SARS-CoV-2 CQ.1 and CQ.1.1 lineage isolates: 100% synonymous base substitution at furin arginine CGG–CGG codons (preprint)

The SARS‐CoV2 spike glycoprotin S1/S2 junction not only has no analogy in other becoronaviruses, including neither the Laos closest known relatives, but also it has two arginine codons CGG–CGG, whose usage is extremely rare in coronaviruses. The 12-nt SARS-CoV-2 S gene insert having that CGG–CGG genetic footprint 100% match to several human mRNA RefSeq transcripts which are located in exons of ubiquitous and highly expressed genes or specific genes of target human tissues of virus infection. An hypothesis for the probable human origin of the S1/S2 junction polybasic motif (originally PRRA) includes that it has been acquired by recombination between the genome of a SARS-CoV-2 progenitor and mRNA transcripts within human infected cells. Since the furin arginine pair is essential for virus infection to human cells and arginine has six codons SARS-CoV-2 can optimize the CGG–CGG codons. Here I show that in a sample of the available GISAID SARS-CoV-2 CQ.1 (516) and CQ.1.1 (117) lineage isolates the 100% of them showed synonymous base substitution at this position. The CGG–CGG footrprint to have changed to CGT–CGG. The location of the CQ.1 lineage isolates was: Asia 8.30% (45 isolates), Europe 76.57% (415), North America 6.64% (36), Oceania 8.30% (45) and South America 0.18% (1). That of CQ.1.1 lineage isolates was: Europe 45.6% (57) and North America 54.4 (68). Based on NCBI Virus database SARS-CoV-2 lineages associated to spike glycoprotein sequences I created a SARS-CoV-2 lineage ranking the from earliest CQ.1 date (August 30, 2022) up to date. Of 418,257 records, there were 1,165 distinct lineages. Ranking positions for CQ.1 and CQ.1.1 lineages were 582 and 506, respectivaly, both in the second quartile. These results coincide with those obtained previosly in SARS-CoV-2 CQ.2 and EE.2 lineage isolates. Everything points to that through evolution SARS-CoV-2 adapts the extremely rare CGG–CGG footprint to its own genomic parameters, thereby also erasing an evidence on its origin.

Synonymous base substitution in SARS-CoV-2 EE.2 lineage furin arginine CGG–CGG codons (preprint)

This work has been carried out within the framework of a study on SARS-CoV-2 spike glycoprotein furin cleavage site evolution. The polybasic motif (originally PRRA) present at the S1/S2 junction has two arginine codons CGG–CGG, whose usage is rare in coronaviruses. It has no analogy in other “linage B” beta‐coronaviruses, including neither the Laos closest known relatives of virus behind covid. An hypothesis for theprobable PRRA human origin includes recombination between the genome of a SARS-CoV-2 progenitor and mRNA transcripts within human infected cells. At protein level, the furin arginine pair is evolutionarily strictly conserved, however, the CGG–CGG code can be optimized by the virus itself through synonymous base substitutions. Here I show that the GISAID SARS-CoV-2 EE.2 lineage isolates (accessed June 29, 2023), 531 out of 1,021 (52.2%) have already optimized this furin CGG–CGG code. It has changed to CGA–CGG. As a new brand of the lineage all these 531 isolates share the same mutation in the third position of the first of the two arginine codons. In addition of the codon usage bias affecting R682 there are also two non-synonymous base substitutions N679K and P681R. The earliest date of the SARS-CoV-2 EE.2 lineage was September 12, 2022. Based on NCBI Virus database, from this date appear 1,028 different SARS-CoV-2 lineages. The majority was XBB.1.5 (64,232 isolates 17.17%). However, the ranking position of the EE.2 lineage was 167 (331 isolates, 0.088%), still in the first quartile. Results provide insight into the change of an extremely rare genetic footprint that within an exogenous 12-nucleotide fragment that was imprinted in the SARS-CoV-2 S gene.

