedAedes aegypti. ISMIS 2002. Abstracts of the Fourth International Symposium on Molecular Insect Science. 70pp. Journal of Insect Science, 2, 17.
15. Adelman ZN, Jasinskiene N, Vally KJ, Peek C, Travanty EA, Olson KE, Brown SE, Stephens JL, Knudson DL, Coates CJ, James AA. Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti. Transgenic Res 2004, 13(5), 411-25.
16. Urschitz J, Kawasumi M, Owens J, Morozumi K, Yamashiro H, Stoytchev I, Marh J, Dee J Kawamoto K, Coates CJ, Kaminski JM, Pelczar P, Yanagimachi R, Moisyadi S. Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects. Proc Natl Acad Sci U S A 2010, 107(18), 8117-22.
17. Galvan DL, Nakazawa Y, Kaja A, Kettlun C, Cooper LJ, Rooney CM, Wilson MH. Genome-wide mapping of PiggyBac transposon integrations in primary human T cells. J Immunother 2009, 32(8), 837-44.
18. Lobo N, Li X and Fraser Jr. MJ. Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti andTrichoplusia ni. Mol Gen Genet 1999: 261: 803-10.
19. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Young JK and Sander JD. High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 2013, 31, 822-6.http://www.ncbi.nlm.nih.gov/pmc/article ... 488397.pdf
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Re: Alarm Spreads in Brazil Over a Virus and a Surge in Malf

Postby admin » Tue Feb 23, 2016 4:58 am

Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg?
by A. S. Oliveira Melo1, G. Malinger2,*, R. Ximenes3, P. O. Szejnfeld4, S. Alves Sampaio5 andA. M. Bispo de Filippis5
Article first published online: 5 JAN 2016
DOI: 10.1002/uog.15831
Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.
Issue Ultrasound in Obstetrics & Gynecology
Ultrasound in Obstetrics & Gynecology
Volume 47, Issue 1, pages 6–7, January 2016

NOTICE: THIS WORK MAY BE PROTECTED BY COPYRIGHT

YOU ARE REQUIRED TO READ THE COPYRIGHT NOTICE AT THIS LINK BEFORE YOU READ THE FOLLOWING WORK, THAT IS AVAILABLE SOLELY FOR PRIVATE STUDY, SCHOLARSHIP OR RESEARCH PURSUANT TO 17 U.S.C. SECTION 107 AND 108. IN THE EVENT THAT THE LIBRARY DETERMINES THAT UNLAWFUL COPYING OF THIS WORK HAS OCCURRED, THE LIBRARY HAS THE RIGHT TO BLOCK THE I.P. ADDRESS AT WHICH THE UNLAWFUL COPYING APPEARED TO HAVE OCCURRED. THANK YOU FOR RESPECTING THE RIGHTS OF COPYRIGHT OWNERS.


An unexpected upsurge in diagnosis of fetal and pediatric microcephaly has been reported in the Brazilian press recently. Cases have been diagnosed in nine Brazilian states so far. By 28 November 2015, 646 cases had been reported in Pernambuco state alone. Although reports have circulated regarding the declaration of a state of national health emergency, there is no information on the imaging and clinical findings of affected cases. Authorities are considering different theories behind the ‘microcephaly outbreak’, including a possible association with the emergence of Zika virus disease within the region, the first case of which was detected in May 2015[1].

Zika virus is a mosquito-borne disease closely related to yellow fever, dengue, West Nile and Japanese encephalitis viruses[2]. It was first identified in 1947 in the Zika Valley in Uganda and causes a mild disease with fever, erythema and arthralgia. Interestingly, vertical transmission to the fetus has not been reported previously, although two cases of perinatal transmission, occurring around the time of delivery and causing mild disease in the newborns, have been described[3].

