The importance of accessory protein variants in the pathogenicity of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS- CoV-2) with an estimated fatality rate of less than 1%. The SARS-CoV-2 accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 possess putative functions to manipulate host immune mechanisms. These involve interferons, which appear as a consensus function, immune signaling receptor NLRP3 (NLR family pyrin domain-containing 3) inflammasome, and inflammatory cytokines such as interleukin 1ß (IL-1ß) and are critical in COVID-19 pathology. Outspread variations of each of the six accessory proteins were observed across six continents of all complete SARS-CoV-2 proteomes based on the data reported before November 2020. A decreasing order of percentage of unique variations in the accessory proteins was determined as ORF3a > ORF8 > ORF7a > ORF6 > ORF10 > ORF7b across all continents. The highest and lowest unique variations of ORF3a were observed in South America and Oceania, respectively. These findings suggest that the wide variations in accessory proteins seem to affect the pathogenicity of SARS-CoV-2.

Periodically aperiodic pattern of SARS-CoV-2 mutations underpins the uncertainty of its origin and evolution.

Various lineages of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have contributed to prolongation of the Coronavirus Disease 2019 (COVID-19) pandemic. Several non-synonymous mutations in SARS-CoV-2 proteins have generated multiple SARS-CoV-2 variants. In our previous report, we have shown that an evenly uneven distribution of unique protein variants of SARS-CoV-2 is geo-location or demography-specific. However, the correlation between the demographic transmutability of the SARS-CoV-2 infection and mutations in various proteins remains unknown due to hidden symmetry/asymmetry in the occurrence of mutations. This study tracked how these mutations are emerging in SARS-CoV-2 proteins in six model countries and globally. In a geo-location, considering the mutations having a frequency of detection of at least 500 in each SARS-CoV-2 protein, we studied the country-wise percentage of invariant residues. Our data revealed that since October 2020, highly frequent mutations in SARS-CoV-2 have been observed mostly in the Open Reading Frame (ORF) 7b and ORF8, worldwide. No such highly frequent mutations in any of the SARS-CoV-2 proteins were found in the UK, India, and Brazil, which does not correlate with the degree of transmissibility of the virus in India and Brazil. However, we have found a signature that SARS-CoV-2 proteins were evolving at a higher rate, and considering global data, mutations are detected in the majority of the available amino acid locations. Fractal analysis of each protein's normalized factor time series showed a periodically aperiodic emergence of dominant variants for SARS-CoV-2 protein mutations across different countries. It was noticed that certain high-frequency variants have emerged in the last couple of months, and thus the emerging SARS-CoV-2 strains are expected to contain prevalent mutations in the ORF3a, membrane, and ORF8 proteins. In contrast to other beta-coronaviruses, SARS-CoV-2 variants have rapidly emerged based on demographically dependent mutations. Characterization of the periodically aperiodic nature of the demographic spread of SARS-CoV-2 variants in various countries can contribute to the identification of the origin of SARS-CoV-2.

SARS-CoV2 envelope protein: non-synonymous mutations and its consequences.

In the NCBI database, as on June 6, 2020, total number of available complete genome sequences of SARS-CoV2 across the world is 3617. The envelope (E) protein of SARS-CoV2 possesses several non-synonymous mutations over the transmembrane and C-terminus domains in 15 (0.414%) genomes among 3617 SARS-CoV2 genomes, analyzed. More precisely, 10(0.386%) out of 2588 genomes from the USA, 3(0.806%) from Asia, 1 (0.348%) from Europe and 1 (0.274%) from Oceania contained the missense mutations over the E-protein of SARS-CoV2 genomes. The C-terminus motif DLLV has been to DFLV and YLLV in the proteins from QJR88103 (Australia: Victoria) and QKI36831 (China: Guangzhou) respectively, which might affect the binding of this motif with the host protein PALS1.

Molecular phylogeny and missense mutations at envelope proteins across coronaviruses.

Envelope (E) protein is one of the structural viroporins (76-109 amino acids long) present in the coronavirus. Sixteen sequentially different E-proteins were observed from a total of 4917 available complete SARS-CoV-2 genomes as on 18th June 2020 in the NCBI database. The missense mutations over the envelope protein across various coronaviruses of the ß-genus were analyzed to know the immediate parental origin of the envelope protein of SARS-CoV-2. The evolutionary origin is also endorsed by the phylogenetic analysis of the envelope proteins comparing sequence homology as well as amino acid conservations.

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 protein 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-CoV2's 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 conservation 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.

Missense mutations in SARS-CoV2 genomes from Indian patients.

