Discussion - 2019-nCoV genetics

[Pathfinder]

La m?me source a produit aussi ceci : https://www.nature.com/articles/s41586-018-0010-9

Here's What Makes Coronavirus and African Swine Fever Different

Jennifer Shike
January 31, 2020 12:40 PM
...
Saif says the best examples of CoV that infect the gut and cause diarrhea and deaths in pigs are porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus that first emerged in the U.S. in 2013-14. Both are still present in pigs.

These viruses are members of two different groups of coronaviruses – alpha and delta CoV. Saif explains they are genetically distinct and do not cross-protect against one another. 2019-nCoV is part of a third distinct group of coronaviruses (beta CoV) and is genetically and antigenically distinct from these two swine coronaviruses, she adds.
...
Could 2019-nCoV Impact the Swine Industry?

There is no evidence from China that 2019-nCoV came from pigs or even that it could infect pigs, Saif says. In addition, there was no previous data that the related SARS CoV infected pigs.
...
https://www.porkbusiness.com/article/heres-what-makes-coronavirus-and-african-swine-fever-different

 

[bertrand789]

Le virus de la peste porcine africaine me semble un virus d'une tique qui d?sormais se multiplie sur animaux ? sang chaud. Il pose des probl?mes car il impose des changements zoo techniques pas simple ? mettre en œuvre... On a expliqu? les pourquoi ou comment, de sa survenue ?

S'il est envisage, que pour le coronavirus de chauve souris, pour par mutation il faudrait 65 ans pour arriver ? celui ?voqu?, je suis surpris qu'en 65 ans, rien n'ai ?t? vu. En tout cas, pour celui qui atteint les porcelets, donc Hku2, on a expliqu? les pourquoi ou comment de sa survenue ?

 

[gsgs]

>An outbreak of respiratory illness caused by a novel coronavirus (nCoV-2019, NC_045512.2 40)
>first identified in Wuhan China has resulted in over seven thousand confirmed cases.
>So far, the nCoV-2019 has been reported to share 96% sequence identity to the RaTG13 genome
>(EPI_ISL_402131). However, the S1 Receptor Binding Domain (RBD) of the nCoV-2019 genome
>was noticeably divergent between the two at amino acid residues 350 to 550 – Figure 1A.
>We aimed to identity coronaviruses related to nCoV-2019 in viral metagenomics datasets
>available in the public domain. In a recently published dataset describing viral diversity in
>Malayan pangolins (PRJNA573298 87) we used VirMAP 38 to reconstruct a coronavirus genome
>(approximately 84% complete from samples SRR10168377 33 and SRR10168378 20) that
>shared 97% amino acid identity across the same RBD segment – Figure 1B. This result
i>ndicates a potential recombination event for nCoV-2019.
>Edit -
>From the coordinates shown in this preprint 121 (Figure 4), it looks like most of the differences between RaTG13 >and nCoV-2019 are restricted to loop 2 of the receptor binding motif (positions ~450-500).

-----------------------------------------------------------------------------------
> t seems unlikely that the receptor binding domain–and especially the receptor binding motif–would be nearly identical to one
> found in pangolin through random chance.
> incidentally, pangolins were sold in the market at the center of all of this 119.

 

[gsgs]

> The 2013 Yunnan bat virus genome sequence {"RaTG13"} is now available
> from GenBank with accession number MN996532 15.

https://ncovinfo.createaforum.com/genetics/ratg13/msg58/#msg58

 

[bertrand789]

Comme ? chaque fois, la situation s'inverse, car, d?sormais, ce sont des humains qui peuvent contaminer des populations d'animaux, peut ?tre, d?j? porteuses d'autres coronavirus. Qui va se charger de suivre cela ? C'est du domaine des instances m?dicales ?

Pour ?tre concret et pr?cis , si la Chine fait un ?norme travail ? f?liciter , il y a des zones d'ombres, vu son organisation interne, en ?volution continue. Pour un autre dossier, celui de la peste porcine africaine, on ne sait rien de ce qui se passe dans ses populations de sangliers et ou de porcs sauvages, ni de sa strat?gie pour ce sujet . Il y a peut ?tre des essais de vaccins plus ou plus volontaire en cours, par exemple .

Mais les autres font vraiment mieux ?
On se sait pas, non plus, grand chose, officiellement, sur les influenzas des populations de dindes sauvages aux U.S.A .

Ce dossier a un m?rite, il a eu et va avoir un co?t tel, que tous les pays vont devoir mieux faire. Parler des ?volutions envisag?es, me semble une fa?on de restaurer la confiance, mise ? mal, ? chaque crise, par la mise en lumi?re des failles des modes organisationnels en place...