SARS-CoV-2 spike glycoprotein hotspots for RNA recombination provide insight into the probable human origin of the PRRA polybasic furin insert (preprint)

The SARS-CoV-2 origin is linked to the acquisition by its closest progenitor of a positive insert in the spike glycoprotein. Itwas the PRRA polybasic motif atthe furin cleavage site. This furin recognition motif has no analogy with other B-lineage beta-coronaviruses, nor with the known Laos bat closest relatives. An hypothesis for probable PRRA human origin includes recombination between a SARS-CoV-2 progenitor genome and mRNA transcripts within human infected cells (previous study). However, whether during the pandemic PRRA-like insertions had occurred in the spike glycoprotein is unknown. Here I show a computational analysis of many large multiple sequence alignments involving 2,315,308SARS-CoV-2 spike glycoprotein sequences downloaded from NCBI Virus database (collection date January 2020 - April 2023). I found theN-terminal domain SRWM insert in 331 sequences from SARS-CoV-2clinical isolates with15 different submitters and organizations, lineages BE.1.1, BQ.1, BQ.1.28, FD.4; USA, 26 states, andcollection date November 2022 up to the analysis period. Both the SRWM S gene 12-nt coding insertand its reverse complement 100% matchedto several human mRNA RefSeq transcripts. The related genes were ubiquitous and highly expressed or specific of virustarget organs or tissues. I have also identified other 50 inserts of four amino acids or more in different structural domains of the protein fromdifferent SARS-CoV-2 clinical isolates. All with the similarcharacteristics. Inconclusion, these SARS-CoV-2 spike glycoprotein inserts seem not to be of viral origin. SARS-CoV-2 is a human virus. Human isthe host. The origin cannot be from another virus. It can be stated that a recombination between SARS-CoV-2 genome and human mRNA transcripts within infected cells would bethe origin of the inserts. The PRRA insert has the same features as those described here. This strongly support the hypothesis that the 12 nucleotides insert imprinted in the S gene encoding PRRA does not have a viral origin either, ratherhuman mRNA transcripts. In this case, SARS-CoV-2 must be changed by its closest ancestor. Viruses swap chunks of RNA through recombination: thus SARS-CoV-2 emerged - thus it also evolves.

On-going evidence on SARS-CoV-2 furin site arginine codon usage bias and new found PRRA-like insertions (preprint)

The origin of SARS-CoV-2 lies in the gain-of-function related to the PRRA spike glycoprotein polybasic furin cleavage site (FCS). The CGG-CGG arginine pair code is unique and rare in the virus. However, novel viruses found in Laos can be considered SARS-CoV-2 ancestors, but they do not have the PRRA. At this point, I have described: (i) the 100% match between the exogenous S gene fragmentencoding PRRA andhuman mRNA RefSeq transcripts; (ii) the related genes are highly expressed in virustarget tissues; (iii) PRRA arginine codon usage bias; (iv) PRRA-like insertions; and (v) the PRRA human origin hypothesis. The aim of this workis to provide further support tothishypothesis. Based on current SARS-CoV-2 sequences (January-March, 2023), here I show that 43 out of 10,644 (0.42 %) GISAID Africa, Asia, Europe and the USA complete genomes and 613 out of 134,022 (0.45 %) NCBI-Virus spike glycoprotein sequences had the CGG-CGG code optimized. The results are in agreement with those in previous works that reveal a viral FCS arginine codon usage bias. Here I also show new found spike glycoprotein PRRA-like insertions (100% match to human mRNA RefSeq transcripts and related genes virus target tissue specifics). In these PRRA-like insertions the human mRNA transcripts should be the origin of the coding S gene fragments. Human is the host of the involved SARS-CoV-2 specimens. They are human viruses isolated from infected human cells. The origin cannot be genetic material from other viruses. Regarding the mechanism, it would be the most common in viruses evolution: recombination. The evolutionary success of the PRRA-like insertions lies in the respect of the reading frame on S gene translation. Results suggest that PRRA spike glycoprotein insertion in a SARS-CoV- ancestor was not an isolated evolutionary event, but rather common in virus evolution.

