SARS-CoV-2 variants show a gradual declining pathogenicity and pro-inflammatory cytokine stimulation and an increasing antigenic and anti-inflammatory cytokine induction

Hyper-transmissibility with decreased disease severity are typical characteristics of Omicron variant. To understand this phenomenon, we used various bioinformatics approaches to analyze randomly selected genome sequences (one each) of the Gamma, Delta, and Omicron variants submitted to NCBI from 15 to 31 December 2021. We show that: (i) Pathogenicity of SARS-CoV-2 variants decreases in the order: Wuhan > Gamma > Delta > Omicron; however, the antigenic property follows the order: Omicron > Gamma > Wuhan > Delta. (ii) Omicron Spike RBD has lower pathogenicity but higher antigenicity than other variants. (iii) Decreased disease severity by Omicron variant may be due to its decreased pro-inflammatory and IL-6 stimulation and increased IFN-{gamma} and IL-4 induction efficacy. (iv) Mutations in N protein are associated with decreased IL-6 induction and human DDX21-mediated increased IL-4 production in Omicron. (v) Due to mutations, the stability of S, M, N, and E proteins decreases in the order: Omicron > Gamma > Delta > Wuhan. (vi) Stronger Spike RBD-hACE2 binding in Omicron is associated with increased transmissibility. However, the lowest stability of the Omicron Spike protein makes Spike RBD-hACE2 interaction weak for systemic infection and for causing severe disease. Finally (vii), the highest instability of Omicron E protein may also be associated with decreased viral maturation and low viral load leading to less severe disease and faster recovery. Our method may be used for other similar viruses, and these findings will contribute to the understanding of the dynamics of SARS-CoV-2 variants and the management of emerging variants.

An Issue of Concern: Unique Truncated ORF8 Protein Variants of SARS-CoV-2

Open reading frame 8 (ORF8) protein is one of the most evolving accessory proteins in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). It was previously reported that the ORF8 protein inhibits presentation of viral antigens by the major histocompatibility complex class I (MHC-I) and interacts with host factors involved in pulmonary inflammation. The ORF8 protein assists SARS-CoV-2 to evade immunity and replication. Among many contributing mutations, Q27STOP, a mutation in the ORF8 protein defines the B.1.1.7 lineage of SARS-CoV-2, which is engendering the second wave of COVID-19. In the present study, 47 unique truncated ORF8 proteins (T-ORF8) due to the Q27STOP mutations were identified among 49055 available B.1.1.7 SARS-CoV-2 sequences. The results show that only one of the 47 T-ORF8 variants spread to over 57 geo-locations in North America, and other continents which includes Africa, Asia, Europe and South America. Based on various quantitative features such as amino acid homology, polar/non-polar sequence homology, Shannon entropy conservation, and other physicochemical properties of all specific 47 T-ORF8 protein variants, a collection of nine possible T-ORF8 unique variants were defined. The question of whether T-ORF8 variants work similarly to ORF8 has yet to be investigated. A positive response to the question could exacerbate future COVID-19 waves, necessitating severe containment measures.

Implications Derived from S-Protein Variants of SARS-CoV-2 from Six Continents

Spike (S) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critical determinants of the infectivity and antigenicity of the virus. Several mutations in the spike protein of SARS-CoV-2 have already been detected, and their effect in immune system evasion and enhanced transmission as a cause of increased morbidity and mortality are being investigated. From pathogenic and epidemiological perspectives, spike proteins are of prime interest to researchers. This study focused on the unique variants of S proteins from six continents Asia, Africa, Europe, Oceania, South America, and North America. In comparison to the other five continents, Africa (29.065%) had the highest percentage of unique S proteins. Notably, only North America had 87% (14046) of the total (16143) specific S proteins available in the NCBI database(across all continents). Based on the amino acid frequency distributions in the S protein variants from all the continents, the phylogenetic relationship implies that unique S proteins from North America were significantly different from those of the other five continents. Overtime, the unique variants originating from North America are most likely to spread to the other geographic locations through international travel or naturally by emerging mutations. Hence it is suggested that restriction of international travel should be considered, and massive vaccination as an utmost measure to combat the spread of COVID-19 pandemic. It is also further suggested that the efficacy of existing vaccines and future vaccine development must be reviewed with careful scrutiny, and if needed, further re-engineered based on requirements dictated by new emerging S protein variants.

