Tracking the evolution of the SARS-CoV-2 Delta variant of concern: analysis of genetic diversity and selection across the whole viral genome.

Background: Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has diversified extensively, producing five highly virulent lineages designated as variants of concern (VOCs). The Delta VOC emerged in India with increased transmission, immune evasion, and mortality, causing a massive global case surge in 2021. This study aims to understand how the Delta VOC evolved by characterizing mutation patterns in the viral population before and after its emergence. Furthermore, we aim to identify the influence of positive and negative selection on VOC evolution and understand the prevalence of different mutation types in the viral genome. Methods: Three groups of whole viral genomes were retrieved from GISAID, sourced from India, with collection periods as follows: Group A-during the initial appearance of SARS-CoV-2; Group B-just before the emergence of the Delta variant; Group C-after the establishment of the Delta variant in India. Mutations in >1% of each group were identified with BioEdit to reveal differences in mutation quantity and type. Sites under positive or negative selection were identified with FUBAR. The results were compared to determine how mutations correspond with selective pressures and how viral mutation profiles changed to reflect genetic diversity before and after VOC emergence. Results: The number of mutations increased progressively in Groups A-C, with Group C reporting a 2.2- and 1.9-fold increase from Groups A and B, respectively. Among all the observed mutations, Group C had the highest percentage of deletions (22.7%; vs. 4.2% and 2.6% in Groups A and B, respectively), and most mutations altered the final amino acid code, such as non-synonymous substitutions and deletions. Conversely, Group B had the most synonymous substitutions that are effectively silent. The number of sites experiencing positive selection increased in Groups A-C, but Group B had 2.4- and 2.6 times more sites under negative selection compared to Groups A and C, respectively. Conclusion: Our findings demonstrated that viral genetic diversity continuously increased during and after the emergence of the Delta VOC. Despite this, Group B reports heightened negative selection, which potentially preserves important gene regions during evolution. Group C contains an unprecedented quantity of mutations and positively selected sites, providing strong evidence of active viral adaptation in the population.

Dinuclear Zn(II) complex promotes cleavage and isomerization of 2-hydroxypropyl alkyl phosphates by a common cyclic phosphate intermediate.

The kinetics and cleavage products of 2-hydroxypropyl p-nitrophenyl phosphate were determined in methanol containing the di-Zn(II) complex of bis-1,3-N1,N1'-(1,5,9-triazacyclododecyl)propane (4). Time-dependent 1H NMR spectra of the reaction mixture at sspH 9.8 +/- 0.1 show that the catalytic reaction proceeds via a cyclic phosphate (4-methylethylene phosphate, 2) that is subsequently cleaved into a kinetic mixture of two isomeric products, 2-hydroxypropyl methyl phosphate (3) and 1-hydroxypropan-2-yl methyl phosphate (3a), in a 29/71 ratio. In the presence of 4, the kinetic mixture of 3/3a is transformed into a thermodynamic mixture of 72/28 3/3a. The time-dependent 1H NMR spectra of 4 and a 22/78 mixture of 3/3a in CD3OH show that the formation of the thermodynamic mixture occurs on the same time scale as replacement of the P-OCH3 group of the 3/3a starting materials with OCD3. Detailed kinetic studies indicate that the dominant process for loss of the OCH3 group and equilibration of 3/3a is via a 4-catalyzed process where each of the isomers cyclizes to methylethylene phosphate (2), which subsequently reforms the 3/3a thermodynamic mixture. The kcatmax for 4-catalyzed cyclization of 3 and three other 2-hydroxypropyl O-alkyl phosphates (alkyl = CF3CH2- (6a), CH2FCH2- (6b), and CH3CH2- (6c)) has been determined, and the Brønsted plot comprising the log kcatmax vs leaving group sspKa that includes several previously studied 2-hydroxypropyl aryl phosphates is linear, following the expression log kcatmax = (-0.85 +/- 0.02) sspKa + (12.8 +/- 0.4). The betalg value of -0.85 suggests that the catalyzed cleavage of the P-OAr/OR bond has progressed to about 45% in the transition state. The combined results are analyzed in terms of two possible processes involving either a concerted reaction leading to the cyclic phosphate 2 from which the thermodynamic mixture of 3/3a is formed or a stepwise one involving a transient phosphorane whose predominant fate is to eliminate methoxide and proceed to 2 rather than partitioning between 3, 3a, and 2.

A simple DNase model system comprising a dinuclear Zn(II) complex in methanol accelerates the cleavage of a series of methyl aryl phosphate diesters by 10(11)-10(13).

The di-Zn(II) complex of 1,3-bis[ N1, N1'-(1,5,9-triazacyclododecyl)]propane with an associated methoxide ( 3:Zn(II) 2: (-)OCH 3) was prepared and its catalysis of the methanolysis of a series of fourteen methyl aryl phosphate diesters ( 6) was studied at s (s)pH 9.8 in methanol at 25.0 +/- 0.1 degrees C. Plots of k obs vs [ 3:Zn(II) 2: (-)OCH 3] free for all members of 6 show saturation behavior from which K(M) and kcat (max) were determined. The second order rate constants for the catalyzed reactions (kcat (max)/K(M)) for each substrate are larger than the corresponding methoxide catalyzed reaction (k 2 (-OMe)) by 1.4 x 10(8) to 3 x 10 (9)-fold. The values of k cat (max) for all members of 6 are between 4 x 10(11) and 3 x 10(13) times larger than the solution reaction at s (s)pH 9.8, with the largest accelerations being given for substrates where the departing aryloxy unit contains ortho-NO 2 or C(O)OCH 3 groups. Based on the linear Brønsted plots of k cat (max) vs s (s)pKa of the phenol, beta lg values of -0.57 and -0.34 are determined respectively for the catalyzed methanolysis of "regular" substrates that do not contain the ortho-NO 2 or C(O)OCH 3 groups, and those substrates that do. The data are consistent with a two step mechanism for the catalyzed reaction with rate limiting formation of a catalyst-coordinated phosphorane intermediate, followed by fast loss of the aryloxy leaving group. A detailed energetics calculation indicates that the catalyst binds the transition state comprising [CH 3O (-): 6], giving a hypothetical [ 3:Zn(II) 2:CH 3O (-): 6] complex, by -21.4 to -24.5 kcal/mol, with the strongest binding being for those substrates having the ortho-NO 2 or C(O)OCH 3 groups.