SARS CoV-2 Variants and Spike Mutations Involved in Second Wave of COVID-19 Pandemic in India

Against the backdrop of the second wave of COVID-19 pandemic in India that started in March 2021, we have monitored the spike (S) protein mutations in all the reported (GISAID portal) whole genome sequences of SARS CoV-2 circulating in India from 1 January 2021 to 31 August 2021. In the 43,102 SARS-CoV-2 genomic sequences analysed, we have identified 24, 260 mutations in the S protein, based on which 265 pango lineages could be categorised. The dominant lineage in most of the 28 states of India and its 8 union territories was B.1.617.2 (the delta variant). However, the states Madhya Pradesh, Jammu & Kashmir, and Punjab had B.1.1.7 (alpha variant) as the major lineage, while the Himachal Pradesh state reported B.1.36 as the dominating lineage. A detailed analysis of various domains of S protein was carried out for detecting mutations having a prevalence of >1%; 70, 18, 7, 3, 9, 4, and 1 (N=112) such mutations were observed in the N -terminal domain, receptor binding domain, C -terminal domain, fusion peptide region, heptapeptide repeat (HR)-1 domains, signal peptide domain, and transmembrane region, respectively. However, no mutations were recorded in the HR-2, and cytoplasmic domains of the S protein. Interestingly, 13.39% (N=15) of these mutations were reported to increase the infectivity and pathogenicity of the virus; 2%(N=3) were known to be vaccine breakthrough mutations; and 0.89%(N=1) were known to escape neutralising antibodies. Biological significance of 82% (N=92) of the reported mutations is yet unknown. As SARS-CoV-2 variants are emerging rapidly, it is critical to continuously monitor local viral mutations to understand national trends of virus circulation. This can tremendously help in designing better preventive regimens in the country, and avoid vaccine breakthrough infections.

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Identification of a novel neutralizing epitope on the N-terminal domain of the HCoV-229E spike protein

Journal of Virology ◽

10.1128/jvi.01955-21 ◽

2021 ◽

Author(s):

Jiale Shi ◽

Yuejun Shi ◽

Ruixue Xiu ◽

Gang Wang ◽

Rui Liang ◽

...

Keyword(s):

Epitope Mapping ◽

Vaccine Development ◽

Selective Pressure ◽

Spike Protein ◽

Neutralizing Epitope ◽

Change Over Time ◽

S Protein ◽

Terminal Domain ◽

Neutralizing Epitopes ◽

Over Time

The receptor binding domain (RBD) of the coronavirus spike protein (S) has been verified to be the main target for potent neutralizing antibodies (nAbs) in most coronaviruses, and the N-terminal domain (NTD) of some betacoronaviruses has also been indicated to induce nAbs. For alphacoronavirus HCoV-229E, its RBD has been shown to have neutralizing epitopes, and these epitopes could change over time. However, whether neutralizing epitopes exist on the NTD and whether these epitopes change like those of the RBD are still unknown. Here, we verified that neutralizing epitopes exist on the NTD of HCoV-229E. Furthermore, we characterized an NTD targeting nAb 5H10, which could neutralize both pseudotyped and authentic HCoV-229E VR740 in vitro. Epitope mapping indicated that 5H10 targeted motif E1 (147-167 aa) and identified F159 as critical for 5H10 binding. More importantly, our results revealed that motif E1 was highly conserved among clinical isolates except for F159. Further data proved that mutations at position 159 gradually appeared over time and could completely abolish the neutralizing ability of 5H10, supporting the notion that position 159 may be under selective pressure during the human epidemic. In addition, we also found that contemporary clinical serum has a stronger binding capacity for the NTD of contemporary strains than historic strains, proving that the epitope on the NTD could change over time. In summary, these findings define a novel neutralizing epitope on the NTD of HCoV-229E S and provide a theoretical basis for the design of vaccines against HCoV-229E or related coronaviruses. Importance Characterization of the neutralizing epitope of the spike (S) protein, the major invasion protein of coronaviruses, can help us better understand the evolutionary characteristics of these viruses and promote vaccine development. To date, the neutralizing epitope distribution of alphacoronaviruses is not well known. Here, we identified a neutralizing antibody that targeted the N-terminal domain (NTD) of the alphacoronavirus HCoV-229E S protein. Epitope mapping revealed a novel epitope that was not previously discovered in HCoV-229E. Further studies identified an important residue, F159. Mutations that gradually appeared over time at this site abolished the neutralizing ability of 5H10, indicating that selective pressure occurred at this position in the spread of HCoV-229E. Furthermore, we found that the epitopes within the NTD also changed over time. Taken together, our findings defined a novel neutralizing epitope and highlighted the role of the NTD in the future prevention and control of HCoV-229E or related coronaviruses.