Clade GR and Clade GH Isolates in Asia Show Highest Amount of SNPs

Clades are monophyletic groups composed of a common ancestor and all its lineal descendants. As the propensity of virulence of a disease depends upon the type of clade the virus belongs to and it causes different fatality rates of disease in different countries, so the clade-wise analysis of SARS-CoV-2 isolates collected from different countries can illuminate the actual evolutionary relationships between them. In this study, 1566 SARS-CoV-2 genome sequences across ten Asian countries are collected, clustered, and characterized based on the clade they belong to. The isolates are compared to the Wuhan reference sequence (Accession no:MN996528.1) to identify the mutations that occurred at different protein regions. Structural changes in amino acids due to mutations lead to functional instability of the proteins. Detailed clade-wise functional assessments are carried out to quantify the stability and vulnerability of the mutations occurring in SARS-CoV-2 genomes which can shade light on personalized prevention and treatment of the disease and encourage towards the invention of clade-specific vaccines.

Variability of Accessory Proteins Rules the SARS-CoV-2 Pathogenicity

The coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) which is pandemic with an estimated fatality rate less than 1% is ongoing. SARS-CoV-2 accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 with putative functions to manipulate host immune mechanisms such as interferons, immune signaling receptor NLRP3 (NOD-, LRR-, and pyrin domain-containing 3) inflammasome, inflammatory cytokines such as interleukin 1{beta} (IL-1{beta}) are critical in COVID-19 pathology. Outspread variations of each of the six accessory proteins of all complete proteomes (available as of October 26, 2020, in the National Center for Biotechnology Information depository) of SARS-CoV-2, were observed across six continents. Across all continents, the decreasing order of percentage of unique variations in the accessory proteins was found to be ORF3a>ORF8>ORF7a>ORF6>ORF10>ORF7b. The highest and lowest unique variations of ORF3a were observed in South America and Oceania, respectively. This finding suggests that the wide variations of accessory proteins seem to govern the pathogenicity of SARS-CoV-2, and consequently, certain propositions and recommendations can be made in the public interest.

Possible transmission flow of SARS-CoV-2 based on ACE2 features

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is engendering the severe coronavirus disease 2019 (COVID-19) pandemic. The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 binds to the three sub-domains viz. amino acids (aa) 22-42, aa 79-84, and aa 330-393 of ACE2 on human cells to initiate entry. It was reported earlier that the receptor utilization capacity of ACE2 proteins from different species, such as cats, chimpanzees, dogs, and cattle, are different. A comprehensive analysis of ACE2 receptors of nineteen species was carried out in this study, and the findings propose a possible SARS-CoV-2 transmission flow across these nineteen species.

Notable sequence homology of the ORF10 protein introspects the architecture of SARS-COV-2

The global public health is endangered due to COVID-19 pandemic, which is caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Despite having similar pathology to MERS and SARS-CoV, the infection fatality rate of SARS-CoV-2 is likely lower than 1%. SARS-CoV-2 has been reported to be uniquely characterized by the accessory protein ORF10, which contains eleven cytotoxic T lymphocyte (CTL) epitopes of nine amino acids length each, across various human leukocyte antigen (HLA) subtypes. In this study, all missense mutations found in sequence databases were examined across twnety-two unique SARS-CoV-2 ORF10 variants that could possibly alter viral pathogenicity. Some of these mutations decrease the stability of ORF10, e.g. I4L and V6I were found in the MoRF region of ORF10 which may also possibly contribute to Intrinsic protein disorder. Furthermore, a physicochemical and structural comparative analysis was carried out on SARS-CoV-2 and Pangolin-CoV ORF10 proteins, which share 97.37% amino acid homology. The high degree of physicochemical and structural similarity of ORF10 proteins of SARS-CoV-2 and Pangolin-CoV open questions about the architecture of SARS-CoV-2 due to the disagreement of these two ORF10 proteins over their sub-structure (loop/coil region), solubility, antigenicity and change from the strand to coil at amino acid position 26, where tyrosine is present. Altogether, SARS-CoV-2 ORF10 is a promising pharmaceutical target and a protein which should be monitored for changes which correlate to change pathogenesis and clinical course of COVID-19 infection.

