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1.
Vet Q ; 40(1): 243-249, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-2315258

ABSTRACT

Several cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection transmitted from human owners to their dogs have recently been reported. The first ever case of SARS-CoV-2 transmission from a human owner to a domestic cat was confirmed on March 27, 2020. A tiger from a zoo in New York, USA, was also reportedly infected with SARS-CoV-2. It is believed that SARS-CoV-2 was transmitted to tigers from their caretakers, who were previously infected with this virus. On May 25, 2020, the Dutch Minister of Agriculture, Nature and Food Quality reported that two employees were infected with SARS-CoV-2 transmitted from minks. These reports have influenced us to perform a comparative analysis among angiotensin-converting enzyme 2 (ACE2) homologous proteins for verifying the conservation of specific protein regions. One of the most conserved peptides is represented by the peptide "353-KGDFR-357 (H. sapiens ACE2 residue numbering), which is located on the surface of the ACE2 molecule and participates in the binding of SARS-CoV-2 spike receptor binding domain (RBD). Multiple sequence alignments of the ACE2 proteins by ClustalW, whereas the three-dimensional structure of its binding region for the spike glycoprotein of SARS-CoV-2 was assessed by means of Spanner, a structural homology modeling pipeline method. In addition, evolutionary phylogenetic tree analysis by ETE3 was used. ACE2 works as a receptor for the SARS-CoV-2 spike glycoprotein between humans, dogs, cats, tigers, minks, and other animals, except for snakes. The three-dimensional structure of the KGDFR hosting protein region involved in direct interactions with SARS-CoV-2 spike RBD of the mink ACE2 appears to form a loop structurally related to the human ACE2 corresponding protein loop, despite of the reduced available protein length (401 residues of the mink ACE2 available sequence vs 805 residues of the human ACE2). The multiple sequence alignments of the ACE2 proteins shows high homology and complete conservation of the five amino acid residues: 353-KGDFR-357 with humans, dogs, cats, tigers, minks, and other animals, except for snakes. Where the information revealed from our examinations can support precision vaccine design and the discovery of antiviral therapeutics, which will accelerate the development of medical countermeasures, the World Health Organization recently reported on the possible risks of reciprocal infections regarding SARS-CoV-2 transmission from animals to humans.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/transmission , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/transmission , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/genetics , COVID-19 , Cats , Coronavirus Infections/prevention & control , Dogs , Humans , Mink , Pandemics/prevention & control , Peptidyl-Dipeptidase A/chemistry , Phylogeny , Pneumonia, Viral/prevention & control , Receptors, Virus/chemistry , Receptors, Virus/genetics , SARS-CoV-2 , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Tigers
2.
J Appl Microbiol ; 130(1): 2-13, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-2299665

ABSTRACT

AIMS: Providing a ready-to-use reverse transcriptase qPCR (RT-qPCR) method fully validated to detect the SARS-CoV-2 with a higher exclusivity than this shown by early published RT-qPCR designs. METHODS AND RESULTS: The specificity of the GPS™ CoVID-19 dtec-RT-qPCR test by analysis of sequence alignments was approached and compared with other RT-qPCR designs. The GPS™ CoVID-19 dtec-RT-qPCR test was validated following criteria of UNE/EN ISO 17025:2005 and ISO/IEC 15189:2012. Diagnostic validation was achieved by two independent reference laboratories, the Instituto de Salud Carlos III, (Madrid, Spain), the Public Health England (Colindale, London, UK), and received the label CE-IVD. The GPS design showed the highest exclusivity and passed all parameters of validation with strict acceptance criteria. Results from reference laboratories 100% correlated with these obtained by using reference methods and showed 100% of diagnostic sensitivity and specificity. CONCLUSIONS: The CE-IVD GPS™ CoVID-19 dtec-RT-qPCR test, available worldwide with full analytical and diagnostic validation, is the more exclusive for SARS-CoV-2 by far. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the CoVID-19 pandemic status, the exclusivity of RT-qPCR tests is crucial to avoid false positives due to related coronaviruses. This work provides of a highly specific and validated RT-qPCR method for detection of SARS-CoV-2, which represents a case of efficient transfer of technology successfully used since the pandemic was declared.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , COVID-19 Nucleic Acid Testing/standards , Computer Simulation , Humans , Pandemics , Real-Time Polymerase Chain Reaction , Reproducibility of Results , SARS-CoV-2/classification , SARS-CoV-2/genetics , Sensitivity and Specificity , Sequence Alignment
3.
J Math Biol ; 86(3): 34, 2023 01 25.
Article in English | MEDLINE | ID: covidwho-2227957

