An analysis of genetic diversity of D614G mutation in SARS-CoV-2
By Dr. Liji Thomas, MDNov 4 2020
Even as the circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains seem to be evolving in different directions, the dominant strain continues to be that which possesses the D614G mutation in the spike protein. A new study published in the preprint server bioRxiv* in November 2020 reports that the effect of the mutation is minimal, in terms of pathogenicity or altered response to neutralizing antibodies elicited against the native virus strain.
Transmission electron micrograph of a SARS-CoV-2 virus particle, isolated from a patient. Image captured and color-enhanced at the NIAID Integrated Research Facility (IRF) in Fort Detrick, Maryland. Credit: NIAID
The D614G Mutation
The Wuhan strain that began to spread across and beyond China, to the rest of the world, within a few short weeks, has given rise to three major variants as of now. Among these, the spike D614G mutation has proved to confer a distinct advantage, becoming rapidly dominant in every area of the world where it has been introduced. This mutation consists of the replacement of the aspartate residue at position 614 of the spike protein (or S-protein) by a glycine residue.
The mutant variant of the spike seems to have increased the infectivity of the virus over that found in the wildtype SARS-CoV-2. It is found to occur in two superclades, namely, S and G, but disease severity and lethality are always associated with only one of them.
At present, the S clade is predominant over the G clade in much of Europe and in Uruguay, indicating that it is more infectious. The D614G mutation involves the substitution of a neutral amino acid, glycine, for a negatively charged one, aspartate. This is in the loop region of the S1 subunit of the spike protein.
This mutation is due to a single nucleotide variant, where A is replaced by G in the viral RNA. It appeared only towards the final weeks of April 2020 in China, but was then found in 90-97% of samples. The mutation has enhanced the transmissibility of the virus, with infections resulting in higher viral loads. The question is whether this is associated with greater lethality and whether this will impact the efficacy of vaccines by making the virus resistant to antibodies that target the original strain of SARS-CoV-2.
If so, the development of vaccines and other antivirals developed with the Wuhan reference strain in mind could be markedly delayed by the need to revise them.
Differences in Viral Effects due to D614G
Some evidence from genetic studies, epidemiology and viral-structural examination already shows that this mutation is associated with a higher incidence of chemosensory deficits relative to the original strain. Additionally, it shows higher binding to the host cell receptor, the angiotensin-converting enzyme 2 (ACE2), allowing more efficient viral entry due to the higher stability of the spike protein and reduced cleavage.
Genetic Diversity and Polymorphism of SARS-CoV-2
The current study aimed to explore the genetic diversity and polymorphisms of the viral spike protein. The researchers found that of all the 18 sequences, obtained from Malaysian and Venezuelan sequences uploaded to the National Center for Biotechnology Information (NCBI), there were 57 polymorphic sites among almost 30,000 analyzed base pairs. These were parsimony-informative sites, with at least two nucleotides showing variation, and at least two of the nucleotide variants showing a frequency of two or more. Further examination of these sites showed the existence of two different groups, and the researchers suspect that these may have common haplotypes in both countries.
Two Genetic Variants Showing Divergent Evolution
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The researchers found that there was marked variation within the population, and a fixation index (FST value) analysis confirmed the presence of two different genetic variants, with distinctly observed divergence both within and between the two groups. However, all Malaysian strains were highly similar genetically, while the Venezuelan strains showed more evolutionary divergence.
All the haplotypes, the sets of genes inherited together from one parent, showed that mutation was actively happening, in the form of transversions and transitions. Insertion-deletion (indel) mutations were found only within haplotype groups in the Venezuelan samples. Both the Tajima and Fs de Fu tests showed that within the Malaysian group, population growth was absent. Mutation rates were stable between different localities.
The time of evolutionary divergence was seen among all populations, using all the earlier methods. However, the researchers summed up the structuring of the two groups, as being slightly higher in degree in the Venezuelan group, perhaps as a result of genetic flow.
Uniform Results by Different Methods Indicate Conservation
The researchers comment that the discontinuous pattern of genetic divergence between the groups “supports the idea of possible isolations resulting from past fragmentation events,” the more so as the number of branches in the tree are few, and these required few steps to be generated.
The few mutations that have occurred seem to have escaped correction by genetic drift or have not experienced the founding effect that is typically found when haplotypes are dispersed or lost over time. When the genetic distance is taken into account, the researchers found that the groups still failed to show much divergence. The researchers think it is important to consider the minimum divergences between the two groups, from Malaysia and Venezuela, at the point of haplotype exchange.
The researchers found from their dendrogram that the most important differences in the virus strains in both countries related to their form rather than their number. They concluded that the 18 strains could be discriminated in the two geographic regions, as a result of the significant variations in the haplotypes, showing a hierarchical structure to all components of covariance; and also by the differences within and between the two groups. The estimated mean divergence between these strains and other countries is non-existent or at least, minimal.
Since many haplotypes were common in the Venezuela group, the researchers did not attempt to find the association between the genetic distance and geographic separation. However, the use of the estimator φ, an indicator that responds to the smallest molecular variation, lends credibility to the results of the various tests used.
They say this estimator “supported the uniformity between the results found by all the methodologies employed, and can be interpreted as a phylogenetic confirmation that there is a consensus in the conservation of the mutant SARS-CoV-2 genome (D614G), in the two countries analyzed).”
The implications are that the small number of polymorphisms found at present probably do not represent any large changes in the proteins expressed by different viral strains. Therefore, despite the differences in the haplotypes, these are not significant in both of these geographical regions. Therefore, it is sound practice to extrapolate these findings to other variants in other countries and conclude that they do not reflect significant differences in the protein products of the wildtype and mutant SARS-CoV-2.
The study concludes, “The analyses made in this study suggest small variations in protein products, especially in those that were targets of this study, bringing tranquility regarding the risk of death by COVID-19, as well as possible problems of adjustments to some molecular targets for vaccines.”
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.