A unique view of SARS-CoV-2 through the lens of ORF8 protein

Immune evasion is one of the unique characteristics of COVID-19 attributed to the ORF8 protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protein is involved in modulating the host adaptive immunity through downregulating MHC (Major Histocompatibility Complex) molecules and innate immune responses by surpassing the interferon mediated antiviral response of the host. To understand the immune perspective of the host with respect to the ORF8 protein, a comprehensive study of the ORF8 protein as well as mutations possessed by it, is performed. Chemical and structural properties of ORF8 proteins from different hosts, that is human, bat and pangolin, suggests that the ORF8 of SARS-CoV-2 and Bat RaTG13-CoV are very much closer related than that of Pangolin-CoV. Eighty-seven mutations across unique variants of ORF8 (SARS-CoV-2) are grouped into four classes based on their predicted effects. Based on geolocations and timescale of collection, a possible flow of mutations was built. Furthermore, conclusive flows of amalgamation of mutations were endorsed upon sequence similarity and amino acid conservation phylogenies. Therefore, this study seeks to highlight the uniqueness of rapid evolving SARS-CoV-2 through the ORF8.

Pathogenetic Perspective of Missense Mutations of ORF3a Protein of SARS-CoV2

One of the most important proteins for COVID-19 pathogenesis in SARS-CoV2 is the ORF3a protein which is the largest accessory protein among others accessory proteins coded by coronavirus genome. The major roles of the protein include virulence, infectivity, ion channel activity, morphogenesis and virus release. The coronavirus, SARS-CoV2 is continuously evolving naturally and thereby the encoded proteins are also mutating rapidly. Therefore, critical study of mutations in ORF3a is certainty important from the pathogenetic perspective. Here, a sum of 175 various non-synonymous mutations in the ORF3a protein of SARS-CoV2 are identified and their corresponding effects in structural stability and functions of the protein ORF3a are studied. Broadly three different classes of mutations, such as neutral, disease and mixed (neutral and disease) type mutations were observed. Consecutive mutations in some ORF3a proteins are established based on timeline of detection of mutations. Considering the amino acid compositions over the ORF3a primary protein sequences, twenty clusters are detected based on K-means clustering method. Our findings on 175 novel mutations of ORF3a proteins will extend our knowledge of ORF3a, a vital accessory protein in SARS-CoV2, which would assist to enlighten on the pathogenicity of this life-threatening COVID-19.

Molecular conservation and Differential mutation on ORF3a gene in Indian SARS-CoV2 genomes

A global emergency due to the COVID-19 pandemic demands various studies related to genes and genomes of the SARS-CoV2. Among other important proteins, the role of accessory proteins are of immense importance in replication, regulation of infections of the coronavirus in the hosts. The largest accessory proteins in the SARS-CoV2 genome is ORF3a which modulates the host response to the virus infection and consequently it plays an important role in pathogenesis. In this study, an attempt is made to decipher the conservation of nucleotides, dimers, codons and amino acids in the ORF3a genes across thirty two genomes of Indian patients. ORF3a gene possesses single and double point mutations in Indian SARS-CoV2 genomes suggesting the change of SARS-CoV2s virulence property in Indian patients. We find that the parental origin of the ORF3a gene over the genomes of SARS-CoV2 and Pangolin-CoV is same from the phylogenetic analysis based on conservations of nucleotides and so on. This study highlights the accumulation of mutation on ORF3a in Indian SARS-CoV2 genomes which may provide the designing therapeutic approach against SARS-CoV2.

On spatial molecular arrangements of SARS-CoV2 genomes of Indian patients

A pandemic caused by the SARS-CoV2 is being experienced by the whole world since December, 2019. A thorough understanding beyond just sequential similarities among the protein coding genes of SARS-CoV2 is important in order to differentiate or relate to the other known CoVs of the same genus. In this study, we compare three genomes namely MT012098 (India-Kerala), MT050493 (India-Kerala), MT358637 (India-Gujrat) from India with NC_045512 (China-Wuhan) to view the spatial as well as molecular arrangements of nucleotide bases of all the genes embedded in these four genomes. Based on different features extracted for each gene embedded in these genomes, corresponding phylogenetic relationships have been built up. Differences in phylogenetic tree arrangement with individual gene suggest that three genomes of Indian origin have come from three different origins or the evolution of viral genome is very fast process. This study would also help to understand the virulence factors, disease pathogenicity, origin and transmission of the SARS-CoV2.