SARS-CoV-2: gone with the evolution (preprint)

Starting at the end, evolution is erasing the imprinted CGG-CGG genetic footrprint on theSARS-CoV-2 acquired with the furin cleavage site. CGG-CGG is the original code ofthe arginine pair inthe polybasic furin cleavage site of the viralS protein. CGG-CGGis the heart of the 12nucleotide length sequence that properly inserted into the S gene encoded the PRRA motif in the early viruses. In Omicron it isHRRA. In terms of functionality, this arginine dimer in the SARS-CoV-2 S proteinis untouchable, but its code is very unfavourable for the virus. So it is plausiblethat asolution for the virus should go through an argininebias codon usage in the furin site. The aimof this study was to going onwith the longitudinal study on the evolution of this furin site arginine dimer, so essential for the virus. However, the surprise has been to discover that at least in the CQ.2 lineage, based on theavailable GISAID genomes, the original CGG-CGG arginine code has been lost and optimized in the 99.65 % of cases. It is worth noting thatthe new codon usage matches the arginine pair of the SARS-CoV-2 furin clevage site and arginine pairsof other viral proteins (e.g., nsp13, nucleocapsid) of both SARS-CoV-2and bat coronaviruses.

A piece in the SARS-CoV-2 genome mosaic: furin site on the spike protein (preprint)

The recent Smriti Mallapaty’work (Nature. News, 10 November 2022) highlights the role of recombination in the evolution of the SARS-CoV-2. It seems hard to deal the origin of the virus as a whole. The SARS-CoV-2genome is a mosaic of regions or parts, each with their own origin. The SARS-CoV-2’s closest known relative is a bat virus found in Laos called BANAL-52, which can therefore infect human cells. Furthermore, BANAL-52 don’t contain the so-called furin cleavage site on the spike protein that further aids the entry of SARS-CoV-2 into human cells. So, the furin site was a key piece in the virus “mosaic”. However, The SARS-CoV-2 furin site “gain-of-function” was being made against the CGG-CGG genetic footprint encoding the polybasic arginine dimer. For SARS-CoV-2 the only output to the CGG-CGGinstability is to proceed with synonymous substitutions (silent mutations) keeping the arginine dimer from the furin site. Theaim of this work is to provide updated data on the plausibility of this evolution in the coding of thatarginine dimer. Using the NCBI Virus database as a source of information, the sample of SARS-CoV-2 spike proteins, with releasedate November 1-10, 2022, shows that 78 out of 27281 (0.28 %) sequences show a synonymous base substitution in the CGG-CGG. This percentage is twice of samples retrieved in August 2022.

SARS-CoV-2 is losing the CGG-CGG genetic footprint. Working sample of August 2022 (preprint)

This work should be seen as a timely delivery of results of a longitudinal study on the arginine codon usage bias in the arginine pair of the SARS-Cov-2 S protein polybasic furin cleavage site. The work sample was made up of virus sequences from NCBI Virus and GISAID databases, with August 2022 as release date. Bats harbour a number of emerging viruses, many of which are highly pathogenic in humans, including the closest relative of the SARS-CoV-2. Everything suggests that progenitor of SARS-CoV-2 jumped into humans, and during undetected human-to-human transmission, a SARS-CoV-2 progenitor acquired from human mRNA transcripts, a fragment of 12-nucleotides that properly inserted in the S gene gave rise to the SARS-CoV-2 polybasic furin cleavage site. This exogenous 12-nt-long sequence has the CGG-CGG genetic footprint that integrated into the S gene coding reading frame, lets encoded the furin site arginine dimer. In this region of S gene and/or S protein, an evolutionary pressure would operates in opposite directions. At the phenotype level, this arginine dimer must remain fixed or consolidated. However, at the genotype level, the virus optimizes the CGG-CGG arginine pair code to achieve more favourable arginine codons. Based on NCBI Virus and GISAID databases as a source of information, and using Perl scripts, here I show that 165 out of 126,727 (0.1302 %) NCBI Virus S protein sequences show synonymous base substitution in the furin site arginine dimer. I have also found this loss of the CGG-CGG code in 21 out of 18,750 (0.1120 %) GISAID S protein sequences. Results provide insight into the evolutionary effect on this strategic regions of the SARS-CoV-2 S protein.

