SARS2 has a 12-nucleotide insert right at the S1/S2 junction.

   Based on reports from hospitals, Dr. Kang, executive director of the Translational Health Science and Technology Institute in India, said, it appeared that B.1.617 was causing more severe disease.   W.H.O. researchers determined that B.1.617 is spreading fast in India, making up over 28 percent of samples from positive tests.  https://www.nytimes.com/live/2021/05/11/world/covid-vaccine-coronavirus-cases

This virus variant is now in about 50 countries and in many places rising in its activity.  

-r.

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5-10-21     “Since 1992 the virology community has known that the one sure way to make a virus deadlier is to give it a furin cleavage site at the S1/S2 junction in the laboratory,” writes Dr. Steven Quay, a biotech entrepreneur interested in the origins of SARS2.  “At least eleven gain-of-function experiments, adding a furin site to make a virus more infective, are published in the open literature, including [by] Dr. Zhengli Shi, head of coronavirus research at the Wuhan Institute of Virology.”…

  SARS2 has a 12-nucleotide insert right at the S1/S2 junction.  The insert is the sequence T-CCT-CGG-CGG-GC.  The CCT codes for proline, the two CGG’s for two arginines, and the GC is the beginning of a GCA codon that codes for alanine.

  There are several curious features about this insert but the oddest is that of the two side-by-side CGG codons.  Only 5% of SARS2’s arginine codons are CGG, and the double codon CGG-CGG has not been found in any other beta-coronavirus.  So how did SARS2 acquire a pair of arginine codons that are favored by human cells but not by coronaviruses?…

  The human-preferred codon is routinely used in labs.  So anyone who wanted to insert a furin cleavage site (FCS) into the virus’s genome would synthesize the PRRA-making sequence in the lab and would be likely to use CGG codons to do so.   -N. Wade  https://science.thewire.in/the-sciences/origins-of-covid-19-who-opened-pandoras-box-at-wuhan-people-or-nature/

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1-8-21   Researchers also investigated whether the SARS-CoV-2 spike protein could bind to the ACE2 receptors from 22 different animals to ascertain which of these, if any, may be susceptible to infection. They demonstrated that bat and bird receptors made the weakest interactions with SARS-CoV-2.

https://www.pirbright.ac.uk/news/2021/01/pirbright-study-shows-how-sars-cov-2-could-have-adapted-bats-humans-and-which-other

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3-25-21  Because the presence and coding sequence of a FCS is important for pathogenesis, host range, and cell tropism (Nagai et al. 1993; Millet et al. 2015), the addition of a FCS into viruses has been an active area of gain-of-function research.  A FCS can be easily inserted using seamless technology (Yount et al. 2002; Sirotkin and Sirotkin 2020) without any need for cell passage, as previously performed in experiments on virulence and host tropism (Cheng et al. 2019).  Insertions to change the properties of SARS-r CoV viruses are documented by Ren et al. (2008) and Wang et al. (2008).  Considering that natural mutations have a very low probability to result in a stretch of 12 amino acids coding for an optimized FCS without any known intermediate form in Sarbecovirus, an artificial insertion of the FCS in SARS-CoV-2 may provide a more parsimonious explanation for its presence than natural evolution.

In summary, the FCS confers SARS-CoV-2 enhanced human pathogenicity and has never been identified in another Sarbecovirus.  At the same time, FCSs have been routinely inserted into coronaviruses in gain-of-function experiments, and we provide a hypothesis through which the specific amino acid sequence of SARS-CoV-2′s FCS may have been generated through cell culture.   https://link.springer.com/article/10.1007/s10311-021-01211-0