ABSTRACT

We propose a novel mathematical paradigm for the study of genetic variation in sequence alignments. This framework originates from extending the notion of pairwise relations, upon which current analysis is based on, to k-ary dissimilarity. This dissimilarity naturally leads to a generalization of simplicial complexes by endowing simplices with weights, compatible with the boundary operator. We introduce the notion of k-stances and dissimilarity complex, the former encapsulating arithmetic as well as topological structure expressing these k-ary relations. We study basic mathematical properties of dissimilarity complexes and show how this approach captures watershed moments of viral dynamics in the context of SARS-CoV-2 and H1N1 flu genomic data.


Subject(s)
COVID-19 , Influenza A Virus, H1N1 Subtype , Humans , SARS-CoV-2/genetics , Sequence Alignment
4.
Viruses ; 15(1)2023 Jan 05.
Article in English | MEDLINE | ID: covidwho-2216949

ABSTRACT

Pseudorabies virus (PRV) generally infects pigs and threatens the pig industry. However, recently we have isolated a PRV strain designated hSD-1/2019 from infected humans. In this study, we compared the complete genome sequence of hSD-1/2019 with those of pig-originated PRV strains. Sequence alignments revealed that the genome sequence of hSD-1/2019 was highly homologous to those of the porcine PRV strains. Phylogenetic analyses found that hSD-1/2019 was the closest related to porcine PRV endemic strains in China, particularly the variant strains circulating recently. We also showed that the glycoproteins important for the multiplication and pathogenesis of hSD-1/2019 were highly similar to those of the pig endemic strains. Diversifying selection analyses revealed that hSD-1/2019 and pig variant strains are under diversifying selection. Recombination analysis indicated that hSD-1/2019 was a recombinant of several PRV variant strains and an earlier PRV classic strain. Finally, we found that both human and pig-originated PRV strains could induce cytopathic effects in cells from humans, pigs, and mice, but only the human PRV and pig-variant PRV formed large syncytia in human cell lines. The data presented in this study contribute to our understanding of the molecular basis for the pathogenesis of human PRV from a genomic aspect.


Subject(s)
Herpesvirus 1, Suid , Pseudorabies , Swine Diseases , Humans , Swine , Animals , Mice , Phylogeny , Genomics , Sequence Alignment
5.
J Biotechnol ; 359: 130-141, 2022 Nov 20.
Article in English | MEDLINE | ID: covidwho-2049401

ABSTRACT

Pattern detection and string matching are fundamental problems in computer science and the accelerated expansion of bioinformatics and computational biology have made them a core topic for both disciplines. The requirement for computational tools for genomic analyses, such as sequence alignment, is very important, although, in most cases the resources and computational power required are enormous. The presented Multiple Genome Analytics Framework combines data structures and algorithms, specifically built for text mining and (repeated) pattern detection, that can help to efficiently address several computational biology and bioinformatics problems, concurrently, with minimal resources. A single execution of advanced algorithms, with space and time complexity O(nlogn), is enough to acquire knowledge on all repeated patterns that exist in multiple genome sequences and this information can be used as input by meta-algorithms for further meta-analyses. For the proof of concept and technology of the proposed Framework scalability, agility and efficiency, a publicly available dataset of more than 300,000 SARS-CoV-2 genome sequences from the National Center for Biotechnology Information has been used for the detection of all repeated patterns. These results have been used by newly introduced algorithms to provide answers to questions such as common patterns among all variants, sequence alignment, palindromes and tandem repeats detection, different organism genome comparisons, polymerase chain reaction primers detection, etc.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Genome, Viral/genetics , Sequence Alignment , Computational Biology
6.
Comput Intell Neurosci ; 2022: 6980335, 2022.
Article in English | MEDLINE | ID: covidwho-2038380

