CoVe-Tracker: An Interactive SARS-CoV-2 Pan Proteome Evolution Tracker.

SARS-CoV-2 has significantly mutated its genome during the past 3 years, leading to the periodic emergence of several variants. Some of the variants possess enhanced fitness advantage, transmissibility, and pathogenicity and can also reduce vaccine efficacy. Thus, it is important to track the viral evolution to prevent and protect the mankind from SARS-CoV-2 infection. To this end, an interactive web-GUI platform, namely, CoVe-tracker (SARS-CoV-2 evolution tracker), is developed to track its pan proteome evolutionary dynamics (https://project.iith.ac.in/cove-tracker/). CoVe-tracker provides an opportunity for the user to fetch the country-wise and protein-wise amino acid mutations (currently, 44139) of SARS-CoV-2 and their month-wise distribution. It also provides position-wise evolution observed in the SARS-CoV-2 proteome. Importantly, CoVe-tracker provides month- and country-wise distributions of 2065 phylogenetic assignment of named global outbreak (PANGO) lineages and their 177564 variants. It further provides periodic updates on SARS-CoV-2 variant(s) evolution. CoVe-tracker provides the results in a user-friendly interactive fashion by projecting the results onto the world map (for country-wise distribution) and protein 3D structure (for protein-wise mutation). The application of CoVe-tracker in tracking the closest cousin(s) of a variant is demonstrated by considering BA.4 and BA.5 PANGO lineages as test cases. Thus, CoVe-tracker would be useful in the quick surveillance of newly emerging mutations/variants/lineages to facilitate the understanding of viral evolution, transmission, and disease epidemiology.

SARS-CoV-2 whole-proteome sequences from environment as an indicator of community viral distribution, evolution and epidemiological dynamics: A cohort analysis of Austria.

Several investigations have been carried out to detect SARS-CoV-2 samples from the environment such as sewage waters and surface swabs. Whole-proteome sequence analysis of 847 SARS-CoV-2 genome sequences collected from the environment in Austria during 2021 and deposited in GISAID indicates that alpha and delta are two dominant variants, coinciding with the human clinical samples with a Pearson correlation coefficient in the range of 0.58 (alpha variant) to 0.82 (delta variant). Both environmental and human samples show that Austrian SARS-CoV-2 alpha variant is found to possess N protein R203K and G204R/P mutations, whereas they are absent in the delta variant. SARS-CoV-2 delta variant is continuously seen in both the environmental and human clinical samples from the month of September 2021 and it spiked in November 2021, which is directly reflected in the increase of the number of SARS-CoV-2 infections and deaths in Austria during November 2021. Thus, the results presented here indicate that the environmental SARS-CoV-2 whole-genome sequences collected from Austria reflect the community viral distribution, evolution and the concomitant epidemiological dynamics. Since SARS-CoV-2 keeps evolving, the results presented here further suggest the need to monitor the environment for the early detection of SARS-CoV-2 variants to take appropriate precautionary measures.

Geographical distribution of SARS-CoV-2 amino acids mutations and the concomitant evolution of seven distinct clades in non-human hosts.

Since its first emergence in December 2019, the world has witnessed the eruption of mutations in the SARS-CoV-2 genome that have led to increased viral transmissibility and pathogenicity due to sustained local viral transmission. Zooanthroponotic and zoonotic transmissions have further raised concerns as they could result in the emergence of viral variants with a novel antigenicity and transmissibility that could jeopardize the vaccine efficacy. To understand the viral evolution during such transmissions, 1016 whole-genome sequences (deposited in GISAID as of March 7, 2022) (from 18 countries) corresponding to mink, cat, deer, dog, hyena, tiger, lion, gorilla, Syrian hamster, leopard cat, fishing cat, bear cat, coati, ferret, snow leopard and green monkey have been analysed here. Intriguingly, phyloproteome analysis indicate that Nsp2:R218C, Nsp2:D268-(deletion), Spike:D614G, Nsp12:P323L, Nsp2:A192V, ORF3a protein:Q57H, N protein:R203K and N protein:G204R/L, Spike:A222V, ORF10 protein:V30L and N protein:A220V are moderate or high recurring and clade decisive mutations, leading to 6 primary clades during the early stage of pandemic. Most interestingly, the human evolved delta variant having a combination of 26 (clade decisive) mutations defines the seventh clade and transmits to non-human hosts across the globe without exhibiting any country-specific mutation(s). Nonetheless, Spike:D614G and Nsp12:P323L together with (i)N protein:R203K,N protein:G204R/L,Spike:V70-, Spike:H69-, Nsp12:T739I, and Nsp1:M85-, (ii)Nsp2:A192V, Nsp3:D178Y, (iii)Nsp2:T85I, N protein:P67S and ORF3a protein:Q57H and (iv)Spike:A222V, ORF10 protein:V30L, N protein:A220V and Spike:F486I are specific to Denmark, Netherlands, USA and Latvia respectively and, (v)Nsp2:D268- and Nsp13:R292C that are devoid of Spike:D614G and Nsp12:P323L is specific to Netherlands. SARS-CoV-2 variants consisting of these mutations are also seen in the human SARS-CoV-2 sequences from the same country. Independent country-specific SARS-CoV-2 variant evolution further indicates distinct epidemiological dynamics during zooanthroponotic and zoonotic transmissions. Thus, the results presented here indicate the need for the surveillance of viral evolution in non-human hosts also during the future pandemic.

