BREAKING! The SARS-CoV-2 Coronavirus Could Have Been Evolving For The Last 6 Years According To Canadian Phylogenetic Studies

SARS-CoV-2 Coronavirus: There is still so much we do not know about the SARS-CoV-2 coronavirus  and it reinforces the fact that there is also so much not known in the field of virology, which continues to challenge the ability of mankind to remain healthy when faced with pathogens. While most known microbes have restricted affinity for specific species, continuing to adapt within the host species, the SARS-CoV-2 has crossed over from an unknown animal reservoir to infect human cells. Such a virus is typically more readily infective and cause more severe disease, as it have not yet adapted fully to the target host.


 
The critical question is how novel coronaviruses acquire the ability to recognize, bind to and enter human cells for the first time whether this is dependent only on viral proteins recognizing host cell proteins, or adaptations in other viral processes that allow replication in a human host.
 
Canadian researchers from the University of Calgary have conducted a detailed study to discover how the spike proteins from the SARS-CoV-2 evolved and adapted to be able to target the human ACE-2 receptors which is responsible for viral entry into the target cell.
 
Interestingly the human ACE2 (hACE2) has some rare variants which make the host more vulnerable to infection. Similarly, the spike protein of this virus has a greater affinity for the receptor than the previous SARS virus, which is another possible explanation for the increased infective potential of the current virus. Using detailed phylogenetic analysis, ancestral sequence reconstruction, and molecular dynamics simulations to examine the Spike-RBD’s functional evolution, the researchers discovered that it has likely possessed high affinity for human cell targets since at least 2013!
 
The study is currently published on an online preprint server and is currently pending peer review. https://www.biorxiv.org/content/10.1101/2020.06.22.165787v1
 
The Canadian research examines the origin of this spike protein variant with its affinity for hACE2, using molecular dynamics (MD) simulations along with sequence reconstruction to identify the adaptation pathway of the virus. The result is a preliminary phylogenetic analysis that agrees with earlier studies – the virus is 96% similar to the bat coronavirus (RaTG13) genome and 90% similar to the Pangolin-CoV genome.


Characterization of SARS-CoV-2 Spike-RBD functional evolution. A. Table of MM/PBSA binding energies between receptor binding domains of SARS-CoV2 evolutionary constructs and hACE2 receptor (note that lower energy indicates tighter binding). Blue cells indicate the presence of the ancestral (N0) state and green cells (with an “x”) indicate the presence of the SARS-CoV-2 state (N1) at a given position. Two values are present for constructs with an ancestral (N0) state at position 498 (which reflect the ambiguity of its ancestral reconstruction), corresponding to h498 and y498 from left to right. Energies are shown as the mean of three replicate simulations with SEM indicated in parenthesis. B. The relative effect of changes in the SARS-CoV-2 receptor-binding domain from ancestral (N0) to SARS-CoV-2 (N1) state on MM/PBSA binding energies. Size of spheres indicates the relative magnitude, with red spheres indicating decreased binding affinity and blue indicating increased binding affinity. Values are averaged for h498 and y498 states (both raw values shown in parentheses). C. Schematic of two possible evolutionary scenarios stemming from the observed evolutionary SARS-CoV-2 Spike-RBD function. In Scenario 1, it is postulated that a zoonotic ancestral SARS-CoV-2 strain possessed the ability to effectively bind hACE2 but was unable to effectively enter human cells, requiring the presence of subsequent mutations to infect humans. In Scenario 2, an ancestral SARS-CoV-2 strain was actively infecting humans prior to the outbreak at low levels, thus escaping public health detection until subsequent mutations lead to increased infectivity and/or severity.

Subsequently a more detailed analysis of 479 sequences collected from December 30, 2019, to March 20, 2020, where they found 16 variants. Of these, 11 were missense mutations occurring in 5% or more of cases, and each had its own phylogenetic route.
 
The study team then tried to recreate the ancestral sequence for the spike-RBD region, so that they could identify the important mutations that specifically drive its recent adaptation to the human host. They reconstructed the hypothetical common ancestor spike-RBD sequence for all human SARS-CoV-2 cases, called N1, and for the common ancestor with the closest animal virus, called N2.
 
Interestingly, N1 is identical to the sequence in the SARS-CoV-2 reference sequence, but the N0 sequence is unique, which shows that this virus has originated uniquely. The two differ at 4 positions. The ancestral protein gave rise to various descendants, one of which is the RaTG13. Since this was around in 2013, the researchers conclude that the ancestral strain existed as early as that year, at least. In other words, the N0-N1 branch has been evolving for at least 6 years before the SARS-CoV-2 made its debut in late 2019.
 
In order to find out the functional differences between N0 and current spike-RBD sequences, the researchers used MD simulations of the spike-RBD-hACE2 complex, beginning with the X-ray crystal structures. The model showed that the free binding energy for this complex decreased as N0 changed to N1. Thus, this actually reduced the binding affinity both in the simulations and in vitro.

However, two of the changes were associated with more significant decreases than the other. This shows that the N0 strain had, unexpectedly, greater binding affinity than the N1 strain. This is the first study to show that the common ancestor of both SARS-CoV-2 and the RaTG13 had the ability to bind to the ACE2 receptor in humans.
 
STUDY IMPLICATIONS:
 
The research findings implications are that firstly, the binding affinity of the spike-RBD to hACE2 is not the primary driver of the highly infectious nature of the current virus since the ancestral virus was capable of doing this too.
 
Secondly, the researchers suggest that this virus was, even then, able to bind tightly to the receptor. Therefore, this was not sufficient to produce the currently observed ability to spread rapidly and widely among humans.
 
Rather this must be due to another set of mutations in the viral genome.
 
Also another implication is that the current virus may not have jumped to humans from an animal origin at all because its affinity for hACE2 was not a recently acquired molecular trait. This may mean that the ability to infect human cells was present over a more extended period in the past, but produced less obvious or fewer clinical symptoms which passed unnoticed.
 
Also another alternative was that it affected only a small number of people, allowing it to remain under the public health radar.
 
All these possibilities can only be tested by a broad-spectrum approach to sequencing all coronavirus strains in human populations, as this will reveal the presence of closely related viruses if such are present.
 
This study is an in silico study, and further validation of these findings is necessary using combinatorial libraries which can be screened to map functionalities to the genomic regions of the virus. This will help understand how the virus evolved in the most recent past.
 
Professor Dr Erin Brintnell from the Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary told Thailand Medical News, “It appears that the SARS-CoV-2 Spike-RBD did not recently evolve binding affinity to a human-specific protein. Instead, that function appears to have been latent, making it clear that the evolution of this disease along with so many other aspects of its etiology is more complex than expected.”
 
Other medical experts are also warning that the SARS-CoV-2 coronavirus is continuing to evolve and mutated ‘smartly’ to the detriment of humankind. These mutations are taking place while it is in a human host.
 
As one virologist who wished to remain anonymous had commented since the beginning of the epidemic, ”The SARS-CoV-2 is a very anomalous virus that is smartly evolving. Not only it is be able to adapt and spread faster that most viruses known as it uses a multiple ways to do so not just by the ACE-receptors but also via the CD147 receptors and furin cleavages and also it is also not going to be like the influenza virus or the HIV virus but rather it has its own self-regulatory bioclocks in the human host and will causes a variety of chronic diseases and their eventual destruction in a preplanned timely manner.”
 
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