ABSTRACT

An area of medical science, that is, gaining prominence, is DNA sequencing. Genetic mutations responsible for the disease have been detected using DNA sequencing. The research is focusing on pattern identification methodologies for dealing with DNA-sequencing problems relating to various applications. A few examples of such problems are alignment and assembly of short reads from next generation sequencing (NGS), comparing DNA sequences, and determining the frequency of a pattern in a sequence. The approximate matching of DNA sequences is also well suited for many applications equivalent to the exact matching of the sequence since the DNA sequences are often subject to mutation. Consequently, recognizing pattern similarity becomes necessary. Furthermore, it can also be used in virtually every application that calls for pattern matching, for example, spell-checking, spam filtering, and search engines. According to the traditional approach, finding a similar pattern in the case where the sequence length is l s and the pattern length is l p occurs in O (l s ∗l p ). This heavy processing is caused by comparing every character of the sequence repeatedly with the pattern. The research intended to reduce the time complexity of the pattern matching by introducing an approach named "optimized pattern similarity identification" (OPSI). This methodology constructs a table, entitled "shift beyond for avoiding redundant comparison" (SBARC), to bypass the characters in the texts that are already compared with the pattern. The table pertains to the information about the character distance to be skipped in the matching. OPSI discovers at most spots of similar patterns occur in the sequence (by ignoring è mismatches). The experiment resulted in the time complexity identified as O (l s . è). In comparison to the size of the pattern, the allowed number of mismatches will be much smaller. Aspects such as scalability, generalizability, and performance of the OPSI algorithm are discussed. In comparison with the hamming distance-based approximate pattern matching algorithm, the proposed algorithm is found to be 69% more efficient.


Subject(s)
Algorithms , Internet , DNA , Sequence Alignment , Sequence Analysis, DNA
7.
PLoS One ; 17(6): e0254736, 2022.
Article in English | MEDLINE | ID: covidwho-1933199

ABSTRACT

In bioinformatics, alignment is an essential technique for finding similarities between biological sequences. Usually, the alignment is performed with the Smith-Waterman (SW) algorithm, a well-known sequence alignment technique of high-level precision based on dynamic programming. However, given the massive data volume in biological databases and their continuous exponential increase, high-speed data processing is necessary. Therefore, this work proposes a parallel hardware design for the SW algorithm with a systolic array structure to accelerate the forward and backtracking steps. For this purpose, the architecture calculates and stores the paths in the forward stage for pre-organizing the alignment, which reduces the complexity of the backtracking stage. The backtracking starts from the maximum score position in the matrix and generates the optimal SW sequence alignment path. The architecture was validated on Field-Programmable Gate Array (FPGA), and synthesis analyses have shown that the proposed design reaches up to 79.5 Giga Cell Updates per Second (GCPUS).


Subject(s)
Algorithms , Computational Biology , Computational Biology/methods , Databases, Factual , Equipment Design , Sequence Alignment , Software
8.
Bioinformatics ; 38(16): 4033-4035, 2022 Aug 10.
Article in English | MEDLINE | ID: covidwho-1922198

ABSTRACT

SUMMARY: gofasta comprises a set of command-line utilities for handling alignments of short assembled genomes in a genomic epidemiology context. It was developed for processing large numbers of closely related SARS-CoV-2 viral genomes and should be useful with other densely sampled pathogen genomic datasets. It provides functions to convert sam-format pairwise alignments between assembled genomes to fasta format; to annotate mutations in multiple sequence alignments, and to extract sets of sequences by genetic distance measures for use in outbreak investigations. AVAILABILITY AND IMPLEMENTATION: gofasta is an open-source project distributed under the MIT license. Binaries are available at https://github.com/virus-evolution/gofasta, from Bioconda, and through the Go programming language's package management system. Source code and further documentation, including walkthroughs for common use cases, are available on the GitHub repository.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/epidemiology , Genomics , Sequence Alignment , Software
9.
BMC Bioinformatics ; 23(1): 239, 2022 Jun 18.
Article in English | MEDLINE | ID: covidwho-1894413