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Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra120.002295 ◽

2020 ◽

pp. mcp.RA120.002295 ◽

Cited By ~ 3

Author(s):

Yong Zhang ◽

Wanjun Zhao ◽

Yonghong Mao ◽

Yaohui Chen ◽

Shisheng Wang ◽

...

Keyword(s):

Insect Cells ◽

Host Cells ◽

Protein Subunit ◽

Genome Sequences ◽

Complex Type ◽

Site Specific ◽

Differential Processing ◽

S Protein ◽

Shed Light

The glycoprotein spike (S) on the surface of SARS-CoV-2 is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunit. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, completely of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells, regardless of the glycosites’ location. Moreover, the N-glycan compositions were conserved among different sizes of subunits. Our study indicate that the S protein N-glycosylation occurs regularly at each site, albeit the occupied N-glycans were diverse and heterogenous. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.

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SARS-CoV-2 B.1.617.2 Delta variant emergence and vaccine breakthrough

10.21203/rs.3.rs-637724/v1 ◽

2021 ◽

Author(s):

Petra Mlcochova ◽

Steven Kemp ◽

Mahesh Shanker Dhar ◽

Guido Papa ◽

Bo Meng ◽

...

Keyword(s):

Severe Disease ◽

Control Measures ◽

Virus Infectivity ◽

Neutralising Antibodies ◽

Post Vaccination ◽

Viral Loads ◽

Infection Control Measures ◽

Alpha Variant ◽

In Vitro Data

Abstract The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and has spread throughout India, displacing the B.1.1.7 (Alpha) variant and other pre-existing lineages. Mathematical modelling indicates that the growth advantage is most likely explained by a combination of increased transmissibility and immune evasion. Indeed in vitro, the delta variant is less sensitive to neutralising antibodies in sera from recovered individuals, with higher replication efficiency as compared to the Alpha variant. In an analysis of vaccine breakthrough in over 100 healthcare workers across three centres in India, the Delta variant not only dominates vaccine-breakthrough infections with higher respiratory viral loads compared to non-delta infections (Ct value of 16.5 versus 19), but also generates greater transmission between HCW as compared to B.1.1.7 or B.1.617.1 (p=0.02). In vitro, the Delta variant shows 8 fold approximately reduced sensitivity to vaccine-elicited antibodies compared to wild type Wuhan-1 bearing D614G. Serum neutralising titres against the SARS-CoV-2 Delta variant were significantly lower in participants vaccinated with ChadOx-1 as compared to BNT162b2 (GMT 3372 versus 654, p<0001). These combined epidemiological and in vitro data indicate that the dominance of the Delta variant in India has been most likely driven by a combination of evasion of neutralising antibodies in previously infected individuals and increased virus infectivity. Whilst severe disease in fully vaccinated HCW was rare, breakthrough transmission clusters in hospitals associated with the Delta variant are concerning and indicate that infection control measures need continue in the post-vaccination era.

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A novel potentially recombinant rodent coronavirus with a polybasic cleavage site in the spike protein

Journal of Virology ◽

10.1128/jvi.01173-21 ◽

2021 ◽

Author(s):

Xin Li ◽

Liang Wang ◽

Peipei Liu ◽

Hongying Li ◽

Shuting Huo ◽

...

