case for lab-made sars-cov-2 spelled out by scientists

3-23-20   If leading up to 1977, the fact that increased research into strains of the (swine) flu were seen to increase the odds that an accidental leak would occur, why isn’t the same logic being applied to our pandemic today?  Why is almost everyone today assuming that the increased pace of research means scientists in fact anticipated this outbreak instead of causing it?

  Wouldn’t an increased pace of research also increase the odds that a leak of a lab-modified coronavirus would occur just like an increased pace of research precipitated the leak of the H1N1 Swine Flu back then?…

  most of this (gain-of-function) work didn’t really raise too many eyebrows until about ten years ago when scientists including some in Stony Brook, NY – not coincidentally also the first place to build a DNA-virus from scratch – took the H5N1 Bird Flu, tweaked its genome in two places and then passed it through a series of ferret hosts in the lab until it became airborne.  This sort of research, a minor alteration and then passage through ferrets, did two things:  Resulted in a virus that would look natural and wouldn’t appear to have been directly genetically altered and also created a virus that was way out on its own branch of the viral family tree since those sequential passages added generations far faster than they’d naturally occur in the wild.  If that sounds familiar, maybe that’s because those traits are also exactly what’s found with COVID-19.  https://harvardtothebighouse.com/2020/03/23/no-monkey-ever-reheated-a-frozen-burrito-what-the-expanse-tells-us-about-the-covid-19-pandemic/

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5-23-20     ‘I haven’t seen a zoonotic virus that has behaved in this way before,’  Prof. Petrovsky of Flinders U, Adelaide, AU, said.   He told The Mail on Sunday that new viruses crossing over from animals normally strengthen as they adapt to human hosts, but for unexplained reasons, this new coronavirus seems perfectly adapted to infect humans without the need to evolve.

  He pointed to the ‘coincidence’ that the most closely related known viruses were being studied in a laboratory in Wuhan, the Chinese city where the pandemic erupted, and insisted that the possibility of a leak, however remote, should not be ignored in the search for its origin.

  ‘The implications may not be good for scientists or global politics, but just because the answers might cause problems, we can’t run away from them,’ he added.  ‘There is currently no evidence of a leak but enough circumstantial data to concern us.  It remains a possibility until it is ruled out.’   https://www.dailymail.co.uk/news/article-8351091/Top-vaccine-scientist-says-coronavirus-come-animal-freak-nature.html

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  According to Israeli geneticist

Dr. Ronen Shemesh, the Furin site is the most unusual finding.  “I believe that the most important issue about the differences between ALL coronavirus types is the insertion of a Furin protease cleavage site at the Spike protein of SARS-CoV-2,” he said.  “Such an insertion is very rare in evolution, the addition of such 4 Amino acids alone in the course of only 20 years is very unlikely.”  Shamesh believes the novel coronavirus was most likely created in a lab and did not evolve in nature.  “There are many reasons to believe that the COVID- generating SARS-CoV-2 was generated in a lab. Most probably by methods of genetic engineering,” he said, adding “I believe that this is the only way an insertion like the FURIN protease cleavage site could have been introduced directly at the right place and become effective.“ Dr Shemesh, who has a PhD in Genetics and Molecular Biology from the Hebrew University in Jerusalem and over 21 years of experience in the field of drug discovery and development, said it is even “more unlikely” that this insertion happened in exactly the right place of the cleavage site of the spike protein – which is where it would need to occur to make the virus more infectious.  “What makes it even more suspicious is that fact that this insertion not only occurred on the right place and in the right time but also turned the cleavage site from an Serine protease cleavage site* to a FURIN cleavage site,” he added.  This protein cleaving protein is highly promiscuous, it’s found in many human tissues and cell types and is involved in many OTHER virus types activation and infection mechanisms (it is involved in HIV, herpes, Ebola and Dengue virus mechanisms).  If I was trying to engineer a virus strain with a higher affinity and infective potential to humans, I would do exactly that: I would add a Furin Cleavage PRRA site directly at the original less effective and more cell specific cleavage site. I believe that this is the only way an insertion like the FURIN protease cleavage site PRRA could have been introduced directly at the right place and become effective.  I believe that the most important issue about the differences between ALL coronavirus types is the insertion of a Furin protease cleavage site at the Spike protein of SARS-CoV-2.”