ABSTRACT

BACKGROUND: This paper presents a new R/Bioconductor package, rprimer, for design of degenerate oligos and PCR assays for sequence variable viruses. A multiple DNA sequence alignment is used as input data, while the outputs consist of comprehensive tables (data frames) and dashboard-like plots. The workflow can be run directly from the R console or through a graphical user interface (Shiny application). Here, rprimer is demonstrated and evaluated by using it to design two norovirus genogroup I (GI) assays: one RT-qPCR assay for quantitative detection and one RT­PCR assay for Sanger sequencing and polymerase-capsid based genotyping. RESULTS: The assays generated were evaluated using stool samples testing positive for norovirus GI. The RT-qPCR assay accurately amplified and quantified all samples and showed comparable performance to a widely-used standardised assay, while the RT-PCR assay resulted in successful sequencing and genotyping of all samples. Merits and limitations of the package were identified through comparison with three similar freely available software packages. Several features were comparable across the different tools, but important advantages of rprimer were its speed, flexibility in oligo design and capacity for visualisation. CONCLUSIONS: An R/Bioconductor package, rprimer, was developed and shown to be successful in designing primers and probes for quantitative detection and genotyping of a sequence-variable virus. The package provides an efficient, flexible and visual approach to degenerate oligo design, and can therefore assist in virus research and method development.


Subject(s)
Norovirus , DNA Primers/genetics , Norovirus/genetics , Real-Time Polymerase Chain Reaction/methods , Sequence Alignment
10.
BMC Genomics ; 23(1): 377, 2022 May 18.
Article in English | MEDLINE | ID: covidwho-1849668

ABSTRACT

BACKGROUND: In the pursuit of a better understanding of biodiversity, evolutionary biologists rely on the study of phylogenetic relationships to illustrate the course of evolution. The relationships among natural organisms, depicted in the shape of phylogenetic trees, not only help to understand evolutionary history but also have a wide range of additional applications in science. One of the most challenging problems that arise when building phylogenetic trees is the presence of missing biological data. More specifically, the possibility of inferring wrong phylogenetic trees increases proportionally to the amount of missing values in the input data. Although there are methods proposed to deal with this issue, their applicability and accuracy is often restricted by different constraints. RESULTS: We propose a framework, called PhyloMissForest, to impute missing entries in phylogenetic distance matrices and infer accurate evolutionary relationships. PhyloMissForest is built upon a random forest structure that infers the missing entries of the input data, based on the known parts of it. PhyloMissForest contributes with a robust and configurable framework that incorporates multiple search strategies and machine learning, complemented by phylogenetic techniques, to provide a more accurate inference of lost phylogenetic distances. We evaluate our framework by examining three real-world datasets, two DNA-based sequence alignments and one containing amino acid data, and two additional instances with simulated DNA data. Moreover, we follow a design of experiments methodology to define the hyperparameter values of our algorithm, which is a concise method, preferable in comparison to the well-known exhaustive parameters search. By varying the percentages of missing data from 5% to 60%, we generally outperform the state-of-the-art alternative imputation techniques in the tests conducted on real DNA data. In addition, significant improvements in execution time are observed for the amino acid instance. The results observed on simulated data also denote the attainment of improved imputations when dealing with large percentages of missing data. CONCLUSIONS: By merging multiple search strategies, machine learning, and phylogenetic techniques, PhyloMissForest provides a highly customizable and robust framework for phylogenetic missing data imputation, with significant topological accuracy and effective speedups over the state of the art.


