Protein D614G Research Articles

Increased viral load in patients infected with severe acute respiratory syndrome coronavirus 2 Omicron variant in the Republic of Korea.

Coronavirus disease 2019 (COVID-19) has been declared a global pandemic owing to the rapid spread of the causative agent, severe acute respiratory syndrome coronavirus 2. Its Delta and Omicron variants are more transmissible and pathogenic than other variants. Some debates have emerged on the mechanism of variants of concern. In the COVID-19 wave that began in December 2021, the Omicron variant, first reported in South Africa, became identifiable in most cases globally. The aim of this study was to provide data to inform effective responses to the transmission of the Omicron variant. The Delta variant and the spike protein D614G mutant were compared with the Omicron variant. Viral loads from 5 days after symptom onset were compared using epidemiological data collected at the time of diagnosis. The Omicron variant exhibited a higher viral load than other variants, resulting in greater transmissibility within 5 days of symptom onset. Future research should focus on vaccine efficacy against the Omicron variant and compare trends in disease severity associated with its high viral load.

Phase I randomized, observer-blinded, placebo-controlled study of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B

Access to vaccines against SARS-CoV-2 virus was limited in poor countries during the COVID-19 pandemic. Therefore, a low-cost mRNA vaccine, PTX-COVID19-B, was produced and evaluated in a Phase 1 trial. PTX-COVID19-B encodes Spike protein D614G variant without the proline-proline (986–987) mutation present in other COVID-19 vaccines. The aim of the study was to evaluate safety, tolerability, and immunogenicity of PTX-COVID19-B vaccine in healthy seronegative adults 18–64 years old. The trial design was observer-blinded, randomized, placebo-controlled, and tested ascending doses of 16-µg, 40-µg, or 100-µg in a total of 60 subjects who received two intramuscular doses, 4 weeks apart. Participants were monitored for solicited and unsolicited adverse events after vaccination and were provided with a Diary Card and thermometer to report any reactogenicity during the trial. Blood samples were collected on baseline, days 8, 28, 42, 90, and 180 for serum analysis of total IgG anti-receptor binding domain (RBD)/Spike titers by ELISA, and neutralizing antibody titers by pseudovirus assay. Titers in BAU/mL were reported as geometric mean and 95% CI per cohort. After vaccination, few solicited adverse events were observed and were mild to moderate and self-resolved within 48 h. The most common solicited local and systemic adverse event was pain at the injection site, and headache, respectively. Seroconversion was observed in all vaccinated participants, who showed high antibody titers against RBD, Spike, and neutralizing activity against the Wuhan strain. Neutralizing antibody titers were also detected against Alpha, Beta, and Delta variants of concerns in a dose dependent manner. All tested doses of PTX-COVID19-B were safe, well-tolerated, and provided a strong immunogenicity response. The 40-µg dose showed fewer adverse reactions than the 100-µg dose, and therefore was selected for a Phase 2 trial, which is currently ongoing.Clinical Trial Registration number: NCT04765436 (21/02/2021). (https://clinicaltrials.gov/ct2/show/NCT04765436).

Molecular epidemiological analysis of SARS-CoV-2 genovariants in Moscow and Moscow region