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  "Notably, this approach surprisingly revealed that the binding energy between SARS-CoV-2 spike protein and ACE2 was highest for humans out of all species tested, suggesting that SARS-CoV-2 spike protein is uniquely evolved to bind and infect cells expressing human ACE2.  This finding is particularly surprising as, typically, a virus would be expected to have highest affinity for the receptor in its original host species, e.g. bat, with a lower initial binding affinity for the receptor of any new host, e.g. humans.  However in this case the affinity of SARS-CoV-2 is higher for humans than for the putative original host species, bats, or for any potential intermediary host species.  Although bats carry many coronaviruses including SARS-CoV, a relative of SARS-CoV-2, direct evidence for existence of SARS-CoV-2 in bats has not been found. As highlighted by our data, the binding strength of SARS-CoV-2 for bat ACE2 is considerably lower than for human ACE2, suggesting that even if SARS-CoV-2 did originally arise from a bat precursor it must later have adapted its spike protein to optimise its binding to human ACE2.  There is no current explanation for how, when or where this might have happened.  Instances of direct human infection by coronaviruses or other bat viruses is rare with transmission typically involving an intermediate host.  For example, lyssaviruses such as Hendra are periodically transmitted from bats to horses and then to humans who contact the infected horse.  Similarly, SARS-CoV was shown to be transmitted from bats to civet cats and from them to humans. To date, a virus identical to SARS-CoV-2 has not been identified in bats or any other non-human species, making its origins unclear.  To date the most closely related coronavirus to SARS-CoV-2, is the bat coronavirus, BatCoV RaTG1, which has 96% whole-genome identity to SARS-CoV-2. 50.  The fact that SARS-CoV-2 has also not been found in any likely intermediate host raises questions of the origins of the original SARS-CoV-2."  

-N. Petrovsky  

  This the original Petrovsky quote:

  "Take a bat coronavirus that is not infectious to humans, and force its selection by culturing it with cells that express human ACE2 receptor, such cells having been created many years ago to culture SARS coronaviruses and you can force the bat virus to adapt to infect human cells via mutations in its spike protein, which would have the effect of increasing the strength of its binding to human ACE2, and inevitably reducing the strength of its binding to bat ACE2.  Viruses in prolonged culture will also develop other random mutations that do not affect its function. The result of these experiments is a virus that is highly virulent in humans but is sufficiently different that it no longer resembles the original bat virus.  Because the mutations are acquired randomly by selection there is no signature of a human gene jockey, but this is clearly a virus still created by human intervention.”  …

  Sørensen statement:  “When we technically describe the virus, we see that it has  not come about in a natural development. It’s done by Americans and Chinese, as part of what’s called “gain of function” studies.  It is done all over the world.  You say you don’t, but it happens all the time in advanced labs.”

  ABSTRACT:  We propose that the dual effect general method of action of this chimeric* virus’s spike, including receptor binding domain, includes membrane components other than the ACE2 receptor, which explains clinical evidence of its infectivity and pathogenicity. We show the non-receptor dependent phagocytic** general method of action to be specifically related to cumulative charge from inserted sections placed on the SARS-CoV-2 Spike surface in positions to bind efficiently by salt bridge formations.

*A virus that has been created by combining cells of more than one distinct genotype.