Revelation of Potent Epitopes Present in Unannotated ORF Antigens of SARS-CoV-2 for Epitope-Based Polyvalent Vaccine Design Using Immunoinformatics Approach.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kills thousands of people worldwide every day, thus necessitating rapid development of countermeasures. Immunoinformatics analyses carried out here in search of immunodominant regions in recently identified SARS-CoV-2 unannotated open reading frames (uORFs) have identified eight linear B-cell, one conformational B-cell, 10 CD4+ T-cell, and 12 CD8+ T-cell promising epitopes. Among them, ORF9b B-cell and T-cell epitopes are the most promising followed by M.ext and ORF3c epitopes. ORF9b40-48 (CD8+ T-cell epitope) is found to be highly immunogenic and antigenic with the highest allele coverage. Furthermore, it has overlap with four potent CD4+ T-cell epitopes. Structure-based B-cell epitope prediction has identified ORF9b61-68 to be immunodominant, which partially overlaps with one of the linear B-cell epitopes (ORF9b65-69). ORF3c CD4+ T-cell epitopes (ORF3c2-16, ORF3c3-17, and ORF3c4-18) and linear B-cell epitope (ORF3c14-22) have also been identified as the candidate epitopes. Similarly, M.ext and 7a.iORF1 (overlap with M and ORF7a) proteins have promising immunogenic regions. By considering the level of antigen expression, four ORF9b and five M.ext epitopes are finally shortlisted as potent epitopes. Mutation analysis has further revealed that the shortlisted potent uORF epitopes are resistant to recurrent mutations. Additionally, four N-protein (expressed by canonical ORF) epitopes are found to be potent. Thus, SARS-CoV-2 uORF B-cell and T-cell epitopes identified here along with canonical ORF epitopes may aid in the design of a promising epitope-based polyvalent vaccine (when connected through appropriate linkers) against SARS-CoV-2. Such a vaccine can act as a bulwark against SARS-CoV-2, especially in the scenario of emergence of variants with recurring mutations in the spike protein.

Global variation in SARS-CoV-2 proteome and its implication in pre-lockdown emergence and dissemination of 5 dominant SARS-CoV-2 clades.

SARS-CoV-2 is currently causing major havoc worldwide with its efficient transmission and propagation. To track the emergence as well as the persistence of mutations during the early stage of the pandemic, a comparative analysis of SARS-CoV-2 whole proteome sequences has been performed by considering manually curated 31,389 whole genome sequences from 84 countries. Among the 7 highly recurring (percentage frequency≥10%) mutations (Nsp2:T85I, Nsp6:L37F, Nsp12:P323L, Spike:D614G, ORF3a:Q57H, N protein:R203K and N protein:G204R), N protein:R203K and N protein: G204R are co-occurring (dependent) mutations. Nsp12:P323L and Spike:D614G often appear simultaneously. The highly recurring Spike:D614G, Nsp12:P323L and Nsp6:L37F as well as moderately recurring (percentage frequency between ≥1 and <10%) ORF3a:G251V and ORF8:L84S mutations have led to4 major clades in addition to a clade that lacks high recurring mutations. Further, the occurrence of ORF3a:Q57H&Nsp2:T85I, ORF3a:Q57H and N protein:R203K&G204R along with Nsp12:P323L&Spike:D614G has led to 3 additional sub-clades. Similarly, occurrence of Nsp6:L37F and ORF3a:G251V together has led to the emergence of a sub-clade. Nonetheless, ORF8:L84S does not occur along with ORF3a:G251V or Nsp6:L37F. Intriguingly, ORF3a:G251V and ORF8:L84S are found to occur independent of Nsp12:P323L and Spike:D614G mutations. These clades have evolved during the early stage of the pandemic and have disseminated across several countries. Further, Nsp10 is found to be highly resistant to mutations, thus, it can be exploited for drug/vaccine development and the corresponding gene sequence can be used for the diagnosis. Concisely, the study reports the SARS-CoV-2 antigens diversity across the globe during the early stage of the pandemic and facilitates the understanding of viral evolution.

The evolving proteome of SARS-CoV-2 predominantly uses mutation combination strategy for survival.

The knowledge about SARS-CoV-2 proteome variations is important to understand its evolutionary tactics and in drug/vaccine design. An extensive analysis of 125,747 whole proteome reveals 7915 recurring mutations (involving 5146 positions) during December2019-November 2020. Among these, 10 and 51 are highly and moderately recurring mutations respectively. Ever since the pandemic outbreak, ∼50% new proteome variants evolve every month, resulting in 5 major clades. Intriguingly, ∼70% of the variants reported in January 2020 are due to the emergence of new mutations, which sharply declines to ∼ 40% in April 2020 and thenceforth, declines steadily till November 2020(∼10%). An exactly opposite trend is seen for variants evolved with cocktail of existing mutations: the lowest in January 2020(∼20%) and the highest in November 2020(80%). This leads to a steady increase in the average number of mutations per sequence. This indicates that the virus has reached the slow pace to accept new mutations. Instead, it uses a mutation combination strategy for survival.