The genetic diversity of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) in several countries sums up to worldwide genetic diversity. In this present study, variations in terms of missense mutations among the SARS-CoV2 genomes from 128 Indian patients, as of May 2020, are accounted and thereby some key findings with some hypotheses were made. These mutations across various genes of these genomes show wide genetic variations in sequence and rapid evolution of SARS-CoV2 virus. The presence of unique mutations in the studied SARS-CoV2 genomes may led to their attenuation. Few Genes such as ORF6, ORF10 are free from any mutations in the Indian context of 339 genomes observed as of 14th July 2020. Further, E protein contains only one mutation. This may suggest that designing a therapeutic approach against ORF6, ORF10 and E genes may have a beneficial effect in controlling COVID-19 pandemic especially in India.

Ranking and clustering of Drosophila olfactory receptors using mathematical morphology.

This article introduces an alignment-free clustering method in order to cluster all the 66 DORs sequentially diverse protein sequences. Two different methods are discussed: one is utilizing twenty standard amino acids (without grouping) and another one is using chemical grouping of amino acids (with grouping). Two grayscale images (representing two protein sequences by order pair frequency matrices) are compared to find the similarity index using morphology technique. We could achieve the correlation coefficients of 0.9734 and 0.9403 for without and with grouping methods respectively with the ClustalW result in the ND5 dataset, which are much better than some of the existing alignment-free methods. Based on the similarity index, the 66 DORs are clustered into three classes - Highest, Moderate and Lowest - which are seen to be best fitted for 66 DORs protein sequences. OR83b is the distinguished olfactory receptor expressed in divergent insect population which is substantiated through our investigation.

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.

Reduced protein sequence patterns in identifying key structural elements of dissimilatory sulfite reductase homologs.

Methanogenic archaea carry homologs of dissimilatory sulfite reductase (Dsr), called Dsr Like proteins (DsrLP). Dsr reduces sulfite to sulfide, a key step in an Earth's ancient metabolic process called dissimilatory sulfate reduction. The DsrLPs do not function as Dsr, and a computational approach is needed to develop hypotheses for guiding wet bench investigations on DsrLP's function. To make the computational analysis process efficient, the DsrLP amino acid sequences were transformed using only eight alphabets functionally representing twenty amino acids. The resultant reduced amino acid sequences were analyzed to identify conserved signature patterns in DsrLPs. Many of these patterns mapped on critical structural elements of Dsr and some were associated tightly with particular DsrLP groups. A search into the UniProtKB database identified several proteins carrying DsrLP's signature patterns; cysteine desulfurase, nucleosidase, and uroporphyrinogen III methylase were such matches. These outcomes provided clues to the functions of DsrLPs and highlighted the utility of the computational approach used.

Non-uniform aspects of the SARS-CoV-2 intraspecies evolution reopen question of its origin.

Several hypotheses have been presented on the origin of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from its identification as the agent causing the current coronavirus disease 19 (COVID-19) pandemic. So far, no solid evidence has been found to support any hypothesis on the origin of this virus, and the issue continue to resurface over and over again. Here we have unfolded a pattern of distribution of several mutations in the SARS-CoV-2 proteins in 24 geo-locations across different continents. The results showed an evenly uneven distribution of the unique protein variants, distinct mutations, unique frequency of common conserved residues, and mutational residues across these 24 geo-locations. Furthermore, ample mutations were identified in the evolutionarily conserved invariant regions in the SARS-CoV-2 proteins across almost all geo-locations studied. This pattern of mutations potentially breaches the law of evolutionary conserved functional units of the beta-coronavirus genus. These mutations may lead to several novel SARS-CoV-2 variants with a high degree of transmissibility and virulence. A thorough investigation on the origin and characteristics of SARS-CoV-2 needs to be conducted in the interest of science and for the preparation of meeting the challenges of potential future pandemics.

Clade GR and clade GH isolates of SARS-CoV-2 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" hCoV-19/Wuhan/WIV04/19″ 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.

Rare mutations in the accessory proteins ORF6, ORF7b, and ORF10 of the SARS-CoV-2 genomes.

A total number of 3080 SARS-CoV-2 genomes from all continents are considered from the NCBI database. Every accessory protein ORF6, ORF7b, and ORF10 of SARS-CoV-2 possess a single missense mutation in less than 1.5% of the 3080 genomes. It has now been observed that different non-synonymous mutations occurred in these three accessory proteins. Most of these rare mutations are changing the amino acids such as hydrophilic to hydrophobic, acidic or basic to hydrophobic, and vice versa etc. So these highly conserved proteins might play an essential role in virus pathogenicity. This study opens a question whether it carries some messages about the virus rapid replications, and virulence.

Characterizing genomic variants and mutations in SARS-CoV-2 proteins from Indian isolates.