SARS-CoV-2, a severe acute respiratory illness virus that emerged in China in late 2019, continues to spread rapidly around the world, accumulating mutations and thus causing serious concern. Five virus variants of concern are currently known: Alpha (lineage B.1.1.7), Beta (lineage B.1.351), Gamma (lineage P.1), Delta (lineage B.1.617.2), and Omicron (lineage B.1.1.529). In this study, we conducted a molecular epidemiological analysis of the most prevalent genovariants in Moscow and the region. The aim of the study is to estimate the distribution of various variants of SARS-CoV-2 in Moscow city and the Moscow Region. 227 SARS-CoV-2 sequences were used for analysis. Isolation of the SARS-CoV-2 virus was performed on Vero E6 cell culture. Sequencing was performed by the Sanger method. Bioinformatic analysis was carried out using software packages: MAFFT, IQ-TREE v1.6.12, jModelTest 2.1.7, Nextstrain, Auspice v2.34. As a result of phylogenetic analysis, we have identified the main variants of the virus circulating in Russia that have been of concern throughout the existence of the pandemic, namely: variant B.1.1.7, which accounted for 30% (9/30), AY.122, which accounted for 16.7% (5/30), BA.1.1 with 20% (6/30) and B.1.1 with 33.3% (10/30). When examining Moscow samples for the presence of mutations in SARS-CoV-2 structural proteins of different genovariants, a significant percentage of the most common substitutions was recorded: S protein D614G (86.7%), P681H/R (63.3%), E protein T9I (20.0%); M protein I82T (30.0%), D3G (20.0%), Q19E (20.0%) and finally N protein R203K/M (90.0%), G204R/P (73.3 %). The study of the frequency and impact of mutations, as well as the analysis of the predominant variants of the virus are important for the development and improvement of vaccines for the prevention of COVID-19. Therefore, ongoing molecular epidemiological studies are needed, as these data provide important information about changes in the genome of circulating SARS-CoV-2 variants.

Sequence analysis of SARS-CoV-2 Delta variant isolated from Makassar, South Sulawesi, Indonesia

IntroductionThis study aimed to perform mutation and phylogenetic analyses of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Delta variants and analyze the characteristic signs and symptoms of patients infected with SARS-CoV-2 Delta variant originated from Makassar during the Delta outbreak.Methods: We collected samples from patients who were infected with coronavirus disease 2019 (COVID-19) between June and October 2021. We selected the Quantitative Reverse Transcription-Polymerase Chain Reaction (PCR)-positive samples with a cycle threshold value of <30 for whole genome sequencing. Total viral ribonucleic acid (RNA) was isolated from 34 PCR-positive nasopharyngeal swab samples, and whole genome sequencing was performed using the Oxford Nanopore GridlON sequencer. Phylogenetic and maximum clade credibility analyses were performed using the Bayesian Markov chain Monte Carlo method. ResultsIt was found that 33 patients were infected with the SARS-CoV-2 Delta variant in this cohort study, among whom 63.6% (21) patients were female. According to the clinical data, 24 (72.7%), 7 (21.2%), and 2 (6.1%) patients had mild, moderate, and severe COVID-19 infections. Phylogenetic analysis based on the spike and RNA-dependent RNA polymerase (RdRp) genes showed that the collected samples were clustered in the main lineage of B.1.617.2 (Delta variant). The Delta variants had a high frequency of distinct mutations in the spike protein region, including T19R (94.12%), L452R (88.23%), T478K (91.17%), D614G (97%), P681R (97%), and D950 N (97%). Other unique mutations found in a smaller frequency in our samples were present in the N-terminal domain, including A27T (2.94%) and A222V (14.70%), and in the receptor-binding domain, including Q414K (5.88%), G446V (2.94%), and T470 N (2.94%). ConclusionThis study revealed the unique mutations in the S protein region of Delta variants. T19R, L452R, T478K/T478R, D614G, P681R, and D950 N were the most common substitutions in Makassar's Delta variant.

Structural effects of spike protein D614G mutation in SARS-CoV-2

A single mutation from aspartate to glycine at position 614 has dominated all circulating variants of the severe acute respiratory syndrome coronavirus 2. D614G mutation induces structural changes in the spike (S) protein that strengthen the virus infectivity. Here, we use molecular dynamics simulations to dissect the effects of mutation and 630-loop rigidification on S-protein structure. The introduction of the mutation orders the 630-loop structure and thereby induces global structural changes toward the cryoelectron microscopy structure of the D614G S-protein. The ordered 630-loop weakens local interactions between the 614th residue and others in contrast to disordered structures in the wild-type protein. The mutation allosterically alters global interactions between receptor-binding domains, forming an asymmetric and mobile down conformation and facilitating transitions toward up conformation. The loss of salt bridge between D614 and K854 upon the mutation generally stabilizes S-protein protomer, including the fusion peptide proximal region that mediates membrane fusion. Understanding the molecular basis of D614G mutation is crucial as it dominates in all variants of concern, including Delta and Omicron.