We have examined recently two pregnant women from the state of Paraiba who were diagnosed with fetal microcephaly and were considered part of the ‘microcephaly cluster’ as both women suffered from symptoms related to Zika virus infection. Although both patients had negative blood results for Zika virus, amniocentesis and subsequent quantitative real-time polymerase chain reaction[4], performed after ultrasound diagnosis of fetal microcephaly and analyzed at the Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, was positive for Zika virus in both patients, most likely representing the first diagnoses of intrauterine transmission of the virus. The sequencing analysis identified in both cases a genotype of Asian origin.

In Case 1, fetal ultrasound examination was performed at 30.1 weeks' gestation. Head circumference (HC) was 246 mm (2.6 SD below expected value) and weight was estimated as 1179 g (21st percentile). Abdominal circumference (AC), femur length (FL) and transcranial Doppler were normal for gestational age as was the width of the lateral ventricles. Anomalies were limited to the brain and included brain atrophy with coarse calcifications involving the white matter of the frontal lobes, including the caudate, lentostriatal vessels and cerebellum. Corpus callosal and vermian dysgenesis and enlarged cisterna magna were observed (Figure 1).

Image
Figure 1. Case 1: (a) Transabdominal axial ultrasound image shows cerebral calcifications with failure of visualization of a normal vermis (large arrow). Calcifications are also present in the brain parenchyma (small arrow). (b) Transvaginal sagittal image shows dysgenesis of the corpus callosum (small arrow) and vermis (large arrow). (c) Coronal plane shows a wide interhemispheric fissure (large arrow) due to brain atrophy and bilateral parenchymatic coarse calcifications (small arrows). (d) Calcifications are visible in this more posterior coronal view and can be seen to involve the caudate (arrows).

In Case 2, fetal ultrasound examination was performed at 29.2 weeks' gestation. HC was 229 mm (3.1 SD below expected value) and estimated fetal weight was 1018 g (19th percentile). AC was below the 3rd percentile but FL was normal. The cerebral hemispheres were markedly asymmetric with severe unilateral ventriculomegaly, displacement of the midline, thinning of the parenchyma on the dilated side, failure to visualize the corpus callosum and almost complete disappearance or failure to develop the thalami. The pons and brainstem were thin and continuous with a non-homogeneous small mass at the position of the basal ganglia. Brain calcifications were more subtle than in Case 1 and located around the lateral ventricles and fourth ventricle. Both eyes had cataracts and intraocular calcifications, and one eye was smaller than the other (Figure 2).

Image
Figure 2. Case 2: (a) Anterior coronal view shows severe asymmetric ventriculomegaly with cystic formation (arrow). (b) Posterior horn of the lateral ventricle (LV) in coronal view is dilated. Note calcifications in the fourth ventricle (arrows). (c) The thalamus is absent (arrow) and the brainstem and pons are thin and difficult to visualize (sagittal view). (d) Axial view shows calcifications in both eyes (arrows). Note that the proximal eye is very small and lacks normal anatomic landmarks.

In the meantime, in Paraiba state, six children diagnosed with Zika virus were born to mothers who were apparently symptomatic during pregnancy, all of them with neonatal HC below the 10th percentile. Fetal neurosonograms showed two cases with cerebellar involvement and three with brain calcifications. One had severe arthrogryposis.

Intrauterine infections affecting the brain are relatively rare; cytomegalovirus (CMV), toxoplasmosis, herpes virus, syphilis and rubella are well known vectors of fetal disease. Among the Flaviviruses there have been only isolated reports linking West Nile encephalitis virus to fetal brain insults[5].

The presence of calcifications was suggestive of an intrauterine infection but severe damage of the cerebellum, brainstem and thalami is rarely associated with intrauterine infection. Both cases showed some similarities to CMV cases but with a more severe and destructive pattern and they lacked the nodules characteristic of toxoplasmosis. Interestingly, the reported case of fetal West Nile virus infection has similar characteristics[5].