SARS-CoV-2 is mutating and creating divergent variants by altering the composition of essential constituent proteins. Pharmacologically, it is crucial to understand the diverse mechanism of mutations for stable vaccine or anti-viral drug design. Our current study concentrates on all the constituent proteins of 469 SARS-CoV-2 genome samples, derived from Indian patients. However, the study may easily be extended to the samples across the globe. We perform clustering analysis towards identifying unique variants in each of the SARS-CoV-2 proteins. A total of 536 mutated positions within the coding regions of SARS-CoV-2 proteins are detected among the identified variants from Indian isolates. We quantify mutations by focusing on the unique variants of each SARS-CoV-2 protein. We report the average number of mutation per variant, percentage of mutated positions, synonymous and non-synonymous mutations, mutations occurring in three codon positions and so on. Our study reveals the most susceptible six (06) proteins, which are ORF1ab, Spike (S), Nucleocapsid (N), ORF3a, ORF7a, and ORF8. Several non-synonymous substitutions are observed to be unique in different SARS-CoV-2 proteins. A total of 57 possible deleterious amino acid substitutions are predicted, which may impact on the protein functions. Several mutations show a large decrease in protein stability and are observed in putative functional domains of the proteins that might have some role in disease pathogenesis. We observe a good number of physicochemical property change during above deleterious substitutions.

Mapping sequence to feature vector using numerical representation of codons targeted to amino acids for alignment-free sequence analysis.

The phylogenetic analysis based on sequence similarity targeted to real biological taxa is one of the major challenging tasks. In this paper, we propose a novel alignment-free method, CoFASA (Codon Feature based Amino acid Sequence Analyser), for similarity analysis of nucleotide sequences. At first, we assign numerical weights to the four nucleotides. We then calculate a score of each codon based on the numerical value of the constituent nucleotides, termed as degree of codons. Accordingly, we obtain the degree of each amino acid based on the degree of codons targeted towards a specific amino acid. Utilizing the degree of twenty amino acids and their relative abundance within a given sequence, we generate 20-dimensional features for every coding DNA sequence or protein sequence. We use the features for performing phylogenetic analysis of the set of candidate sequences. We use multiple protein sequences derived from Beta-globin (BG), NADH dehydrogenase subunit 5 (ND5), Transferrins (TFs), Xylanases, low identity (<40%) and high identity (⩾40%) protein sequences (encompassing 533 and 1064 protein families) for experimental assessments. We compare our results with sixteen (16) well-known methods, including both alignment-based and alignment-free methods. Various assessment indices are used, such as the Pearson correlation coefficient, RF (Robinson-Foulds) distance and ROC score for performance analysis. While comparing the performance of CoFASA with alignment-based methods (ClustalW, ClustalΩ, MAFFT, and MUSCLE), it shows very similar results. Further, CoFASA shows better performance in comparison to well-known alignment-free methods, including LZW-Kernal, jD2Stat, FFP, spaced, and AFKS-D2s in predicting taxonomic relationship among candidate taxa. Overall, we observe that the features derived by CoFASA are very much useful in isolating the sequences according to their taxonomic labels. While our method is cost-effective, at the same time, produces consistent and satisfactory outcomes.

Pathogenic perspective of missense mutations of ORF3a protein of SARS-CoV-2.

One of the most important proteins for COVID-19 pathogenesis in SARS-CoV-2 is the ORF3a which is the largest accessory protein among others coded by the SARS-CoV-2 genome. The major roles of the protein include virulence, infectivity, ion channel activity, morphogenesis, and virus release. The coronavirus, SARS-CoV-2 is mutating rapidly, therefore, critical study of mutations in ORF3a is certainly important from the pathogenic perspective. Here, a sum of 175 non-synonymous mutations in the ORF3a of SARS-CoV-2 were identified from 7194 complete genomes of SARS-CoV-2 available from NCBI database. Effects of these mutations on structural stability, and functions of ORF3a were also studied. Broadly, three different classes of mutations, such as neutral, disease, and mixed (neutral and disease) types of mutations were observed. Consecutive phenomena of mutations in ORF3a protein were studied based on the timeline of detection of the mutations. Considering the amino acid compositions of the ORF3a protein, twenty clusters were detected using the K-means clustering method. The present findings on 175 novel mutations of ORF3a proteins will extend our knowledge on ORF3a, a vital accessory protein in SARS-CoV-2, to enlighten the pathogenicity of this life-threatening virus.