Keyword(s):

Rural Areas ◽

Urban Areas ◽

Cleavage Site ◽

One Health ◽

Hepatitis Virus ◽

Southern China ◽

Genome Sequences ◽

Murine Hepatitis Virus ◽

S Protein ◽

Pcr Screening

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reignited global interest in animal coronaviruses and their potential for human transmission. While bats are thought to be the wildlife reservoir of SARS-CoV and SARS-CoV-2, the widespread human coronavirus OC43 is thought to have originated in rodents. We sampled 297 rodents and shrews representing eight species in three municipalities of southern China. We report coronavirus prevalence of 23.3% and 0.7% in Guangzhou and Guilin, respectively, with samples from urban areas having significantly higher coronavirus prevalence than those from rural areas. We obtained three coronavirus genome sequences from Rattus norvegicus , including a Betacoronavirus RCoV-GCCDC3, an Alphacoronavirus RCoV-GCCDC5 and a novel Betacoronavirus RCoV-GCCDC4. Recombination analysis suggests that there was a potential recombinant event involving RCoV-GCCDC4, Murine hepatitis virus and Longquan Rl rat coronavirus. Furthermore, we uncovered a polybasic cleavage site RARR in the spike (S) protein of RCoV-GCCDC4, which is dominant in RCoV. These findings provide further information on the potential for inter-species transmission of coronaviruses and demonstrate the value of a One Health approach to virus discovery. Importance Surveillance of viruses among rodents within rural and urban areas of South China identified three rodent coronaviruses RCoV-GCCDC3, 4 and 5, one of which was identified as a novel potentially recombinant coronavirus with a polybasic cleavage site in the spike (S) protein. Through reverse transcription PCR screening of coronaviruses, we found that coronavirus prevalence in urban areas is much higher than that in rural areas. Subsequently, we obtained three coronavirus genome sequences by deep sequencing. After different method-based analyses, we found that RCoV-GCCDC4 was a novel potentially recombinant coronavirus with a polybasic cleavage site in S protein, dominant in RCoV. This newly identified coronavirus RCoV-GCCDC4 with its potentially recombinant genome and polybasic cleavage site provides a new insight into the evolution of coronaviruses. Furthermore, our results provide further information on the potential for inter-species transmission of coronaviruses and demonstrate the necessity of a One Health approach for zoonotic disease surveillance.

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Relative Consolidation of the Kappa Variant Pre-Dates the Massive Second Wave of COVID-19 in India

Genes ◽

10.3390/genes12111803 ◽

2021 ◽

Vol 12 (11) ◽

pp. 1803

Author(s):

Jitendra Singh ◽

Anvita Gupta Malhotra ◽

Debasis Biswas ◽

Prem Shankar ◽

Leena Lokhande ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Spike Protein ◽

Whole Genome ◽

The Novel ◽

Genome Sequences ◽

Second Wave ◽

Insight Into

India experienced a tragic second wave after the end of March 2021, which was far more massive than the first wave and was driven by the emergence of the novel delta variant (B.1.617.2) of the SARS-CoV-2 virus. In this study, we explored the local and national landscape of the viral variants in the period immediately preceding the second wave to gain insight into the mechanism of emergence of the delta variant and thus improve our understanding of the causation of the second wave. We randomly selected 20 SARS-CoV-2 positive samples diagnosed in our lab between 3 February and 8 March 2021 and subjected them to whole genome sequencing. Nine of the 20 sequenced genomes were classified as kappa variant (B.1.617.1). The phylogenetic analysis of pan-India SARS-CoV-2 genome sequences also suggested the gradual replacement of the α variant with the kappa variant during this period. This relative consolidation of the kappa variant was significant, since it shared 3 of the 4 signature mutations (L452R, E484Q and P681R) observed in the spike protein of delta variant and thus was likely to be the precursor in its evolution. This study demonstrates the predominance of the kappa variant in the period immediately prior to the second wave and underscores its role as the “bridging variant” between the α and delta variants that drove the first and second waves of COVID-19 in India, respectively.