 

[Emily]

Here's an opinion that RaTG13 is not the proximal source:

http://virological.org/t/tackling-rumors-of-a-suspicious-origin-of-ncov2019/384

 

[gsgs]

> he TMRCA of the two sequences nCoV2019 and RaTG13 of 69.5 years ago – roughly 1950 +/- 10 years or so.

that's surprising, given that the others are even much more distant

I'm not familiar with coronaviruses ...if it were avian flu, I'd calculate ~6years for 96.5% identity

They have probably calculated the mutation rate in the current outbreak ... maybe I can find it

-----------------------------------------------------------

> Coalescent model Estimated rate 95% interval
> Constant size 1.21x10^-3 0.60x10^-3 – 1.96x10^-3
> Exponential growth 9.81x10^-3 0.25x10^-3 – 1.59x10^-3
> Table 1 | The estimated rate of evolution (substitutions per site per year) of the sampled nCoV-2019 genomes.

so let's say 1*10^-3 mutations per nucleotide position per year
or 30 mutations per year on the 30000-nucleotides-genome.
That would be ~1/3 of the flu-rate, so my 6 years above grow to 18.
No way to get 69.5 years

----------------------------------------------------
I'm puzzled about all the hills and valleys in the mutation-pictures, are these all
due to recombinations ?
e.g. http://magictour.free.fr/wuhrat6.GIF

 

[JJackson]

gs if you use the nextstrain link and set the 'tree type' to clock and the 'divergence/time' button to time it will give you the calculated rate for the GISAID data set currently it reads 27.817 subs/y

 

[JJackson]

The current TWIV is very good, it is 1 hour on SARS-CoV-2 with Vincent, an American Infectious disease specialist and an Italian researcher working in Switzerland. The researchers team are testing a nano-particle with common viral receptors on their surfaces as a viral 'fly-paper' with some success on a wide range of viruses inc. SARS-CoV-2. This is at a very early stage and they have not even verified their first test yet so it is unlikely to help this time round. Wouldn't it have been nice to have a useful broadly acting anti-viral before, rather than after, this sort of thing happened? (note: rhetorical)

http://www.microbe.tv/twiv/

 

[Kathy]

Regarding the mysterious sequence RaTG13, I got from another blog the hint that I should look closer at the sequence KP876546 that is cited in the article:
Ge, X., Wang, N., Zhang, W. et al. Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft. Virol. Sin. 31, 31–40 (2016). https://doi.org/10.1007/s12250-016-3713-9

The sequence KP876546 in NCBI is very short (only 370 bp) and is defined as Rhinolophus bat coronavirus BtCoV/4991 partial RNA-dependent RNA polymerase. This sequence is also analysed in the article Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats [url]https://doi.org/10.3390/v11040379[/URL].

I blasted the KP876546 sequence in NCBI and I got 100% identities with RaTG13 and 99% identities with MT039890 Severe acute respiratory syndrome coronavirus 2 isolate SNU01, complete genome (South Korea). Next closer sequence not from SARS-CoV2 is the pangolin sequence MT084071.
To my opinion the sequence KP876546 could be the first evidence of the RaTG13 sequence or a sequence even closer to SARs-CoV2. In Ge et al., it is stated that the 370 bp sequence was prolonged of 816 bp and the spike protein was sequenced but this information for this sample has been not made public.

I have found a publication on the comparison of KP876546 with SARS-CoV2 before that RaTG13 was submitted to NCBI:

Liangjun Chen, Weiyong Liu, Qi Zhang, Ke Xu, Guangming Ye, Weichen Wu, Ziyong Sun, Fang Liu, Kailang Wu, Bo Zhong, Yi Mei, Wenxia Zhang, Yu Chen, Yirong Li, Mang Shi, Ke Lan & Yingle Liu (2020) RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak, Emerging Microbes & Infections, 9:1, 313-319, DOI: 10.1080/22221751.2020.1725399

The author writes that: “further sequencing of the corresponding PCR product (from SARS-CoV2) surprisingly suggested that the virus discovered is more closely related to BtCoV/4991” (KP876546) “(97.35%) but not SARS-CoV. The genomes of the 2019-nCoV were further analysed to determine its origin and evolutionary history. Full genome comparisons indicated that 2019-nCoV is close to CoVs circulating in Rhinolophus (Horseshoe bats). For example, it shared 98.7% nucleotide identity to bat coronavirus strain BtCoV/4991 (GenBank KP876546, only 370 nt sequence of RdRp gene) and 87.9% nucleotide identity to bat CoV strain bat-SLCoVZC45 and bat-SL-CoVZXC21, indicating that it was quite divergent from the currently known human CoV, including SARS-CoV (79.7%). The close relationship with BtCoV/4991 is quite essential in tracing the potential reservoir host of 2019-nCoV. Unfortunately, the BtCoV/4991 sequence was only partial (373bp in length) and thus no comparisons can be made for the rest of genomes.”