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

Immune evasion is one of the unique characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attributed to its ORF8 protein. This protein modulates the adaptive host immunity through down-regulation of MHC-1 (Major Histocompatibility Complex) molecules and innate immune responses by surpassing the host's interferon-mediated antiviral response. To understand the host's immune perspective in reference to the ORF8 protein, a comprehensive study of the ORF8 protein and mutations possessed by it have been performed. Chemical and structural properties of ORF8 proteins from different hosts, such as human, bat, and pangolin, suggest that the ORF8 of SARS-CoV-2 is much closer to ORF8 of Bat RaTG13-CoV than to that of Pangolin-CoV. Eighty-seven mutations across unique variants of ORF8 in SARS-CoV-2 can be grouped into four classes based on their predicted effects (Hussain et al., 2021) [1]. Based on the geo-locations and timescale of sample collection, a possible flow of mutations was built. Furthermore, conclusive flows of amalgamation of mutations were found upon sequence similarity analyses and consideration of the amino acid conservation phylogenies. Therefore, this study seeks to highlight the uniqueness of the rapidly evolving SARS-CoV-2 through the ORF8.

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

The current Coronavirus Disease 19 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) shows similar pathology to MERS and SARS-CoV, with a current estimated fatality rate of 1.4%. Open reading frame 10 (ORF10) is a unique SARS-CoV-2 accessory protein, which contains eleven cytotoxic T lymphocyte (CTL) epitopes each of nine amino acids in length. Twenty-two unique SARS-CoV-2 ORF10 variants have been identified based on missense mutations found in sequence databases. Some of these mutations are predicted to decrease the stability of ORF10 in silico physicochemical and structural comparative analyses were carried out on SARS-CoV-2 and Pangolin-CoV ORF10 proteins, which share 97.37% amino acid (aa) homology. Though there is a high degree of ORF10 protein similarity of SARS-CoV-2 and Pangolin-CoV, there are differences of these two ORF10 proteins related to their sub-structure (loop/coil region), solubility, antigenicity and shift from strand to coil at aa position 26 (tyrosine). SARS-CoV-2 ORF10, which is apparently expressed in vivo since reactive T cell clones are found in convalescent patients should be monitored for changes which could correlate with the pathogenesis of COVID-19.

Autoimmunity roots of the thrombotic events after COVID-19 vaccination.

Although vaccination represents the most promising way to stop or contain the coronavirus disease 2019 (COVID-19) pandemic and safety and effectiveness of available vaccines were proven, a small number of individuals who received anti-SARS-CoV-2 vaccines developed a prothrombotic syndrome. Vaccine-induced immune thrombotic thrombocytopenia (VITT) can be triggered by the adenoviral vector-based vaccine, whereas lipid nanoparticle-mRNA-based vaccines can induce rare cases of deep vein thrombosis (DVT). Although the main pathogenic mechanisms behind this rare phenomenon have not yet been identified, both host and vaccine factors might be involved, with pathology at least in part being related to the vaccine-triggered autoimmune reaction. In this review, we are considering some aspects related to pathogenesis, major risk factors, as well as peculiarities of diagnosis and treatment of this rare condition.

Implications derived from S-protein variants of SARS-CoV-2 from six continents.

The spike (S) protein is a critical determinant of the infectivity and antigenicity of SARS-CoV-2. Several mutations in the S 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, S 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 had the highest percentage of unique S proteins (29.1%). The phylogenetic relationship implies that unique S proteins from North America are significantly different from those of the other five continents. They are most likely to spread to the other geographic locations through international travel or naturally by emerging mutations. It is suggested that restriction of international travel should be considered, and massive vaccination as an utmost measure to combat the spread of the 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.

Analysis of Purines and Pyrimidines distribution over miRNAs of Human, Gorilla, Chimpanzee, Mouse and Rat.

Meaningful words in English need vowels to break up the sounds that consonants make. The Nature has encoded her messages in RNA molecules using only four alphabets A, U, C and G in which the nine member double-ring bases (adenine (A) and Guanine (G)) are purines, while the six member single-ring bases (cytosine (C) and uracil (U)) are pyrimidines. Four bases A, U, C and G of RNA sequences are divided into three kinds of classifications according to their chemical properties. One of the three classifications, the purine-pyrimidine class is important. In understanding the distribution (organization) of purines and pyrimidines over some of the non-coding RNAs, all miRNAs of the three species (human, gorilla and chimpanzee) of Hominidae family and two species (mouse and rat) from of Muridae family are considered. The distribution of purines and pyrimidines over miRNA shows deviation from randomness. Based on the quantitative metrics (fractal dimension, Hurst exponent, Hamming distance, distance pattern of purine-pyrimidine, density distribution of purine-pyrimidine and Shannon entropy) five different clusters have been made for the five species. We have observed some couple of results including the closeness of different clusters among the five species.