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The N-terminal domain of Spike protein contributes to antigenicity difference between porcine epidemic diarrhea virus genotypes G1 and G2

10.22541/au.161460452.27398224/v1 ◽

2021 ◽

Author(s):

Qi-long Qiao ◽

Ning Li ◽

Ming-zhen Song ◽

Jing Chen ◽

Pan pan Yang ◽

...

Keyword(s):

Amino Acid ◽

Porcine Epidemic Diarrhea Virus ◽

Enzyme Linked Immunosorbent Assay ◽

S Protein ◽

Diarrhea Virus ◽

Terminal Domain ◽

Porcine Epidemic Diarrhea ◽

Amino Acid Mutations ◽

Multiple Amino Acid ◽

S Proteins

Porcine epidemic diarrhea virus (PEDV) strains have been clarified into two genotypes, G1 and G2, based on the sequence of the spike (S) gene. Amino acid mutations that distinguish the two PEDV genotypes were mostly located in the N-terminal domain (NTD) (aa 1-380) of S protein. The fact of increased outbreaks of G2 subtype PEDV and the failure of G1 subtype PEDV strain (CV777)-based vaccine in China since 2010 suggested that multiple amino acid mutations located in the NTD altered the antigenicity of S protein. To determine the role of the NTD of S protein in the antigenicity difference, the NTD of the CV777 vaccine strain (G1) and CH/ZMDZY/11 strain (G2) was expressed in E. coli, respectively. polyclonal antibodies (PAbs) against genotype-specific S proteins were prepared by immunizing BALB/c mice using purified S proteins. Antigenicity was systematically compared by detection of PAbs against two genotype PEDV strains and purified S proteins using Western blot, indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and serum cross-neutralization assay (SN). Consistent with the multiple amino acid mutations in the NTD of S protein, different antigenic cross-reactivity between the two genotypes was demonstrated. There was six-fold and more than twenty-fold difference in ELISA and SN titer between anti-CV777 S protein antibodies against G1 and G2 subtype strains, respectively. There was twofold and eight-fold difference in ELISA and SN titer between anti-ZMDZY S protein antibodies against G1 and G2 genotype strains, respectively. The results proved that the NTD of S protein contributes to the antigenicity difference between PEDV genotypes G1 and G2, and highlighted a G2 strain should be used to develop a vaccine for providing better protection against prevalent genotype of PEDV.

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Immunogenicity of SCB-2019 COVID-19 Vaccine Compared to Four Approved Vaccines

10.21203/rs.3.rs-902086/v1 ◽

2021 ◽

Author(s):

Donna Ambrosino ◽

Htay Htay Han ◽

Branda Hu ◽

Joshua Liang ◽

Ralf Clemens ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Vaccine Efficacy ◽

Antibody Responses ◽

Original Strain ◽

S Protein ◽

Binding Antibody ◽

Alpha Variant ◽

Efficacy Studies

Abstract A significant correlation has been shown between the binding antibody responses against original SARS-CoV-2-S-protein all performed in one laboratory and vaccine efficacy of four approved COVID-19 vaccines. We therefore assessed the immune response against original SARS-CoV-2 elicited by the adjuvanted S-Trimer vaccine, SCB-2019 + CpG/alum, in the same assay and laboratory. When compared with four approved vaccines immune responses to SCB-2019 predicted 81% − 94% efficacy against the original strain and 75–94% against the Alpha variant (B.1.1.7). Immunogenicity comparisons to original strain and variants of concern (VOC) should be considered as a basis for authorization of vaccines because efficacy studies now have predominantly VOC cases.