Long-term, infection-acquired immunity against the SARS-CoV-2 Delta variant in a hamster model.

The emergence of the SARS-CoV-2 Delta variant (B.1.617.2) raises concerns about potential reduced sensitivity of the virus to antibody neutralization and subsequent vaccine breakthrough infections. Here, we use a live virus neutralization assay with sera from Pfizer- and Moderna-vaccinated individuals to examine neutralizing antibody titers against SARS-CoV-2 and observe a 3.9- and 2.7-fold reduction, respectively, in neutralizing antibody titers against the Delta variant compared with an early isolate bearing only a D614G substitution in its spike protein. We observe similar reduced sensitivity with sera from hamsters that were previously infected with an early isolate of SARS-CoV-2. Despite this reduction in neutralizing antibody titers against the Delta variant, hamsters previously infected (up to 15 months earlier) with an early isolate are protected from infection with the Delta variant, suggesting that the immune response to the first infection is sufficient to provide protection against subsequent infection with the Delta variant.

Prediction of the Effects of Nonsynonymous Variants on SARS-CoV-2 Proteins.

Background:SARS-CoV-2 virus isa highly transmissible pathogen that causesCOVID-19. Theoutbreak originatedinWuhan, China in December 2019. A number of nonsynonymous mutations located atdifferentSARS-CoV-2 proteinshavebeen reportedby multiple studies.However, therearelimitedcomputationalstudies onthebiological impacts of these mutations on the structure and function of the proteins. Methods:In our study nonsynonymous mutations oftheSARS-CoV-2 genome and their frequencieswere identifiedfrom30,229sequences.Subsequently, theeffects ofthetop10nonsynonymous mutations of different SARS-CoV-2 proteinswere analyzed using bioinformatics toolsincluding co-mutation analysis,prediction oftheprotein structure stabilityand flexibilityanalysis,andprediction oftheprotein functions. Results:Atotal of 231 nonsynonymous mutations were identified from 30,229 SARS-CoV-2 genome sequences. The top10nonsynonymous mutationsaffectingnineamino acid residueswere ORF1a nsp5 P108S, ORF1b nsp12 P323L and A423V, S protein N501Y and D614G, ORF3a Q57H, N protein P151L, R203K and G204R. Many nonsynonymous mutations showedahigh concurrence ratio, suggesting these mutations may evolve together and interact functionally. Our result showedthatORF1a nsp5P108S,ORF3aQ57H and N protein P151Lmutations may be deleterious to thefunctionof SARS-CoV-2 proteins.In addition,ORF1a nsp5P108Sand S proteinD614Gmaydestabilizethe protein structureswhileS protein D614Gmay have a more open conformationcompared to the wild type. Conclusion:The biological consequences of these nonsynonymous mutations of SARS-CoV-2 proteins should be further validated byin vivoandin vitroexperimental studies in the future.

COVID-19 Outcomes and Genomic Characterization of SARS-CoV-2 Isolated From Veterans in New England States: Retrospective Analysis.

