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 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.

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