Tracking evolutionary pressure on the SARS-CoV-2 polybasic furin cleavage site arginine dimer: 25-31 July, 2022, sample (preprint)

This work is part of a longitudinal study on the evolutionary pressure at the arginine dimer of the SARS-CoV-2 S-protein polybasic furin cleavage site. When a virus precursor acquired that furin site, it was not only a huge gain-of-function, but it was also the origin of a species (SARS-CoV-2). At molecular level, the furin site opened up two conceptually highly opposed landscapes. First (favourable for the virus), at protein level, the arginine dimer within the furin site provides an additional positive charge to the S protein that is crucial for the human furin activity. Second (no favourable for the virus), at gene level, this arginine dimer is CGG-CGG encoded. The CGG arginine codon is the arginine codon less favourable in the virus genome (arginine has six codons). The arginine codon usage bias could be a viral strategy to optimize that arginine dimer code. Results shown here thus suggest. They refer to a set of SARS-CoV-2 genomes (0.18 %), from a sample the last days of July, 2022, with a genetically optimized furin site.

SARS-CoV-2 codon usage bias at furin site clear up the origin. Landscape of Omicron sub-variants BA.4 and BA.5 (a July, 2022 sample) (preprint)

SARS-CoV-2 has a 12-nt-long exogenous sequence into the S gene, encoding the polybasic furin cleavage site, including the CGG-CGG arginine dimer. In this regard, I recently posted two facts: (i) this 12-nt and the reverse complement sequences are identical to a many highly expressed sequences in human cells, suggesting that a progenitor of SARS-CoV-2 jumped into humans and recombination with human cell mRNA produced the pandemic virus; and (ii) the identification of a representative group of SARS-CoV-2 specimens with arginine codon usage bias in the furin site. Are these two independent facts? To address the issue, the aim of this work is to trace the arginine codon bias in the dimers of this amino acid in the furin site from virus updated genomes. At S protein level, the acquisition of the furin site was enormously favourable for the virus. For the humanity was (or is) the new and unprecedented covid pandemic. Moreover, at S gene level the CGG-CGG arginine code was enormously unfavourable for the virus. Since arginine has six codons, the virus can overcome the situation through a codon usage bias. Here I show some SARS-CoV-2 Omicron sub-variants BA.4 and BA.5 (a July, 2022, sample) with furin site arginine dimer codon optimized: 8 out of 4,986 (0.16 %). In SARS-CoV-2 the synonymous base substitutions are not common, however, in this particular case of the arginine, it is the only way that the virus has to stabilize its genome. On the other hand, from science, this arginine codon usage bias can support the hypothesis of recombination for the SARS-CoV-2 polybasic furin cleavage site origin. Finally, this work opens the door to monitor the course of that codon bias in the new virus genomes that are coming.

Probable human origin of the SARS-CoV-2 polybasic furin cleavage site (preprint)