BackgroundClinical and virologic characteristics of COVID-19 infections in veterans in New England have not been described. The average US veteran is a male older than the general US population. SARS-CoV-2 infection is known to cause poorer outcomes among men and older adults, making the veteran population an especially vulnerable group for COVID-19.ObjectiveThis study aims to evaluate clinical and virologic factors impacting COVID-19 outcomes.MethodsThis retrospective chart review included 476 veterans in six New England states with confirmed SARS-CoV-2 infection between April and September 2020. Whole genome sequencing was performed on SARS-CoV-2 RNA isolated from these veterans, and the correlation of genomic data to clinical outcomes was evaluated. Clinical and demographic variables were collected by manual chart review and were correlated to the end points of peak disease severity (based on oxygenation requirements), hospitalization, and mortality using multivariate regression analyses.ResultsOf 476 veterans, 274 had complete and accessible charts. Of the 274 veterans, 92.7% (n=254) were men and 83.2% (n=228) were White, and the mean age was 63 years. In the multivariate regression, significant predictors of hospitalization (C statistic 0.75) were age (odds ratio [OR] 1.05, 95% CI 1.03-1.08) and non-White race (OR 2.39, 95% CI 1.13-5.01). Peak severity (C statistic 0.70) also varied by age (OR 1.07, 95% CI 1.03-1.11) and O2 requirement on admission (OR 45.7, 95% CI 18.79-111). Mortality (C statistic 0.87) was predicted by age (OR 1.06, 95% CI 1.01-1.11), dementia (OR 3.44, 95% CI 1.07-11.1), and O2 requirement on admission (OR 6.74, 95% CI 1.74-26.1). Most (291/299, 97.3%) of our samples were dominated by the spike protein D614G substitution and were from SARS-CoV-2 B.1 lineage or one of 37 different B.1 sublineages, with none representing more than 8.7% (26/299) of the cases.ConclusionsIn a cohort of veterans from the six New England states with a mean age of 63 years and a high comorbidity burden, age was the largest predictor of hospitalization, peak disease severity, and mortality. Non-White veterans were more likely to be hospitalized, and patients who required oxygen on admission were more likely to have severe disease and higher rates of mortality. Multiple SARS-CoV-2 lineages were distributed in patients in New England early in the COVID-19 era, mostly related to viruses from New York State with D614G mutation.

Site-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains.

The SARS-CoV-2 spike protein is heavily glycosylated, having 22 predicted N-glycosylation sites per monomer. It is also O-glycosylated, although the number of O-glycosites is less defined. Recent studies show that spike protein glycans play critical roles in viral entry and infection. The spike monomer has two subdomains, S1 and S2, and a receptor-binding domain (RBD) within the S1 domain. In this study, we have characterized the site-specific glycosylation patterns of the HEK293 recombinant spike RBD and S1 domains as well as the intact spike derived from the whole virus produced in Vero cells. The Vero cell-derived spike from the WA1 strain and a D614G variant was analyzed. All spike proteins, S1, and RBDs were analyzed using hydrophilic interaction chromatography (HILIC) and LC-MS/MS on an Orbitrap Eclipse Tribrid mass spectrometer. N-glycans identified in HEK293-derived S1 were structurally diverse. Those found in the HEK293-derived RBD were highly similar to those in HEK293 S1 where N-glycosites were shared. Comparison of the whole cell-derived WA1 and D614G spike proteins revealed that N-glycosites local to the mutation site appeared to be more readily detected, hinting that these sites are more exposed to glycosylation machinery. Moreover, recombinant HEK293-derived S1 was occupied almost completely with complex glycan, while both WA1 and D614G derived from the Vero E6 cell whole virus were predominantly high-mannose glycans. This stands in stark contrast to glycosylation patterns seen in both CHO- and HEK cell-derived recombinant S1, S2, and the whole spike previously reported. Concerning O-glycosylation, our analyses revealed that HEK293 recombinant proteins possessed a range of O-glycosites with compositions consistent with Core type 1 and 2 glycans. The O-glycosites shared between the S1 and RBD constructs, sites T323 and T523, were occupied by a similar range of Core 1 and 2 type O-glycans. Overall, this study reveals that the sample nature and cell substrate used for production of these proteins can have a dramatic impact on the glycosylation profile. SARS-CoV-2 spike glycans are associated with host ACE2 receptor interaction efficiency. Therefore, understanding such differences will serve to better understand these host–pathogen interactions and inform the choice of cell substrates to suite downstream investigations.