Subject(s)
Algorithms , DNA , Amino Acids , Phylogeny , Sequence Alignment
11.
Front Public Health ; 10: 889973, 2022.
Article in English | MEDLINE | ID: covidwho-1847248

ABSTRACT

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays are the most widely used molecular tests for the detection of SARS-CoV-2 and diagnosis of COVID-19 in clinical samples. PCR assays target unique genomic RNA regions to identify SARS-CoV-2 with high sensitivity and specificity. In general, assay development incorporates the whole genome sequences available at design time to be inclusive of all target species and exclusive of near neighbors. However, rapid accumulation of mutations in viral genomes during sustained growth in the population can result in signature erosion and assay failures, creating situational blind spots during a pandemic. In this study, we analyzed the signatures of 43 PCR assays distributed across the genome against over 1.6 million SARS-CoV-2 sequences. We present evidence of significant signature erosion emerging in just two assays due to mutations, while adequate sequence identity was preserved in the other 41 assays. Failure of more than one assay against a given variant sequence was rare and mostly occurred in the two assays noted to have signature erosion. Assays tended to be designed in regions with statistically higher mutations rates. in silico analyses over time can provide insights into mutation trends and alert users to the emergence of novel variants that are present in the population at low proportions before they become dominant. Such routine assessment can also potentially highlight false negatives in test samples that may be indicative of mutations having functional consequences in the form of vaccine and therapeutic failures. This study highlights the importance of whole genome sequencing and expanded real-time monitoring of diagnostic PCR assays during a pandemic.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Mutation , Polymerase Chain Reaction , SARS-CoV-2/genetics , Sequence Alignment
12.
Biomolecules ; 12(4)2022 04 06.
Article in English | MEDLINE | ID: covidwho-1809687

ABSTRACT

The continuous development of sequencing technologies has enabled researchers to obtain large amounts of biological sequence data, and this has resulted in increasing demands for software that can perform sequence alignment fast and accurately. A number of algorithms and tools for sequence alignment have been designed to meet the various needs of biologists. Here, the ideas that prevail in the research of sequence alignment and some quality estimation methods for multiple sequence alignment tools are summarized.


Subject(s)
Algorithms , Software , Sequence Alignment , Sequence Analysis
13.
PLoS One ; 17(3): e0264640, 2022.
Article in English | MEDLINE | ID: covidwho-1731599

ABSTRACT

The SARS-CoV-2 is the third coronavirus in addition to SARS-CoV and MERS-CoV that causes severe respiratory syndrome in humans. All of them likely crossed the interspecific barrier between animals and humans and are of zoonotic origin, respectively. The origin and evolution of viruses and their phylogenetic relationships are of great importance for study of their pathogenicity and development of antiviral drugs and vaccines. The main objective of the presented study was to compare two methods for identifying relationships between coronavirus genomes: phylogenetic one based on the whole genome alignment followed by molecular phylogenetic tree inference and alignment-free clustering of triplet frequencies, respectively, using 69 coronavirus genomes selected from two public databases. Both approaches resulted in well-resolved robust classifications. In general, the clusters identified by the first approach were in good agreement with the classes identified by the second using K-means and the elastic map method, but not always, which still needs to be explained. Both approaches demonstrated also a significant divergence of genomes on a taxonomic level, but there was less correspondence between genomes regarding the types of diseases they caused, which may be due to the individual characteristics of the host. This research showed that alignment-free methods are efficient in combination with alignment-based methods. They have a significant advantage in computational complexity and provide valuable additional alternative information on the genomes relationships.


Subject(s)
Comparative Genomic Hybridization/methods , Coronavirus/genetics , Genome, Viral , Chromosome Mapping , Cluster Analysis , Coronavirus/classification , Humans , Phylogeny , SARS-CoV-2/classification , SARS-CoV-2/genetics , Sequence Alignment
14.
Protein J ; 39(3): 198-216, 2020 06.
Article in English | MEDLINE | ID: covidwho-1718840

ABSTRACT

The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Betacoronavirus/genetics , COVID-19 , Coronavirus Envelope Proteins , Coronavirus Infections/drug therapy , Coronavirus Infections/metabolism , Coronavirus Nucleocapsid Proteins , Drug Discovery/methods , Genome, Viral , Host-Pathogen Interactions/drug effects , Humans , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/drug therapy , Pneumonia, Viral/metabolism , Polyproteins , Protein Interaction Maps/drug effects , SARS-CoV-2 , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Proteins/genetics , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Viroporin Proteins
15.
J Med Virol ; 94(4): 1627-1632, 2022 04.
Article in English | MEDLINE | ID: covidwho-1718407

