Peturban

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Once again, welcome, and happy editing! -- Treck08 (Diskussion) 15:13, 3. Jan. 2022 (CET)

Omicron_mutationen

Hallo Peturban, zu Diskussion:SARS-CoV-2-Variante Omikron#Omicron mutationen habe ich drei Fragen:

• Möchtest Du mit oder zu der langen Liste der Studien zu den Mutationen eine Aussage treffen? Wenn ja, welche?
• Hast Du die Studien nur so aufgelistet oder bist Du inhaltlich tiefer drin? Falls ja, wäre jeweils ein Kurzhinweis je Mutation hilfreich, welche Studie am zielführendsten ist.
• Und/oder hast Du die Auflistung gemacht, weil Dir im Artikel etwas fehlt? Wenn ja, was?

Du kannst entweder hier auf Deiner Diskussionsseite antworten oder auf der Omikron-Diskussionsseite Diskussion:SARS-CoV-2-Variante Omikron ergänzen. Liebe Grüße, --Treck08 (Diskussion) 23:12, 2. Jan. 2022 (CET)

I answer in English because I do not wish to translate the English citations from the original papers to German.

My intention was to provide original scientific records, which help to understand the properties of the omicron variant for those who take the time to read them. My intention was not to draw a a subsequent “pre-made” conclusions due to the reasons described below. As a molecular geneticist, who contributed from the late 70s to the development of molecular biology and recombinant DNA research tools, I think that without critical reading of original scientific publications it is difficult to comment on the conclusions of popular newspaper articles (like Spektrum, NCB etc. references used in the Omicron draft), especially those, which discuss the three current theories on the evolution of Omicron variant.

To answer your second question:

Of course, I have read all the papers for which I provided the references, and even tested the conclusions of the first cited paper by performing sequence homology comparisons and building from them an evolutionary tree. Like most other scientist, I also failed to conclude that the new variant derived by spontaneous mutations from the previously known pandemic variants.

Therefore, to start with, I provided in my mail the

“Wei et al. (2021) Evidence for a mouse origin of the SARS-CoV-2 Omicron variant. J Genet Genomics. 2021 Dec 23:cS1673-8527(21)00373-8. doi: 10.1016/j.jgg.2021.12.003. Epub ahead of print. PMID: 34954396; PMCID: PMC8702434.”

reference, which contains several other citations that follow this reference in the list.

Wei et al. say that:

“we identified 45 point mutations that Omicron acquired since divergence from the B.1.1 lineage. We found that the Omicron spike protein sequence was subjected to stronger positive selection than that of any reported SARS-CoV-2 variants known to evolve persistently in human hosts, suggesting the possibility of host-jumping. The molecular spectrum (i.e., the relative frequency of the twelve types of base substitutions) of mutations acquired by the progenitor of Omicron was significantly different from the spectrum for viruses that evolved in human patients, but was highly consistent with spectra associated with evolution in a mouse cellular environment… Collectively, our results suggest that the progenitor of Omicron jumped from humans to mice, rapidly accumulated mutations conducive to infecting that host, then jumped back into humans, indicating an inter-species evolutionary trajectory for the Omicron outbreak.”

However, Wei et al. do not mention that mouse-adaptation of the SARS-COC-2 virus was not a natural process, because the human SARS-COV-2 virus is either not or very badly infects and replicates in wild and laboratory mice. So, the mouse adaptation procedure means multiple (sometimes 16 to 28 times) injection of mice with an infective human SARS-COV-2 stock to isolate a mutation in the virus, which permits better replication in mice and plaque formation in mouse tissue culture). To overcome this problem, for example,

“Roy et al. (2021) Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19. bioRxiv [Preprint]. 2021 Apr 21:2021.04.20.440676. doi: 10.1101/2021.04.20.440676.”

applied the following procedure, which I cite here from their paper:

“Isolation of mouse-adapted virus.

Mice are naturally resistant to infection with SARS-CoV-2, resulting from incompatibility between the viral surface (S) glycoprotein and mouse angiotensin converting enzyme 2 (mACE2). To address this incompatibility, human ACE2 (hACE2) has been provided by genetic manipulation of the mouse genome or exogenously using viral vectors. Transgenic expression of hACE26-9, complete or partial replacement of mACE2 with hACE2 (hACE2-KI)10 or administration of viral vectors expressing hACE2 all sensitize mice for infection. Alternatively, the spike protein (S) of SARS-CoV-2 has been altered using reverse genetics to enable binding to mACE213,14. SARSCoV-2 has been adapted to mice by targeting amino acids at positions 498/499 (Q498Y/P499T) or 501 (N501Y). The resulting viruses can infect mice, although the resulting infection is very mild. However, additional virus passage through mouse lungs results in increased virulence….