Emergence and expansion of highly infectious spike protein D614G mutant SARS-CoV-2 in central India

COVID-19 has emerged as global pandemic with largest damage to the public health, economy and human psyche.The genome sequence data obtained during the ongoing pandemic are valuable to understand the virus evolutionary patterns and spread across the globe. Increased availability of genome information of circulating SARS-CoV-2 strains in India will enable the scientific community to understand the emergence of new variants and their impact on human health. The first case of COVID-19 was detected in Chambal region of Madhya Pradesh state in mid of March 2020 followed by multiple introduction events and expansion of cases within next three months. More than 5000 COVID-19 suspected samples referred to Defence Research and Development Establishment, Gwalior, Madhya Pradesh were analyzed during the nation -wide lockdown and unlock period. A total of 136 cases were found positive over a span of three months that included virus introduction to the region and its further spread. Whole genome sequences employing Oxford nanopore technology were generated for 26 SARS-CoV-2 circulating in 10 different districts in Madhya Pradesh state of India. This period witnessed index cases with multiple travel histories responsible for introduction of COVID-19 followed by remarkable expansion of virus. The genome wide substitutions including in important viral proteins were identified. The detailed phylogenetic analysis revealed the circulating SARS-CoV-2 clustered in multiple clades including A2a, A4 and B. The cluster-wise segregation was observed, suggesting multiple introduction links and subsequent evolution of virus in the region. This is the first comprehensive whole genome sequence analysis from central India, which revealed the emergence and evolution of SARS-CoV-2 during thenation-wide lockdown and unlock.

Comparative MD Study of Inhibitory Activity of Opaganib and Adamantane-Isothiourea Derivatives toward COVID-19 Main Protease Mpro.

In this study, the inhibitory potency of four adamantly‐ isothiourea derivatives (compounds 1 [4‐bromobenzyl (Z)‐N′‐(adamantan‐1‐yl)‐4‐phenylpiperazine‐1‐carbothioimidate], 2 [3,5‐bis(trifluoromethyl)benzyl (Z)‐N′‐(adamantan‐1‐yl)‐4‐phenylpiperazine‐1‐carbothioimidate], 3 [4‐bromobenzyl (Z)‐N‐(adamantan‐1‐yl)morpholine‐4‐carbothioimidate] and 4 [3,5‐bis(trifluoromethyl)benzyl (Z)‐N‐(adamantan‐1‐yl)morpholine‐4‐carbothioimidate]) was evaluated against SARS‐CoV‐2 targeted proteins. The investigated compounds 1–4 possess a similar structure to opaganib, which is used in studies like a potential drug for COVID‐19 treatment. Since examined adamantly‐isothiourea derivatives (1–4) shown broad‐spectrum of antibacterial activity and significant in vitro cytotoxic effects against five human tumor cell lines and shown similarity in structure with opaganib, it was of interest to study their inhibitory potency toward some SARS‐CoV‐2 proteins such as SARS‐CoV‐2 main protease Mpro and mutation of SARS‐CoV‐2 Spike (S) Protein D614G. The inhibitory potency of studied compounds is examined using molecular docking and molecular dynamic simulations. The results of molecular docking simulations indicate compound 1 as the most prominent candidate of inhibition of SARS‐CoV‐2 main protease Mpro (▵Gbind=11.24 kcal/mol), while almost the same inhibition potency of all studied compounds is exhibited toward D614G. Regarding the results obtained by molecular dynamic simulations, compounds 1 and 4 possess similar inhibitory potency toward SARS‐CoV‐2 main protease Mpro as opaganib (▵Gbind ≈ 40 kcal/mol).