It is difficult to explain why there have been no fetal cases of Zika virus infection reported until now but this may be due to the underreporting of cases, possible early acquisition of immunity in endemic areas or due to the rarity of the disease until now. As genomic changes in the virus have been reported[6], the possibility of a new, more virulent, strain needs to be considered. Until more cases are diagnosed and histopathological proof is obtained, the possibility of other etiologies cannot be ruled out.

Earliest AIDS Cases

One important consideration remained before the basic premise of manmade HIV development could be reconciled. The literature held several accounts of alleged AIDS cases and HIV discoveries predating the work of Gallo, Hilleman, Duesberg, et aI., that is, prior to the 1960s or early 1970s.

A literature search conducted to investigate such claims revealed several interesting discoveries.

I first read a series of reports published in The Lancet regarding the earliest alleged AIDS case -- a "25-year-old former naval seaman" who died of cytomegalovirus and pneumocystis infections in 1959.34 The claim was made by University of Manchester researcher Gerald Corbitt and coworkers. Having meticulously extracted the seaman's tissues from paraffin blocks initially developed to make autopsy slides, DNA amplification methods were used to "search for very small quantities of HIV proviral DNA in target cells." The group found the suspected particles in several of the recaptured tissues, and the news media broadly heralded the event.

Several years later, Drs. David Ho and Tuofu Zhu of the Aaron Diamond Research Center in New York reexamined Corbitt's tissues in an effort to investigate the theory that HIV evolved somehow from tainted poliovaccines during the 1950s. Ho and Zhu determined that the DNA sequences found by Corbitt and his colleagues were essentially identical to a strain of HIV circulating in the United States during the late 1980s. This raised "the spectre of specimen contamination."35

This contamination, the evidence showed, "was more likely to be [caused] by another clinical specimen" than by improper DNA probing techniques. And when additional methods were used to learn more, the New York researchers concluded that the tissues examined were "derived from at least two individuals."

Thus, the Corbitt group's finding was invalidated.

Corbitt, allegedly mystified by what Steve Connor of London's daily The Independent called a calculated hoax or mammoth error, later requested the material be reexamined by a third party.36,37

HIV in Zaire in 1959?

Another study supported by grants obtained by Robert GaUo's and Don Francis's coUeague Max Essex and his Harvard associate P. Kanki, alleged to have found one HTLV-III (HIV)-positive plasma specimen among 1213 from central Africa dating back to 1959.38 Much ado accompanied this find especially since it was said to have come from Zaire. Essex's group also reported that the presence of HIV in the sample had been confirmed by three other laboratories using "different techniques." According to the scientific consensus, however, an independent confirmatory test was never done.39

The "three other laboratories" that Essex alleged checked his group's work, may not have been impartial. These included: Gallo's lab in Bethesda, Dr. C. Schable's lab at the CDC, and Abbott Laboratories.38

Abbott Labs are best known for having licensed and produced: the ELISA screening test for HIV This test had also been used by Saxinger, Gallo, and others who claimed 60 percent of Ugandan children were infected with HIV by the early 1970s.40 This report was later debunked by several research groups that noted the esteemed NCI researchers had failed to use HIV specific testing methods or materials.41-44

Abbott also licensed and marketed the hepatitis core antigen test purchased by New York City Blood Center officials, following years of delay, and before the ELISA test was available, to help identify blood units suspected of HIV infection. The company had also supplied expertise and the radioactive experimental reagents Szmuness required for his NewYork homosexual hepatitis B vaccine trial. Furthermore, Abbott Labs ended up commerciaUy marketing MSDs hepatitis B vaccine.45, 46, 47

Moreover, the hepatitis B vaccines suspected of having transmitted HIV to American homosexuals, was researched by Abbott's L. R. Overby who was intimately connected to NYUMC hepatitis B chief Saul Krugman. Together, they evaluated hepatitis B susceptibility and vaccination methods in the New York subjects during the mid-1970s. 48

As it turned out, additional confirmatory studies could not be carried out by independent investigators as the specimens containing the "Leopoldville strain" HIV had been lost by the Essex team.39

AIDS Case in 1968?