Background: the origin of the current SARS-CoV-2 virus is still elusive. However, the finding that Laos bats host closest known relatives of virus behind Covid that even without the furin site they can infect human cells, it is a giant stride forward to known the progenitor of the virus. The mystery focus on the CGG-CGG code of the SARS-CoV-2 furin site arginine dimer. This genetic footprint has not been observed in natural coronaviruses.  Results: based in a bioinformatic approach I found that the two possible 12-nucleotide fragment (and they reverse complement) properly inserted within the codon S680 in the S gene of the closest relative to the virus, encoding the PRRA furin site motif, 100% match to severe human NCBI RefSeq curated mRNA protein-coding transcripts. The genes to which these transcripts belong are ubiquitously and highly expressed genes, e.g., the alpha subunit of the mitochondrial ATP synthase F1 (ATP5F1) and/or the ubiquitin specific peptidase 21 (USP21); or specific and highly expressed genes in tissues such as small intestine, duodenum, brain, kidney and gonads which are the gateway of the SARS-CoV-2 infection. On the other hand, a significant group of virus specimens have been discovered in which the arginine dimer of the furin site is codon-optimized by the SARS-CoV-2 codon usage bias. Another main finding was to discover other PRRA-like insertions in the S protein from some SARS-CoV-2 specimens.  Conclusions: the first conclusion of this work is that the SARS-CoV-2 is deleting the non-viral origin of the furin site CGG-CGG arginine pair through codon optimization. Based on the results shown here together with the new Laos coronaviruses the main conclusion of this work is that a genetic recombination of unrelated RNA sequences between the a closest relative to SARS-CoV-2 and human transcripts, during an undetected viral circulation in humans, could be the origin of the polybasic furin cleavage site.

SARS-CoV-2 and MERS-CoV Share the Furin Site CGG-CGG Genetic Footprint (preprint)

The SARS-CoV-2 polybasic furin cleavage site is still a missing link. Remarkably, the two arginine residues of this protease recognition site are encoded by the CGG codon, which is rare in Betacoronavirus. However, the arginine pair is common at viral furin cleavage sites, but are not CGG-CGG encoded. The question is: Is this genetic footprint unique to the SARS-CoV-2? To address the issue, using Perl scripts, here I dissect in detail the NCBI Virus database in order to report the arginine dimers of the Betacoronavirus proteins. The main result reveals that a group of Middle East respiratory syndrome-related coronavirus (MERS-CoV) (isolates: camel/Nigeria/NVx/2016, host: Camelus dromedarius) also have the CGG-CGG arginine pair in the spike protein polybasic furin cleavage region. In addition, CGG-CGG encoded arginine pairs were found in the orf1ab polyprotein from HKU9 and HKU14 Betacoronavirus, as well as, in the nucleocapsid phosphoprotein from few SARS-CoV-2 isolates. To quantify the probability of finding the arginine CGG-CGG codon pair in Betacoronavirus, the likelihood ratio (LR) and a Markov model were defined. In conclusion, it is highly unlikely to find this genetic marker in betacoronaviruses wildlife, but they are there. Collectively, results shed light on recombination as origin of the virus CGG-CGG arginine pair in the S1/S2 cleavage site.

SARS-CoV-2 and the Secret of the Furin Site (preprint)

The SARS-CoV-2 high infectivity is due to the functional polybasic furin cleavage site in the S protein. How it was acquired is unknown. There are two challenges to face: (i) an evolutionary model, to fit the origin of the coronavirus; and (ii) a molecular mechanism for the site acquisition. Here we show genomic fingerprints which are specific of Pangolin-CoVs, Bat-SARS-like (CoVZC45, CoVZXC21), bat RatG13 and human SARS-CoV-2 coronaviruses. This, along with phylogenetic analysis, we found that these species have the same evolutionary origin in the bat, including a genetic recombination of S gene between Pangolin-CoV (2017) and RatG13 ancestors. However, this does not explain why SARS-CoV-2 is the only of them with the furin site, which consists in four amino acid (PRRA) motif. The Arginine doublet is encoded by CGGCGG codons. Surprisingly, none of the Arginine doublet of other furin site of viral proteins from several type of viruses, are encoded by the CGGCGG codons. This makes it difficult to consider a virus recombination as mechanism for the PRRA acquisition. The origin of SARS-CoV-2, is the origin of the recognition cleavage site. The bat coronavirus RaTG13 appears to be the closest relative of the SARS-CoV-2, but was isolated in 2013. So, new RatG13 samples would provide insights into the acquisition of the polybasic motif.