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Phosphorylation Status of the Parvovirus Minute Virus of Mice Particle: Mapping and Biological Relevance of the Major Phosphorylation Sites

Journal of Virology ◽

10.1128/jvi.74.23.10892-10902.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 10892-10902 ◽

Cited By ~ 39

Author(s):

Beatriz Maroto ◽

Juan C. Ramı́rez ◽

José M. Almendral

Keyword(s):

Amino Acids ◽

Nuclear Translocation ◽

Biological Significance ◽

Dimensional Structure ◽

Phosphorylation Sites ◽

Phenotypic Effect ◽

Minute Virus Of Mice ◽

Terminal Domain ◽

Capsid Shell ◽

Minute Virus

ABSTRACT The core of the VP-1 and VP-2 proteins forming the T=1 icosahedral capsid of the prototype strain of the parvovirus minute virus of mice (MVMp) share amino acids sequence and a common three-dimensional structure; however, the roles of these polypeptides in the virus infection cycle differ. To gain insights into this paradox, the nature, distribution, and biological significance of MVMp particle phosphorylation was investigated. The VP-1 and VP-2 proteins isolated from purified empty capsids and from virions containing DNA harbored phosphoserine and phosphothreonine amino acids, which in two-dimensional tryptic analysis resulted in complex patterns reproducibly composed by more than 15 unevenly phosphorylated peptides. Whereas secondary protease digestions and comigration of most weak peptides in the fingerprints revealed common phosphorylation sites in the VP-1 and VP-2 subunits assembled in capsids, the major tryptic phosphopeptides were remarkably characteristic of either polypeptide. The VP-2-specific peptide named B, containing the bulk of the32P label of the MVMp particle in the form of phosphoserine, was mapped to the structurally unordered N-terminal domain of this polypeptide. Mutations in any or all four serine residues present in peptide B showed that the VP-2 N-terminal domain is phosphorylated at multiple sites, even though none of them was essential for capsid assembly or virus formation. Chromatographic analysis of purified wild-type (wt) and mutant peptide B digested with a panel of specific proteases allowed us to identify the VP-2 residues Ser-2, Ser-6, and Ser-10 as the main phosphate acceptors for MVMp capsid during the natural viral infection. Phosphorylation at VP-2 N-terminal serines was not necessary for the externalization of this domain outside of the capsid shell in particles containing DNA. However, the plaque-forming capacity and plaque size of VP-2 N-terminal phosphorylation mutants were severely reduced, with the evolutionarily conserved Ser-2 determining most of the phenotypic effect. In addition, the phosphorylated amino acids were not required for infection initiation or for nuclear translocation of the expressed structural proteins, and thus a role at a late stage of MVMp life cycle is proposed. This study illustrates the complexity of posttranslational modification of icosahedral viral capsids and underscores phosphorylation as a versatile mechanism to modulate the biological functions of their protein subunits.

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A first-in-human evaluation of the safety and immunogenicity of SCB-2019, an adjuvanted, recombinant SARS-CoV-2 trimeric S-protein subunit vaccine for COVID-19 in healthy adults; a phase 1, randomised, double-blind, placebo-controlled trial

10.1101/2020.12.03.20243709 ◽

2020 ◽

Author(s):

Peter Richmond ◽

Lara Hatchuel ◽

Min Dong ◽

Brenda Ma ◽

Branda Hu ◽

...

Keyword(s):