ABSTRACT

Following the discovery of the SARS-CoV-2 Omicron variant (B.1.1.529), the global COVID-19 outbreak has resurfaced after appearing to be relentlessly spreading over the past 2 years. This new variant showed marked degree of mutation, compared with the previous SARS-CoV-2 variants. This study investigates the evolutionary links between Omicron variant and recently emerged SARS-CoV-2 variants. The entire genome sequences of SARS-CoV-2 variants were obtained, aligned using Clustal Omega, pairwise comparison was computed, differences, identity percent, gaps, and mutations were noted, and the identity matrix was generated. The phylogenetics of Omicron variants were determined using a variety of evolutionary substitution models. The ultrametric and metric clustering methods, such as UPGMA and neighbor-joining (NJ), using nucleotide substitution models that allowed the inclusion of nucleotide transitions and transversions as Kimura 80 models, revealed that the Omicron variant forms a new monophyletic clade that is distant from other SARS-CoV-2 variants. In contrast, the NJ method using a basic nucleotide substitution model such as Jukes-Cantor revealed a close relationship between the Omicron variant and the recently evolved Alpha variant. Based on the percentage of sequence identity, the closest variants were in the following order: Omicron, Alpha, Gamma, Delta, Beta, Mu, and then the SARS-CoV-2 USA isolate. A genome alignment with other variants indicated the greatest number of gaps in the Omicron variant's genome ranging from 43 to 63 gaps. It is possible, given their close relationship to the Alpha variety, that Omicron has been around for much longer than predicted, even though they created a separate monophyletic group. Sequencing initiatives in a systematic and comprehensive manner is highly recommended to study the evolution and mutations of the virus.


Subject(s)
Evolution, Molecular , Genome, Viral/genetics , Phylogeny , SARS-CoV-2/genetics , Base Sequence , COVID-19/epidemiology , COVID-19/virology , Humans , Mutation , Sequence Alignment
16.
Microbiol Spectr ; 10(1): e0278021, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1700612

ABSTRACT

Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8+ T-cell epitopes across the nucleocapsid (N), spike (S), and open reading frame (ORF)3a proteins of SARS-CoV-2 and five CD8+ T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8+ T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad preexisting immunity in the community. IMPORTANCE T cells play a critical role in protection against SARS-CoV-2. Despite being highly topical, the protective role of preexisting memory CD8+ T cells, induced by prior exposure to circulating common coronavirus strains, remains less clear. In this study, we established a robust approach to specifically assess T cell responses to highly conserved regions within SARS-CoV-2. Consistent with recent observations we demonstrate that recognition of these highly conserved regions is associated with an increased likelihood of milder disease. However, extending these observations we observed that recognition of these conserved regions is rare in both exposed and unexposed volunteers, which we believe is associated with the low abundance of these proteins in SARS-CoV-2 infected cells. These observations have important implications for the likely role preexisting immunity plays in controlling severe disease, further emphasizing the importance of vaccination to generate the immunodominant T cells required for immune protection.


Subject(s)
COVID-19/immunology , Epitopes, T-Lymphocyte/immunology , SARS-CoV-2/immunology , Amino Acid Sequence , CD8-Positive T-Lymphocytes/immunology , COVID-19/genetics , COVID-19/virology , Conserved Sequence , Coronavirus/chemistry , Coronavirus/classification , Coronavirus/genetics , Coronavirus/immunology , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cross Reactions , Epitope Mapping , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/genetics , Humans , Memory T Cells/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
17.
Nat Commun ; 13(1): 317, 2022 01 14.
Article in English | MEDLINE | ID: covidwho-1671550

ABSTRACT

Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.