In the article:
Zhou, P., Yang, X., Wang, X. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020). https://doi.org/10.1038/s41586-020-2012-7

where for the first time RaTg13 appears, it is written:

“We then found a short RdRp region from a bat coronavirus termed BatCoV RaTG13 which we previously detected in Rhinolophus affinis from Yunnan Province showed high sequence identity to nCoV-2019. We did full-length sequencing to this RNA sample. Simplot analysis showed that nCoV-2019 was highly similar throughout the genome to RaTG13, with 96.2% overall genome sequence identity.”

Interestingly, the article of Ge et al. is not part of the bibliography of this work. To my opinion further sequencing of KP876546 was so interesting that the results were kept secret and manipulations of this virus was carried over until the outbreak of SARS-CoV2.

 

[JJackson]

I have been trying to work out what I can make the nextstrain tool do and was fairly sure it was capable of more than I had found by trial and error. My search led me to a lecture which I will come back to and link below.

re rosmarina's post I don't think there has been anything close to evidence that humans were involved in this viruses evolutionary history, beyond being unwitting hosts. I suspect the short fragment was not deemed worth uploading at the time but once COVID arrived it, and bits like it, suddenly became a lot more interesting. I expect more partial sequences but few full genomes. I looked at the human sequence data using nextstrain and looked at the AA mutation frequency across the full genome and its entropy (first image below) to get a feel for which parts were conserved and which changing. In the second image I zoom in to just the short section that matches the fragment (note the little black triangles at the bottom) and there are a few AA changes at random, with very low entropies, indicating they have little impact on the phylogenic tree's structure.

The sequence covers the region around 15,340 to 15,709 which is part of the RdRp gene which in turn is part of ORF1b. This accounts 1/75th of the genome and so would be expected to show high homology in a conserved region, and when the full sequence is blasted against the NCBI data set I get 89% homology in a range of bat and SARS-1 sequences. This is lower than I expected. It came up in today's TWiV (link below) that when the civit intermediate host for SARS-1 was found those sequences had 99% homology with the human strain (presumably across the full genome, based on context, all though not stated explicitly).

In the top image I highlighted one spike in green, this is C14408T (therefore outside KP876546) resulting in ORF1b P314L and creating clade A2a which is active in Northern Europe, hence its high entropy score.

The promised lecture link by Richard Neher, University of Basel on 6 March 2019 https://www.youtube.com/watch?v=YxTUF10redQ
He was a co-developer of Nextstrain and uses it as research tool. He starts with an intro on flu and then starts using it to analyse H3N2 data. Unfortunately in later parts of the video he is not at the podium and the sound is variable also he is pointing out features on graphics for the audience which we can not see which makes it trickier to follow. However if you persevere he looks at the predictive ability of the tree structures and how well their predictions for H3N2 over the years have compared with what actually occurred. The system is obviously making a better than average estimate of which branches would show mutations and become dominant.

This is a plug for the current TWiV which the panel, and I, both thought was Awesome! I am not even going to try list the areas covered in detail as the list of items not covered would be shorter. It is 2hrs long but I doubt you could improve you understanding of this epidemic with 2hrs spent in any other way. TWiV 591 http://www.microbe.tv/twiv/

 

[Kathy]

JJackson: Thank you for your comments and links. I still belive that is very suspicious that the authors Zhou et al, in the article published by Nature "forgot" to cite that the RaTG13 was first identified and named KP876546 in the work of Ge et al on the coexistence of multiple coronaviruses in bat colonies. I can't believe that they did not go on sequencing and working on such an interesting sequence, that they define a possible novel betacoronavirus species. I stay with my opinion: the backbone of SARS-CoC2 was discovered with that study, they went on working on it, possibly manipulating the spike protein using a CoV from an animal such pangolin, that has a better affinity for ACE2 receptor. The furin cleavage site is extremely unnatural in this group of viruses and must probably added to the virus. I recently read that SARS-CoV2 has four ways to attack human cells and that looks to me not natural.

https://www.thailandmedical.news/ne...-specific-preference-of-attacking-human-host-

We also need to keep in mind that the sequence RaTG13 could have been edited before to submit it to NCBI.
I will go on with my research on the topic and try to find virologists that could help me writing a publication on the possible human manipulation of SARS-CoV2. Too many people are dying because of it, I can't accept it.

 

[JJackson]

Has anyone seen anything on ORF1a L3606F? I was looking at the NTs which have been regularly cropping up and this one is interesting because it has occurred several times in different parts of the tree and the subsequent cases are holding their own against the wild type. It is the only change I have seen so far that appears to show signs of host adaption. As I do not even know which protein this changes I have no idea what functional change it may make. This is what I have been looking for but a search on ORF1a L3606F only found a few references none of which were particularly helpful.