             To generate a virulent mouse-adapted SARS-CoV-2, we inserted the N501Y mutation into the SARS-CoV-2 genome (rSARS2-N501YP0) using reverse genetics as previously described. As expected, this mutation rendered mice susceptible to SARS-CoV-2 infection but BALB/c mice lost only a minimal amount of weight even after administration of 105 PFU of virus….

             We sequenced viruses after 10, 20 and 30 passages. Virus from these passages became progressively more lethal …. Focusing on changes in the S protein, passage 10 virus contained a Q498R mutation, which likely enhanced binding to mACE213... By passage 20, a mutation at position 493 (Q493R) was detected. By passage 30, mutations in residues 417 and 484 (K417M, E484K) also arose. E484K does not appear to be required for mouse virulence since it was variably expressed by different isolates that had equivalent lethality…

             Two additional spike mutations (Q498R and Q493R) further increased affinity and stability, mainly driven by R493 which can create a salt bridge with D38 in mACE2 (previously a hydrogen bond between Q493 in spike and D38 in mACE2).”

Rathnasinghe et al. (2021) The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and aged mice and is neutralized by convalescent and post-vaccination human sera. medRxiv [Preprint]. 2021 Jan 20:2021.01.19.21249592. doi: 10.1101/2021.01.19.21249592.

describe a similar approach in their paper:

“The initially available SARS-CoV-2 isolates require adaptation in order to use the mouse angiotensin converting enzyme 2 (mACE-2) entry receptor and to productively infect the cells of the murine respiratory tract. We have “mouse-adapted" SARS-CoV-2 by serial passaging a clinical virus isolate in the lungs of mice. We then used low doses of this virus in mouse models for advanced age, diabetes and obesity. Similar to SARS-CoV-2 infection in humans, the outcome of infection with mouse-adapted SARS-CoV-2 resulted in enhanced morbidity in aged and diabetic obese mice. Mutations associated with mouse adaptation occurred in the S, M, N and ORF8 genes….

             Many of these models rely on transgenic mice that express hACE-2 in epithelial cells or sensitize mice to SARS-CoV-2 infection by adenovirus mediated transduction of the hACE-2 gene (Ad-hACE-2) in the respiratory tract…(see further references in the paper).

             In this study, we have developed and characterized a MA-SARS-CoV-2 strain after serially passaging a clinical virus isolate (USA-WA1/2020)… We mapped mutations associated with mouse adaptation in the SARSCoV-2 genome and observed that one of them is the N501Y mutation…with potentially enhanced human transmission potential. This mouse-adapted SARS-CoV-2 strainwith N501Y mutation causes enhanced morbidity in mouse models…

             The USA-WA1/2020-SARS-CoV-2 (termed WT-SARS-CoV-2) virus isolate was passaged eleven times in the lungs of various strains of mice… The virus was then further passaged three times in BALB/c mice and four times in 129 mice. The 129 mice were chosen for mouse adaptation as they have been shown to be more susceptible to SARS-CoV, a virus that uses the same hACE-2 receptor as SARS-CoV-2. After eleven passages, the virus was plaque-purified and clonal virus stocks of the MA-SARS-CoV-2 were prepared in mACE-2 expressing Vero-E6 for further infection experiments." (Note1: WIKIPEDIA: Bei Vero-Zellen handelt es sich um eine etablierte kontinuierliche Zelllinie, die aus normalen Nierenzellen von Grünen Meerkatzen gewonnen wurde. Die Grünen Meerkatzen (Chlorocebus) sind eine Primatengattung (früher „Affen“ genant) aus der Familie der Meerkatzenverwandten (Cercopithecidae). Note2: Das an Mäuse adaptierte Virus war also wahrscheinlich an Primatenzellen "rückadaptiert".)

Montagutelli et al. (2021) A mouse-adapted SARS-CoV-2 strain replicating in standard laboratory mice bioRxiv 2021.07.10.451880; doi: https://doi.org/10.1101/2021.07.10.451880 write:

“Mouse infection with SARS-CoV-2 is limited by poor affinity between the virus spike protein and its cellular receptor ACE2. We have developed by serial passages the MACo3 virus strain which efficiently replicates in the lungs of standard mouse strains and induces age-dependent lung lesions… After 15 and 16 passages, two viral variants named MACo1 and MACo2 were isolated. Sequencing revealed a shared mutation Q493R (A22040G) located in the RBD region that directly interfaces with the receptor…. MACo2 was used for further passaging on BALB/c mice with the aim of favoring variants which would more efficiently replicate in standard laboratory strains. After 11 passages, another variant was isolated, cloned and named MACo3. Sequencing revealed the presence of a second mutation in the RBD: Q498R (A23055G). Two other amino-acid substitutions and one small deletion were also identified…