Subject(s)
Arthropod Proteins/chemistry , Arthropod Proteins/immunology , Complement Inactivating Agents/chemistry , Complement Inactivating Agents/immunology , Properdin/immunology , Rhipicephalus/immunology , Amino Acid Sequence , Animals , Arthropod Proteins/genetics , Complement Activation , Complement C3/chemistry , Complement C3/immunology , Complement Pathway, Alternative , Humans , Kinetics , Properdin/chemistry , Properdin/genetics , Rhipicephalus/chemistry , Rhipicephalus/genetics , Sequence Alignment
18.
Nat Commun ; 12(1): 7325, 2021 12 16.
Article in English | MEDLINE | ID: covidwho-1585854

ABSTRACT

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Crystallography, X-Ray , Receptors, Antigen/chemistry , Receptors, Antigen/immunology , Sharks/immunology , Angiotensin-Converting Enzyme 2 , Animals , COVID-19 , Epitopes , Mutation , Phylogeny , Protein Binding , SARS-CoV-2 , Sequence Alignment , Single-Domain Antibodies , Spike Glycoprotein, Coronavirus/immunology
19.
Proc Natl Acad Sci U S A ; 118(52)2021 12 28.
Article in English | MEDLINE | ID: covidwho-1565770

ABSTRACT

The constant emergence of COVID-19 variants reduces the effectiveness of existing vaccines and test kits. Therefore, it is critical to identify conserved structures in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes as potential targets for variant-proof diagnostics and therapeutics. However, the algorithms to predict these conserved structures, which simultaneously fold and align multiple RNA homologs, scale at best cubically with sequence length and are thus infeasible for coronaviruses, which possess the longest genomes (∼30,000 nt) among RNA viruses. As a result, existing efforts on modeling SARS-CoV-2 structures resort to single-sequence folding as well as local folding methods with short window sizes, which inevitably neglect long-range interactions that are crucial in RNA functions. Here we present LinearTurboFold, an efficient algorithm for folding RNA homologs that scales linearly with sequence length, enabling unprecedented global structural analysis on SARS-CoV-2. Surprisingly, on a group of SARS-CoV-2 and SARS-related genomes, LinearTurboFold's purely in silico prediction not only is close to experimentally guided models for local structures, but also goes far beyond them by capturing the end-to-end pairs between 5' and 3' untranslated regions (UTRs) (∼29,800 nt apart) that match perfectly with a purely experimental work. Furthermore, LinearTurboFold identifies undiscovered conserved structures and conserved accessible regions as potential targets for designing efficient and mutation-insensitive small-molecule drugs, antisense oligonucleotides, small interfering RNAs (siRNAs), CRISPR-Cas13 guide RNAs, and RT-PCR primers. LinearTurboFold is a general technique that can also be applied to other RNA viruses and full-length genome studies and will be a useful tool in fighting the current and future pandemics.


Subject(s)
Algorithms , RNA, Viral/chemistry , SARS-CoV-2/chemistry , Betacoronavirus/chemistry , Betacoronavirus/genetics , Conserved Sequence , Genome, Viral , Mutation , Nucleic Acid Conformation , RNA Folding , RNA, Viral/genetics , SARS-CoV-2/genetics , Sequence Alignment
20.
Sci Rep ; 11(1): 23622, 2021 12 08.
Article in English | MEDLINE | ID: covidwho-1559938

ABSTRACT

Spike glycoprotein (Sgp) is liable for binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the host receptors. Since Sgp is the main target for vaccine and drug designing, elucidating its mutation pattern could help in this regard. This study is aimed at investigating the correspondence of specific residues to the SgpSARS-CoV-2 functionality by explorative interpretation of sequence alignments. Centrality analysis of the Sgp dissects the importance of these residues in the interaction network of the RBD-ACE2 (receptor-binding domain) complex and furin cleavage site. Correspondence of RBD to threonine500 and asparagine501 and furin cleavage site to glutamine675, glutamine677, threonine678, and alanine684 was observed; all residues are exactly located at the interaction interfaces. The harmonious location of residues dictates the RBD binding property and the flexibility, hydrophobicity, and accessibility of the furin cleavage site. These species-specific residues can be assumed as real targets of evolution, while other substitutions tend to support them. Moreover, all these residues are parts of experimentally identified epitopes. Therefore, their substitution may affect vaccine efficacy. Higher rate of RBD maintenance than furin cleavage site was predicted. The accumulation of substitutions reinforces the probability of the multi-host circulation of the virus and emphasizes the enduring evolutionary events.


Subject(s)
SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Cluster Analysis , Humans , Markov Chains , Mutation , Protein Binding , Protein Domains/genetics , SARS-CoV-2/isolation & purification , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
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