Detection of SARS-CoV-2 spike protein D614G mutation by qPCR-HRM analysis

ObjectivesMonitoring the spread of the G614 in specific locations is critical as this variant is highly transmissible and can trigger the emergence of other mutations. Therefore, a rapid and accurate method that can reliably detect the D614G mutation will be beneficial. This study aims to analyze the potential use of the two-step Reverse Transcriptase quantitative polymerase chain reaction - high resolution melting analysis (RT-qPCR-HRM) to detect a specific mutation in the SARS-CoV-2 genome. MethodsSix SARS-CoV-2 RNA samples were synthesized into cDNA and analyzed with the qPCR-HRM method in order to detect the D614G mutation in Spike protein of SARS-CoV-2. The primers are designed to target the specific Spike region containing the D614G mutation. The qPCR-HRM analysis was conducted simultaneously, and the identification of the SARS-CoV-2 variant was confirmed by conventional PCR and Sanger sequencing methods. ResultsThe results showed that the melting temperature (Tm) of the D614 variant was 79.39 ± 0.03 °C, which was slightly lower than the Tm of the G614 variant (79.62 ± 0.015 °C). The results of the HRM analysis, visualized by the normalized melting curve and the difference curve were able to discriminate the D614 and G614 variant samples. All samples were identified as G614 variants by qPCR-HRM assay, which was subsequently confirmed by Sanger sequencing. ConclusionsThis study demonstrated a sensitive method that can identify the D614G mutation by a simple two-step RT-qPCR-HRM assay procedure analysis, which can be useful for active surveillance of the transmission of a specific mutation.

Implications of the Novel Mutations in the SARS-CoV-2 Genome for Transmission, Disease Severity, and the Vaccine Development.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of the coronavirus disease 2019 (COVID-19), has been identified in China in late December 2019. SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA betacoronavirus of the Coronaviridae family. Coronaviruses have genetic proofreading mechanism that corrects copying mistakes and thus SARS-CoV-2 genetic diversity is extremely low. Despite lower mutation rate of the virus, researchers have detected a total of 12,706 mutations in the SARS-CoV-2 genome, the majority of which were single nucleotide polymorphisms. Sequencing data revealed that the SARS-CoV-2 accumulates two-single nucleotide mutations per month in its genome. Recently, an amino acid aspartate (D) to glycine (G) (D614G) mutation due to an adenine to guanine nucleotide change at position 23,403 at the 614th amino-acid position of the spike protein in the original reference genotype has been identified. The SARS-CoV-2 viruses that carry the spike protein D614G mutation have become dominant variant around the world. The D614G mutation has been found to be associated with 3 other mutations in the spike protein. Clinical and pseudovirus experimental studies have demonstrated that the spike protein D614G mutation alters the virus phenotype. However, the impact of the mutation on the rate of transmission between people, disease severity and the vaccine and therapeutic development remains unclear. Three variants of SARS-CoV-2 have recently been identified. They are B.1.1.7 (UK) variant, B.1.351 (N501Y.V2, South African) variant and B.1.1.28 (Brazilian) variant. Epidemiological data suggest that they have a higher transmissibility than the original variant. There are reports that some vaccines are less efficacious against the B.1.351 variant. This review article discusses the effects of novel mutations in the SARS-CoV-2 genome on transmission, clinical outcomes and vaccine development.

Comparative analysis of mutational hotspots in the spike protein of SARS-CoV-2 isolates from different geographic origins

The spike (S) protein mutations of SARS-CoV-2 are of major concern in terms of viral transmission and pathogenesis. Hence, we developed a PCR-based method to rapidly detect the 6 mutational hotspots (H49Y, G476S, V483A, H519Q, A520S, and D614G) in the S protein and applied this method to analyze the hotspots in the viral isolates from different geographical origins. Here, we identified that there was only the D614G mutation in the viral isolates. As of September 30, 2020, the analysis of 113,381 sequences available from the public repositories revealed that the SARS-CoV-2 variant carrying G614 has become the most prevalent form globally. Our results support recent epidemiological and genomic data demonstrating that the viral infectivity and transmission are enhanced by the S protein D614G mutation.

Structural phylogenetic analysis reveals lineage-specific RNA repetitive structural motifs in all coronaviruses and associated variations in SARS-CoV-2.