             CC strains CC071 and CC001 were instrumental to adapting the virus to mice. They showed some permissiveness to the replication of an early isolate of SARS-CoV-2 (Pango lineage B), unlike C57BL/6 mice in which no virus was detected upon infection (Fig S1). Although others were successful, we also attempted twice to adapt a B.1 virus (carrying the D614G mutation) on aged BALB/c mice, without success…”

Corollary, Schmidt et al. (2021) High genetic barrier to SARS-CoV-2 polyclonal neutralizing antibody escape. Nature. 2021 Dec;600(7889):512-516. doi: 10.1038/s41586-021-04005-0.

report that:

“By combining plasma selected spike substitutions, we generated synthetic ‘polymutant’ spike protein pseudotypes that resisted polyclonal antibody neutralization to a similar degree as circulating variants of concern… By aggregating variant of concern-associated and antibody-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in the SARS-CoV-2 spike protein are sufficient to generate pseudotypes with near-complete resistance to the polyclonal neutralizing antibodies generated by individuals who are convalescent or recipients who received an mRNA vaccine.”

In their very recent paper,

Schmidt et al. (2022) Plasma neutralization properties of the SARS-CoV-2 Omicron variant. medRxiv [Preprint]. 2021 Dec 13:2021.12.12.21267646. doi: 10.1101/2021.12.12.21267646. PMID: 34931199; PMCID: PMC8687470.https://www.nejm.org/doi/full/10.1056/NEJMc2119641

write:

“Previously, we showed that approximately 20 changes introduced into a synthetic polymutant spike protein (PMS20) are sufficient for substantial evasion of the polyclonal neutralizing antibodies elicited in the majority of persons who have recovered from coronavirus disease 2019 (Covid-19) or have received two doses of an mRNA vaccine.2 Of note, several changes in the PMS20 spike protein are the same as or similar to changes in the omicron variant (Fig. S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org).

Due to copyright considerations, I cannot add here their supplementary Fig S1, but you can view it by clicking on:https://www.nejm.org/doi/full/10.1056/NEJMc2119641

So far, these papers tell that at least 21 from the 35 omicron amino acid changes have been studied in the lab and published before the first report on the omicron variant from South-Africa.

As mentioned by Wei et al. (2021) in the starting reference, the majority of amino acid exchanges in the omicron variant are so-called non-synonym exchanges. This means that they arose by so-called transversions, i.e., mutations, which change either adenine (A) or guanine (G) to either cytosine (C) or uracil (U) and vice versa. When considering potential natural evolution of omicron by random mutations from previously detected variants, we must consider that the probability of occurrence of spontaneous transversions is much (at least 10 times) lower compared to the so-called transitions, i. e., mutations that change adenine (A) to guanine (G) or uracil (U) to cytosine (C) and vice versa. These long established experiment-based observations/predictions of the mutation theory are supported, for example by the following papers (one of which deals also the specific situation of mutational changes in influenza and HIV viruses):

Park L. Relative mutation rates of each nucleotide for another estimated from allele frequency spectra at human gene loci. Genet Res (Camb). 2009 Aug;91(4):293-303. doi: 10.1017/S0016672309990164. PMID: 19640324

Lyons DM, Lauring AS. Evidence for the Selective Basis of Transition-to-Transversion Substitution Bias in Two RNA Viruses. Mol Biol Evol. 2017 Dec 1;34(12):3205-3215. doi: 10.1093/molbev/msx251. PMID: 29029187; PMCID: PMC5850290.

From the latter paper we also learn that:

“…selection is a major contributor to the transition:transversion substitution bias in viruses and that this effect is only partially explained by the greater likelihood of transversion mutations to cause radical as opposed to conservative amino acid changes.

             The distribution of fitness effects of transversions is shifted toward more detrimental effects compared with transitions at some points along the fitness distribution in each gene, and transitions are never more detrimental. Three of the most commonly cited radical/conservative distinctions are predictive of mutational fitness effects. However, transversions are more detrimental than transitions even when controlling for their greater likelihood to be radical.”

So, we have a puzzling problem because the many transversions present in omicron do not confirm the above prediction and do not fit the mutation theory predictions on the frequency of spontaneous mutations because the nearly all amino acid exchanges caused by transversions in omicron result in either i) an increase in the strength of virus binding to the ACE2 receptor or ii) to destroy or weaken antibody binding to the virus (as it was shown by the above mentioned laboratory experiments using mouse-adapted virus mutants).