**Phagocytes are cells that protect the body by ingesting harmful foreign particles, bacteria, and dead or dying cells.

  These data reveal the biological structure of SARS-CoV-2 Spike and confirm that accumulated charge from inserts and salt bridges are in surface positions capable of binding with cell membrane components other than the ACE2 receptor ...Cumulative data suggests that the general method of action of this chimeric virus includes membrane components other than the ACE2 receptor, which may explain clinical evidence of its infectivity and pathogenicity. Data shows the non-spike receptor binding domain dependent phagocytic general method of action to be specifically related to cumulative charge from inserted sections on the SARS-CoV-2 Spike (see Fig 1) poised to form salt bridges with attachment receptors. This suggests that attachment to such previously reported membrane proteins has been enhanced directly due to the basic and positive charged inserts in the Spike protein together with other basic and positive charged amino acid substitutions enabling formation of salt bridges with the receptor CLEC4M/DC-SIGNR or, indirectly, by the additional salt bridges formed between the positive charged amino acids and negative charged phospholipids on the cell membrane. Positive charged amino acids are inserted into peptides and proteins to enhance cell affinity and can also be used for transport of peptides and proteins through the cell wall (Richard et al., 2003; Thorén et al., 2000; Åmand et al., 2011). In addition, these positive charges may be used for co-receptor binding where the opposite negative charge is available. It is a matter of fact that there are unique inserts in the SARS-CoV-2 spike protein when they are aligned with other SARS-CoV sequences as shown in Fig. 2. …////////////

  The researchers note that RmYN02 does not closely resemble SAR-CoV-2 in the region of the genome that encodes the key receptor binding domain that binds to the human ACE2 receptor that SARS-CoV-2 uses to infect host cells [POLYBASIC FURIN CLEAVAGE SITE PRRA]. This means it's not likely to infect human cells. The key similarity between SARS-CoV-2 and RmYN02, is the finding that RmYN02 also contains amino acid insertions at the point where the two subunits of its spike protein meet. SARS-CoV-2 is characterized by a four-amino-acid insertion at the junction of S1 and S2; this insertion is unique to the virus and has been present in all SARS-CoV-2 sequenced so far. The insertions in RmYN02 are not the same as those in SARS-CoV-2, which indicates that they occurred through independent insertion events." -WEIFENG SHI, Mqy 2020.

  "Neither RaTG13 nor RmYN02 is the direct ancestor of SARS-CoV-2, because there is still an evolutionary gap between these viruses. But our study strongly suggests that sampling of more wildlife species will reveal viruses that are even more closely related to SARS-CoV-2 and perhaps even its direct ancestors, which will tell us a great deal about how this virus emerged in humans."  -Dr. Shi Zhengli  …

  Dr. Ebright reports he observed no evidence of cutting and pasting genes by humans in SARS-CoV-2.  "Labs around the globe have been creating synthetic viruses like SARS-CoV-2 for years and its genome would not necessarily contain hallmarks of human manipulation:  modern genetic engineering tools permit cutting and pasting genomic fragments without leaving a trace. It can be done quickly, too:  it took a Swiss team less than a month to create a synthetic clone of SARS-CoV-2."  

  SHING HEI ZHAN, BENJAMIN E. DEVERMAN AND YUJIA ALINA CHAN:  May 2, 2020 "…we were surprised to find that SARS-CoV-2 resembles SARS-CoV in the late phase of the 2003 epidemic after SARS-CoV had developed several advantageous adaptations for human transmission.  Our observations suggest that by the time SARS-CoV-2 was first detected in late 2019, it was already pre-adapted to human transmission to an extent similar to late epidemic SARS-CoV.  However, no precursors or branches of evolution stemming from a less human-adapted SARS-CoV-2-like virus have been detected…. It would be curious if no precursor or branches of SARS-CoV-2 evolution are discovered in humans or animals….Even the possibility that a non-genetically-engineered precursor could have adapted to humans while being studied in a laboratory should be considered, regardless of how likely or unlikely.  It is important to note that no intermediary host has yet been identified for the SARS-CoV-2 virus.