 

[JJackson]

This paper is excellent and provides a ton of data I had not seen elsewhere on the coding regions and their proteins, functions, biological conformations. Not an easy read but worth the effort.
https://www.futuremedicine.com/doi/10.2217/fvl-2018-0008

 

[Shiloh]

Source: https://phys.org/news/2020-04-sars-cov-genomes-austria-openly.html

First SARS-CoV-2 genomes in Austria openly available
by CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences

...Initial sequence analysis of the 29,900 nucleotide-long SARS-CoV-2 genomes from Austria revealed on average 6 mutations different to the reference genome isolated in Wuhan, the capital city of the province of Hubei, China in December 2019. The observed number of mutations is in line with other recently reported SARS-CoV-2 genomes. Most of the observed mutations lead to changes in viral proteins, providing evidence for positive selection pressure and evolution within the human population. Assessing the actual impact of these mutations for the virus life cycle and its interactions with both the host and the immune system will be within the scope of future investigations. Ongoing in-depth genomic analyses focus on mutational hotspots, dissect viral diversity between the Austrian strains and the strains from other countries as well as study of the mutational dynamics of pandemic SARS-CoV-2...

 

[JJackson]

The link is a useful lecture on SARS-2 and looks into how the virus persuades the host translational system to read its ORFs.
https://www.youtube.com/watch?v=8_bOhZd6ieM

 

[JJackson]

This is an interesting paper in terms of the technique and the interactive heat-map tool for looking at the data. The experiment produced engineered yeast cells each of which has a different SARS-CoV-2 spike RBD displayed on its surface. They replace every nucleotide at every position in the RBD and then measure, for each AA change, the effect on protein expression and binding affinity. They also show if that AA is in direct contact with the ACE2 receptor and what the consensus AA was at that position for each of SARS-1, RaTG13 and Pangolins. All of this data can be accessed by hovering the mouse over the squares on the heat map. This is useful data for vaccine formulation, especially mono or poly-clonal antibodies, as it shows functional biological constrained AA positions which will be more resistant to vaccine escape mutations.
https://www.biorxiv.org/content/10.1....157982v1.full
https://jbloomlab.github.io/SARS-CoV-2-RBD_DMS/ - Interactive heat-map tool.
https://www.microbe.tv/twievo/twievo-57/ - TWiEVO podcast discussion with the authors.

The heat map works well as an adjunct to the Nextstrain tool, discussed earlier, to examine how well the experimental data matches the actual mutational frequencies. It may also be helpful to have a look at my first post in this thread which looks at additional SL BetaCoV RBDs and the S1 protein structure around the RBD pocket.
https://flutrackers.com/forum/forum/...rsonal-opinion

 

[Pathfinder]

Mining coronavirus genomes for clues to the outbreak’s origins

By Jon CohenJan. 31, 2020 , 6:20 PM
...
“One of the biggest takeaway messages [from the viral sequences] is that there was a single introduction into humans and then human-to-human spread,” says Trevor Bedford, a bioinformatics specialist at the University of Washington and Fred Hutchinson Cancer Research Center.
...
The longer a virus circulates in a human populations, the more time it has to develop mutations that differentiate strains in infected people, and given that the 2019-nCoV sequences analyzed to date differ from each other by seven nucleotides at most, this suggests it jumped into humans very recently. But it remains a mystery which animal spread the virus to humans.
...
According to Xinhua, the state-run news agency, “environmental sampling” of the Wuhan seafood market has found evidence of 2019-nCoV. Of the 585 samples tested, 33 were positive for 2019-nCoV and all were in the huge market’s western portion, which is where wildlife were sold. “The positive tests from the wet market are hugely important,” says Edward Holmes, an evolutionary biologist at the University of Sydney ...
...
Yet there have been no preprints or official scientific reports on the sampling, so it’s not clear which, if any, animals tested positive. “Until you consistently isolate the virus out of a single species, it’s really, really difficult to try and determine what the natural host is,” says Kristian Andersen, an evolutionary biologist at Scripps Research.
...
It’s not just a “curious interest” to figure out what sparked the current outbreak, Daszak says. “If we don't find the origin, it could still be a raging infection at a farm somewhere, and once this outbreak dies, there could be a continued spillover that’s really hard to stop. But the jury is still out on what the real origins of this are.”

https://www.sciencemag.org/news/2020...reak-s-origins

Jimmy Tobias

@JamesCTobias
Very interesting email from the Fauci documents obtained by
@JasonLeopold
:

1:33 PM · Jun 1, 2021·Twitter Web App

 

[JJackson]

The location of the viral sequences in the market were documented in the WHO origins report. Most were from the drains as the procedure at the market was to hose down the stalls, where animals were slaughtered for food, at the end of the day making it almost impossible to pin point any one stall as being contaminated.