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2.1.  Serial Passage and Its Molecular Signatures    Although serial passage mimics many of the natural zoonotic processes that occur during a natural zoonotic jump, because serial passage artificially condenses a natural phenomenon into a small temporal window, some subtle differences can be found.  In addition to the inexplicable genetic distance from its sister strains, which screams out for an intermediate relative to complete the phylogenetic picture, SARS-CoV-2 has a remarkably strong affinity for spike-protein binding to ACE2--some 10– 20 times higher than SARS-CoV’s.[18]  That affinity may have emerged after mutational events either in an intermediate natural host or after a zoonotic jump into humans that theoretically could have occurred earlier than the first documented infection, which would give it time to increase that significantly.  So logically, it could also have emerged via selection after serial passage through laboratory cell cultures or laboratory animals as well.  And regarding the second distinctive feature found in the novel coronavirus:  If other viruses have been observed to acquire furin cleavage sites by passage under experimental laboratory conditions, then such a mechanism is theoretically possible for SARSCoV-2 as well.[2]  In the case of influenza viruses like those mentioned above, their gain-of-function furin cleavage sites are thought to be a result of two different molecular processes.  The first is either nucleotide insertions or substitutions that are able to be rescued and then eventually selected for due to the high multiplicity of infection found in serial passage protocols.[19] And the second is the recombination of multiple viral RNAs inside a host cell,[20] which may also include additional viruses introduced through accidental laboratory co-infections.  Unlike influenza viruses, serial passage through ferrets has not been recorded in the literature for coronaviruses.  However, since several branches of coronavirus have furin cleavage sites, a molecular pathway for their emergence must exist and may reemerge during serial passage.  Several factors weigh into the probability that coronaviruses can gain furin cleavage sites following serial passage:  The frequency of evolutionary motifs meant to deal with virus–host interactions that are often shared between viruses, the observations that when the infectious bronchitis coronavirus (IBV) coronavirus is serially passed through chickens it developed notable mutations along its spike-protein genes,[21] and the fact that when a lineage A bovine coronavirus was subject to in vitro serial passage through cell lines, a 12- nucleotide insert found within only a small minority of the pooled viruses spike-protein region was strongly selected for and quickly emerged as the dominate strain.[22]  These findings all point to the possibility that SARS-CoV-2 may have gained its furin cleavage site the same way influenza viruses do—through the in vivo serial passage between the live hosts that presents the immune challenges and intense selective pressure necessary for the recombination and mutations that lead to its emergence to occur. And just like influenza viruses are only able to preserve their furin cleavages in artificial environments since the heightened virulence they impart kills their hosts before they can propagate in a natural setting, based on the known taxonomy lineage B coronaviruses do not appear to be able to support furin cleavages in nature.  There is no doubt that the acquisition of the furin cleavage site was one of the key adaptations that enable SARS-CoV-2 to efficiently spread in the human populations compared to other lineage B coronaviruses, and provides a gain-of-function.[23] In addition to the possibility of obtaining a furin cleavage site through natural recombination in a secondary host or through serial passage either in a laboratory or on a commercial farm, one could have been spliced directly into the novel coronavirus’s backbone in a laboratory using classic recombinant DNA technology that has been available for nearly 20 years.  This allows for the removal of the restriction site junctions that are the telltale sign of direct genetic manipulation and permits reassembly without introducing nucleotide changes--creating a virus without any evidence of manipulation using the aptly named “No See’m technology.”[24]  So although the entire spike-protein RBD was not assembled from scratch, it is certainly plausible that the 12-nucleotide-long furin cleavage site could have been spliced directly into SARS-CoV-2. Furin cleavages already have been successfully spliced into other coronaviruses, including the IBV,[25] and even into SARS-CoV, where it increased cell-to-cell fusion in in vitro experiments that only examined only the spike-protein’s function, which would presumably heighten its infectivity in vivo.[26]  Moreover, when a furin cleavage site was introduced to the IBV coronavirus spike-protein via recombination, just like influenza viruses hosting this feature, it appeared to impart it with increased lethality as well as inflict neurological symptoms that had never previously been reported in studies of the murine IBV coronavirus.[25]  The presence of this cleavage site also increased damage to the respiratory and urinary systems, paralleling SARSCoV-2 systemic multiorgan symptoms--especially reports that infection with the novel coronavirus not only targets the lungs where it binds to ACE2 receptors, but also the entire cardiovascular system,[27] the nervous system,[28] and our kidneys as well.[29] It might be more than a coincidence that the Vero cells often used in serial passage are derived from kidney epithelial cells extracted from African green monkeys, which have ACE2 receptors very similar to those found in humans and would be shared by the humanized mice that are also used for serial passage research.  

2.2. Natural Origin, or Gain-of-Function Lab Escape?  Gain-of-function research on bat-borne coronaviruses has been ongoing for nearly a decade everywhere from the University of North Carolina to the Wuhan’s Institute of Virology, which is supported by related facilities such as Wuhan’s Center for Disease Control and Prevention as well as Wuhan University.  A coronavirus that targets the ACE2 receptor like SARS-CoV-2 was first isolated from a wild bat in 2013 by a team out of Wuhan.  This research was funded in part by EcoHealth Alliance,[30] and set the stage for the manipulation of bat-borne coronavirus genomes that target this receptor and can become airborne.  Many more viruses have been collected in Wuhan over the years    -Sirotkin and Sirotkin, summer 2020   https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/bies.202000091

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