In many single-stranded (ss) RNA viruses, the cis-acting packaging signal that confers selectivity genome packaging usually encompasses short structured RNA repeats. These structural units, termed repetitive structural motifs (RSMs), potentially mediate capsid assembly by specific RNA–protein interactions. However, general knowledge of the conservation and/or the diversity of RSMs in the positive-sense ssRNA coronaviruses (CoVs) is limited. By performing structural phylogenetic analysis, we identified a variety of RSMs in nearly all CoV genomic RNAs, which are exclusively located in the 5′-untranslated regions (UTRs) and/or in the inter-domain regions of poly-protein 1ab coding sequences in a lineage-specific manner. In all alpha- and beta-CoVs, except for Embecovirus spp, two to four copies of 5′-gUUYCGUc-3′ RSMs displaying conserved hexa-loop sequences were generally identified in Stem-loop 5 (SL5) located in the 5′-UTRs of genomic RNAs. In Embecovirus spp., however, two to eight copies of 5′-agc-3′/guAAu RSMs were found in the coding regions of non-structural protein (NSP) 3 and/or NSP15 in open reading frame (ORF) 1ab. In gamma- and delta-CoVs, other types of RSMs were found in several clustered structural elements in 5′-UTRs and/or ORF1ab. The identification of RSM-encompassing structural elements in all CoVs suggests that these RNA elements play fundamental roles in the life cycle of CoVs. In the recently emerged SARS-CoV-2, beta-CoV-specific RSMs are also found in its SL5, displaying two copies of 5′-gUUUCGUc-3′ motifs. However, multiple sequence alignment reveals that the majority of SARS-CoV-2 possesses a variant RSM harboring SL5b C241U, and intriguingly, several variations in the coding sequences of viral proteins, such as Nsp12 P323L, S protein D614G, and N protein R203K-G204R, are concurrently found with such variant RSM. In conclusion, the comprehensive exploration for RSMs reveals phylogenetic insights into the RNA structural elements in CoVs as a whole and provides a new perspective on variations currently found in SARS-CoV-2.

Research Article Temporal data series and logistic models reveal the dynamics of SARS-CoV-2 spike protein D614G variant in the COVID-19 pandemic

The COVID-19 pandemic started in Wuhan, China, and caused the worldwide spread of the RNA virus SARS-CoV-2, the causative agent of COVID-19. Because of its mutational rate, wide geographical distribution, and host response variance this coronavirus is currently evolving into an array of strains with increasing genetic diversity. Most variants apparently have neutral effects for disease spread and symptoms severity. However, in the viral Spike protein, which is responsible for host cell attachment and invasion, an emergent variant, containing the amino acid substitution D to G in position 614 (D614G), was suggested to increase viral infection capability. To test whether this variant has epidemiological impact, the temporal distributions of the SARS-CoV-2 samples bearing D or G at position 614 were compared in the USA, Asia and Europe. The epidemiological curves were compared at early and late epidemic stages. At early stages, where containment measures were still not fully implemented, the viral variants are supposed to be unconstrained and its growth curves might approximate the free viral dynamics. Our analysis shows that the D614G prevalence and the growth rates of COVID-19 epidemic curves are correlated in the USA, Asia and Europe. Our results suggest a selective sweep that can be explained, at least in part, by a propagation advantage of this variant, in other words, that the molecular level effects of D614G have sufficient impact on population transmission dynamics as to be detected by differences in rate coefficients of epidemic growth curves.

Spike protein D614G and RdRp P323L: the SARS-CoV-2 mutations associated with severity of COVID-19.