In 1984, researchers published evidence that "Robert R.," a fifteen-year-old, black, male, heterosexual, born and reared in St. Louis, had died of AIDS-like illnesses.49 Later, in 1987, scientists provided evidence that the boy's blood contained antibodies to HIV. 50

"If a virus related to HIV," they concluded, "has been present in the United States, Africa, or elsewhere for several decades, its failure to spread in an epidemic fashion earlier may reflect a recent genetic change in the virus and/or sociocultural factors involving sexual practices or numbers of sexual partners."50

By 1990, advanced gene analysis techniques allowed scientists to reexamine this case, at which time they determined that "Robert R." had not died of AIDS. His blood never contained HIV. 39

This evidence and more led the consensus of international AIDS researchers to conclude that some radical event between 1970 and 1975 had changed the HIV-I progenitor from a virtually harmless germ, essentially noninfectious to humans, to a silent killer.

Dr. Gerald Myers, chief of the special HIV Sequence Database AIDS Project of the U.S. Government's Los Alamos National Laboratory, may have articulated this position best when he said "the preponderance of evidence still argues for an explosive event in the mid-1970s." Furthermore, regarding the origin of AIDS, he insisted HIV-I was a fairly new virus, surely only a few decades young.39

Alternatively, Ho and other virologists proposed that HIV-I may have been ancient, but the mass of epidemiological evidence indicates a dramatic change occurred in the 1970s, most likely due to human events more than biological ones in the natural evolution of the virus.39

What might those human events have been?


Obviously, they would have needed to take place simultaneously in North America and Africa. An iatrogenic event, involving vaccines being tested on both continents on monkeys and people offers the most plausible, and in fact only, explanation being advanced.39

-- Emerging Viruses: AIDS & Ebola: Nature, Accident or Intentional?, by Leonard G. Horowitz, DMD, MA, MPH


As with other intrauterine infections, it is possible that the reported cases of microcephaly represent only the more severely affected children and that newborns with less severe disease, affecting not only the brain but also other organs, have not yet been diagnosed.

If patients diagnosed in other states are found to be seropositive for Zika virus, this represents a severe health threat that needs to be controlled expeditiously. The Brazilian authorities reacted rapidly by declaring a state of national health emergency. As there is no known medical treatment for this disease, a serious attempt will be needed to eradicate the mosquito and prevent the spread of the disease to other Brazilian states and across the border[7].

References

1 Campos GS, Bandeira AC, Sardi SI. Zika Virus Outbreak, Bahia, Brazil. Emerg Infect Dis 2015; 21: 1885–1886.

2 Ioos S, Mallet HP, Leparc Goffart I, Gauthier V, Cardoso T, Herida M. Current Zika virus epidemiology and recent epidemics. Medecine et maladies infectieuses 2014; 44: 302–307.

3 Besnard M, Lastere S, Teissier A, Cao-Lormeau V, Musso D. Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014. Euro Surveill 2014; 19.

4 Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, Stanfield SM, Duffy MR. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 2008; 8: 1232–1239.

5 Centers for Disease Control and Prevention (CDC). Intrauterine West Nile virus infection--New York, 2002. MMWR Morb Mortal Wkly Rep 2002; 51: 1135–1136.

6 Faye O, Freire CC, Iamarino A, Faye O, de Oliveira JV, Diallo M, Zanotto PM, Sall AA. Molecular evolution of Zika virus during its emergence in the 20(th) century. PLoS Negl Trop Dis 2014; 8: e2636.

7 Goenaga S, Kenney JL, Duggal NK, Delorey M, Ebel GD, Zhang B, Levis SC, Enria DA, Brault AC. Potential for Co-Infection of a Mosquito-Specific Flavivirus, Nhumirim Virus, to Block West Nile Virus Transmission in Mosquitoes. Viruses 2015; 7: 5801–5812.
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