  MAY 13, 2020:  Two Wuhan Institute of Virology scientists, Xing-Yi Ge and Zhengli-Li Shi, use reverse genetics to generate a chimeric virus (one that has been created by combining cells of more than one distinct genotype) closely resembling the novel coronavirus SARS-CoV-2:  “On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo.”  …

 

Luc Montagnier:  We have no experiments, but the experience comes from the disease itself; from the measures that are made currently in labs and on patients. Well, we came to the conclusion that there has been a manipulation regarding this virus. In fact, part of the virus, not the whole, is manipulated. Well, the virus follows a classic model that comes from bats; but on top of this model they have added sequences of HIV, the AIDS virus.  It’s not natural.  It’s a lab work of professional molecular biologists.  It’s a very accurate work…we can say a work of watchmaker.  I accuse nobody.  I don’t know who did it; neither why.  The possibility is probably they wanted to make a vaccine against the Aids.  So they took small sequences of the virus and they installed them on the larger sequence of the coronavirus.  The genetic material of the virus is a long tape of RNA…as in DNA but it’s RNA. On this tape; in a certain place of it, they have planted small sequences of HIV. And these sequences are not small for nothing; they have the possibility to modify what we call, for example, the antigens sites. This means that if we want to make a vaccine, we can modify the protein subject to the vaccine by a small sequence coming from another virus.  There’s a will to suppress the works on the subject.  We are not the first.  A group of renowned Indian researchers have published the same thing.   But they forced them to retract it. It has been cancelled.  If you check their work you find a cancellation band.  I’m out of age and I’m a Nobel laureate so I can work freely; so no pressure can be exerted on me.”  …

  India paper—  “We found four new insertions in the S protein of SARS-CoV-2 when compared to its nearest relative, SARS CoV. The genome sequence from the recent 28 clinical isolates showed that the sequence coding for these insertions are conserved amongst all these isolates.  This indicates that these insertions have been preferably acquired by the SARS-CoV-2, providing it with additional survival and infectivity advantage.  Delving deeper we found that these insertions were similar to HIV-1.

  "Our results highlight an astonishing relation between the gp120 and Gag protein of HIV, with SARS-CoV-2 spike glycoprotein.  These proteins are critical for the viruses to identify and latch on to their host cells and for viral assembly (Beniac et al., 2006).  Since surface proteins are responsible for host tropism, changes in these proteins imply a change in host specificity of the virus.   According to reports from China, there has been a gain of host specificity in case SARS-CoV-2 as the virus was originally known to infect animals and not humans but after the mutations, it has gained tropism to humans as well.  Moving ahead, 3D modelling of the protein structure displayed that these insertions are present at the binding site of SARS-CoV-2.  Due to the presence of gp120 motifs in SARS-CoV-2 spike glycoprotein at its binding domain, we propose that these motif insertions could have provided an enhanced affinity towards host cell receptors.  Further, this structural change might have also increased the range of host cells that SARS-CoV-2 can infect.  To the best of our knowledge, the function of these motifs is still not clear in HIV and need to be explored. The exchange of genetic material among the viruses is well known and such critical exchange highlights the risk and the need to investigate the relations between seemingly unrelated virus families.

  "Conclusions:  Our analysis of the spike glycoprotein of SARS-CoV-2 revealed several interesting findings:  First, we identified 4 unique inserts in the SARS-CoV-2 spike glycoprotein that are not present in any other coronavirus reported till date.  To our surprise, all the 4 inserts in the SARS-CoV-2 mapped to short segments of amino acids in the HIV-1 gp120 and Gag among all annotated virus proteins in the NCBI database.  This uncanny similarity of novel inserts in the SARS-CoV-2 spike protein to HIV-1 gp120 and Gag is unlikely to be fortuitous.  Further, 3D modelling suggests that at least 3 of the unique inserts which are non-contiguous in the primary protein sequence of the SARS-CoV-2 spike glycoprotein converge to constitute the key components of the receptor binding site.  Of note, all the 4 inserts have pI values of around 10 that may facilitate virus-host interactions. Taken together, our findings suggest unconventional evolution of SARS-CoV-2 that warrants further investigation.  Our work highlights novel evolutionary aspects of the SARS-CoV-2 and has implications on the pathogenesis and diagnosis of this virus."    -Prashant Pradhan, Ashutosh Kumar Pandey, Akhilesh Mishra, Parul Gupta, Praveen Kumar Tripathi, Manoj Balakrishnan Menon, James Gomes, Perumal Vivekanandan, Bishwajit Kundu

doi: https://doi.org/10.1101/2020.01.30.927871

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