So, the omicron case gives to geneticists, molecular biologist, evolution researchers, and most people educated in biology a real puzzle. It is not by chance that another recent online paper

Kanika Bansal and Sanjeet Kumar (2021) Mutational cascade of SARS-CoV-2 leading to evolution 1 and emergence of omicron 2 variant. bioRxiv preprint doi: https://doi.org/10.1101/2021.12.06.471389; December 7, 2021

concludes that:

“Mutational analysis detected 18,261 mutations in the omicron variant (i.e. in all known so far sequenced omicron variants), majority of which were non-synonymous mutations in spike (A67, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H), followed by RNA dependent RNA polymerase (rdrp) (A1892T, I189V, P314L, K38R, T492I, V57V), ORF6 (M19M) and nucleocapsid protein (RG203KR). Conclusion: Delta and omicron have evolutionary diverged into distinct phylogroups and do not share a common ancestry. While, omicron shares common ancestry with VOI lambda and its evolution is mainly derived by the non-synonymous mutations.”

A typical question on an university genetics exam is that:

“What is the probability of multiple mutations?”

The classical answer is that “The probability of multiple random mutations is the product of probabilities of individual mutations.”

Of course, there are many deviations from this classical definiton, which are caused by differences and various loss of function changes in the repair systems of different organisms. In the case of RNA viruses, recombination by template switch of RNA polymerase also adds an extra option, which could incorporate novel sequences (e.g., from a related influenza or corona virus) into a new virus variant product. However, the frequency of “mistakes”, namely nucleotide exchanges made by the RNA polymerase probably remain constant even if virus recombination occurred. An excellent review on this subject was published by:

Simon-Loriere E, Holmes EC. Why do RNA viruses recombine? Nat Rev Microbiol. 2011 Jul 4;9(8):617-26. doi: 10.1038/nrmicro2614. PMID: 21725337; PMCID: PMC3324781.

So, what is the conclusion or message?

1. We cannot explain the natural evolution of omicron by assuming random and successive mutations using the existing tools of mutation theory. In this sense, the proposed three models, which are cited in the omicron Wikipedia draft are rather anecdotal and not supported by any core data.

2. Although we know that many (at least 20 or more) mutations present in omicron were independently generated in different laboratories around the world, WE HAVE YET NO VERIFIABLE EVIDENCE for that the virus was released from a laboratory due to an accident (which potentially happened in the case of SARS-COV-1 according to the Wikipedia site: https://en.wikipedia.org/wiki/SARS)

3. However, the latter possibility raises again the question about the risks and regulations of gain of function experiments because unfortunate lab accidents could happen. Consequently, while stopping with all efforts the pandemics, we must find out and verify by further intensive international investigation the origin of the omicron variant.

4. Finally, from genetic point of view, understanding the origin and evolution of omicron also implies that we might discover something new, which gives potentially more insight into mutational events that contribute to the evolution of RNA viruses, which for scientists is a very exciting motivation. --Peturban (Diskussion) 14:41, 3. Jan. 2022 (CET)

Hello! Sorry, I didn't know you would like to conversate in English. I've added a new table of contents for Wikipedia in English above. Thank you very much for your efforts. Now I can understand your aspects. At Wikipedia, we have to use the established state of science, especially for subjects like this. We can't do “theory finding” on Wikipedia, so we have to use medical textbooks or printed journals and we better don't use preprints. For SARS-CoV-2, the WHO launched the Scientific Advisory Group for the Origins of Novel Pathogens (SAGO) in 2021 to examine the origins of SARS-CoV-2. For Omicron, we used the sources that met the criterias to some extent. You mentioned a lot of sources. I'm not sure if we'll find the right one which mets the criterias. The less in high quality, the better – for Wikipedia. --Treck08 (Diskussion) 15:13, 3. Jan. 2022 (CET)

This is a real problem. On this way some new taken from e.g., FoxNews and other similar sites, might find their way to Wikipedia.

Scientific preciseness is absolutely essential when talking about a problem, which influences the life of the whole world population.

And the language cannot be a problem given the excellent translation options, e.g. Goggle English German translator. If you wish,

I can translate my letter to German. --Peturban (Diskussion) 15:21, 3. Jan. 2022 (CET)

I don't think that the language will be a problem. I'm sure, that FoxNews or similar sites won't be suitable sites for the german Wikipedia, especially for medical subjects. Maybe something like Nature or Science would met the criterias, or UKHSA, CDC or WHO. --Treck08 (Diskussion) 15:41, 3. Jan. 2022 (CET)

I also hope so very much! No more comment. --2003:E7:4F0F:3D09:F0CE:9A30:38F9:109C 15:43, 3. Jan. 2022 (CET)

Thank you for your long comments and the literature. I have taken a closer look at the mice study. According to your literature collection, there are incidences for a gain-of-function hypothesis for the Omicron origin. I'm sure there will be a scientific discussion, would be good if we had papers here for this hypothesis --Empiricus (Diskussion) 22:01, 16. Jan. 2022 (CET)