The severity of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), greatly varies from patient to patient. In the present study, we explored and compared mutation profiles of SARS-CoV-2 isolated from mildly affected and severely affected COVID-19 patients in order to explore any relationship between mutation profile and disease severity. Genomic sequences of SARS-CoV-2 were downloaded from Global Initiative on Sharing Avian Influenza Data (GISAID) database. With the help of Genome Detective Coronavirus Typing Tool, genomic sequences were aligned with the Wuhan seafood market pneumonia virus reference sequence and all the mutations were identified. Distribution of mutant variants was then compared between mildly and severely affected groups. Among the numerous mutations detected, 14408C>T and 23403A>G mutations resulting in RNA-dependent RNA polymerase (RdRp) P323L and spike protein D614G mutations, respectively, were found predominantly in severely affected group (>82%) compared with mildly affected group (<46%, p < 0.001). The 241C>T mutation in the non-coding region of the genome was also found predominantly in severely affected group (p < 0.001). The 3037C>T, a silent mutation, also appeared in relatively high frequency in severely affected group compared with mildly affected group, but the difference was not statistically significant (p = 0.06). We concluded that spike protein D614G and RdRp P323L mutations in SARS-CoV-2 are associated with severity of COVID-19. Further studies will be required to explore whether these mutations have any impact on the severity of disease.

SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

SARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (SG614) with the original (SD614). We report here pseudoviruses carrying SG614 enter ACE2-expressing cells more efficiently than those with SD614. This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion.

Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection After Recovery from Mild Coronavirus Disease 2019.

BackgroundPositive results from real-time reverse-transcription polymerase chain reaction (rRT-PCR) in recovered patients raise concern that patients who recover from coronavirus disease 2019 (COVID-19) may be at risk of reinfection. Currently, however, evidence that supports reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been reported. MethodsWe conducted whole-genome sequencing of the viral RNA from clinical specimens at the initial infection and at the positive retest from 6 patients who recovered from COVID-19 and retested positive for SARS-CoV-2 via rRT-PCR after recovery. A total of 13 viral RNAs from the patients’ respiratory specimens were consecutively obtained, which enabled us to characterize the difference in viral genomes between initial infection and positive retest. ResultsAt the time of the positive retest, we were able to acquire a complete genome sequence from patient 1, a 21-year-old previously healthy woman. In this patient, through the phylogenetic analysis, we confirmed that the viral RNA of positive retest was clustered into a subgroup distinct from that of the initial infection, suggesting that there was a reinfection of SARS-CoV-2 with a subtype that was different from that of the primary strain. The spike protein D614G substitution that defines the clade “G” emerged in reinfection, while mutations that characterize the clade “V” (ie, nsp6 L37F and ORF3a G251V) were present at initial infection. ConclusionsReinfection with a genetically distinct SARS-CoV-2 strain may occur in an immunocompetent patient shortly after recovery from mild COVID-19. SARS-CoV-2 infection may not confer immunity against a different SARS-CoV-2 strain.

Higher binding affinity of furin for SARS-CoV-2 spike (S) protein D614G mutant could be associated with higher SARS-CoV-2 infectivity

ObjectiveThe coronavirus disease 2019 (COVID-19) pandemic has caused an exponential rise in death rates and hospitalizations. The aim of this study was to characterize the D614G substitution in the severe acute respiratory syndome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S protein), which may affect viral infectivity. MethodsThe effect of D614G substitution on the structure and thermodynamic stability of the S protein was analyzed with use of DynaMut and SCooP. HDOCK and PRODIGY were used to model furin protease binding to the S protein RRAR cleavage site and calculate binding affinities. Molecular dynamics simulations were used to predict the S protein apo structure, the S protein–furin complex structure, and the free binding energy of the complex. ResultsThe D614G substitution in the G clade of SARS-CoV-2 strains introduced structural mobility and decreased the thermal stability of the S protein (ΔΔG = −0.086 kcal mol−1). The substitution resulted in stronger binding affinity (Kd = 1.6 × 10−8) for furin, which may enhance S protein cleavage. The results were corroborated by molecular dynamics simulations demonstrating higher binding energy of furin and the S protein D614G mutant (−61.9 kcal mol−1 compared with −56.78 kcal mol−1 for wild-type S protein). ConclusionsThe D614G substitution in the G clade induced flexibility of the S protein, resulting in increased furin binding, which may enhance S protein cleavage and infiltration of host cells. Therefore, the SARS-CoV-2 D614G substitution may result in a more virulent strain.