Older Adults ◽

Immune Responses ◽

Subunit Vaccine ◽

Phase 1 ◽

Intracellular Cytokine Staining ◽

High Dose ◽

Protein Subunit ◽

Neutralising Antibodies ◽

S Protein ◽

Human Dose

ABSTRACTBackgroundAs part of the accelerated development of prophylactic vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) we report a first-in-human dose-finding and adjuvant justification study of SCB-2019, a novel protein subunit vaccine candidate composed of a stabilised trimeric form of the spike (S)-protein produced in CHO-cells, combined with two different adjuvants.MethodsThis phase 1 study was done in one centre in Western Australia in 151 healthy adult volunteers in two age groups (18–54 and 55–75 years), allocated to 15 groups (nine young and six older adults) to receive two doses, 21 days apart, of placebo, or 3 μg, 9 μg or 30 μg SCB-2019, alone or adjuvanted with AS03 or CpG/Alum. Reactogenicity was assessed for 7 days after each vaccination. Humoral responses were measured as SCB-2019 binding and ACE2-competitive binding IgG antibodies by ELISA, and as neutralising antibodies by wild-type SARS-CoV-2 microneutralisation assay; cellular responses to pooled S-protein peptides were measured by flow-cytometric intracellular cytokine staining.FindingsWe report on 148 participants with at least 4 weeks follow-up post dose 2. Three participants withdrew, two for personal reasons and one with an unrelated SAE (pituitary adenoma). Vaccination was well tolerated, with few Grade 3 solicited adverse events (AE). Most local AEs were mild injection site pain, which were more frequent with formulations containing AS03 than CpG/Alum or unadjuvanted SCB-2019. Systemic AEs, mostly transient headache, fatigue or myalgia, were more frequent in young adults than older adults after the first dose, but similar after second doses. Unadjuvanted SCB-2019 elicited minimal immune responses, but SCB-2019 with fixed doses of AS03 or CpG/Alum induced high titres and seroconversion rates of binding and neutralising antibodies in both young and older adults. Titres were higher than those observed in a panel of COVID-19 convalescent sera in all AS03 groups and high dose CpG/Alum groups. Both adjuvanted formulations elicited Th1-biased CD4+ T cell responses.InterpretationSCB-2019 trimeric protein formulated with AS03 or CpG/Alum adjuvants elicited robust humoral and cellular immune responses against SARS-CoV-2 with high viral neutralising activity. Both adjuvanted formulations were well tolerated and are suitable for further clinical development.Clinical trial registrationClinicalTrials.gov identifier NCT04405908.

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Wave-wise comparative genomic study for revealing the complete scenario and dynamic nature of COVID-19 pandemic in Bangladesh

PLoS ONE ◽

10.1371/journal.pone.0258019 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0258019

Author(s):

Ishtiaque Ahammad ◽

Mohammad Uzzal Hossain ◽

Anisur Rahman ◽

Zeshan Mahmud Chowdhury ◽

Arittra Bhattacharjee ◽

...

Keyword(s):

Average Rate ◽

Spike Protein ◽

Comparative Genomic ◽

Genome Sequences ◽

Dynamic Nature ◽

Genomic Study ◽

The World ◽

Comparative Genomic Study ◽

Second Wave ◽

Successful Prevention

As the COVID-19 pandemic continues to ravage across the globe and take millions of lives and like many parts of the world, the second wave of the pandemic hit Bangladesh, this study aimed at understanding its causative agent, SARS-CoV-2 at the genomic and proteomic level and provide precious insights about the pathogenesis, evolution, strengths and weaknesses of the virus. As of Mid-June 2021, over 1500 SARS-CoV-2 genomesequences have been deposited in the GISAID database from Bangladesh which were extracted and categorized into two waves. By analyzing these genome sequences, it was discovered that the wave-2 samples had a significantly greater average rate of mutation/sample (30.79%) than the wave-1 samples (12.32%). Wave-2 samples also had a higher frequency of deletion, and transversion events. During the first wave, the GR clade was the most predominant but it was replaced by the GH clade in the latter wave. The B.1.1.25 variant showed the highest frequency in wave-1 while in case of wave-2, the B.1.351.3 variant, was the most common one. A notable presence of the delta variant, which is currently at the center of concern, was also observed. Comparison of the Spike protein found in the reference and the 3 most common lineages found in Bangladesh namely, B.1.1.7, B.1.351, B.1.617 in terms of their ability to form stable complexes with ACE2 receptor revealed that B.1.617 had the potential to be more transmissible than others. Importantly, no indigenous variants have been detected so far which implies that the successful prevention of import of foreign variants can diminish the outbreak in the country.

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